WO2023052447A2 - Methods and compositions - Google Patents

Methods and compositions Download PDF

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Publication number
WO2023052447A2
WO2023052447A2 PCT/EP2022/077016 EP2022077016W WO2023052447A2 WO 2023052447 A2 WO2023052447 A2 WO 2023052447A2 EP 2022077016 W EP2022077016 W EP 2022077016W WO 2023052447 A2 WO2023052447 A2 WO 2023052447A2
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Prior art keywords
coiled
seq
coil
domain
cargo
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PCT/EP2022/077016
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French (fr)
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WO2023052447A3 (en
Inventor
Laura Von Schantz
Barnabas NYESIGA
Karin HÄGERBRAND
Laura Varas
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Alligator Bioscience Ab
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Publication of WO2023052447A2 publication Critical patent/WO2023052447A2/en
Publication of WO2023052447A3 publication Critical patent/WO2023052447A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)

Definitions

  • the present invention relates to the field of targeted delivery of cargo.
  • Therapeutic cancer vaccines have a poor clinical track record, mainly due to the choice of vaccination antigens, which mostly have been tumor-associated self-antigens that do not generate a strong tumor-specific T cell response.
  • the breakthroughs in immunotherapy have shown that for a vaccine to generate a strong T cell response, it is critical to target tumor-specific antigens, e.g. neoantigens.
  • neoantigen-load (mutational burden) of patients' tumors correlate with favorable clinical outcome.
  • tumor neoantigen-specific T cells are expanded by successful immunotherapies (targeting PD- 1, CTLA-4).
  • neoantigen-specific T cells are responsible for the anti-tumor effect of successful immunotherapies.
  • the main challenges to address when developing immune cell-targeted therapies are to: i) provide the right type of immune activation that generates an effective antitumor T cell response, and ii) target tumor antigens, such as neoantigens, that can induce a strong antitumor response.
  • an issue with current methods of antibody-targeted vaccination is that a new antigenbinding molecule, i.e. antibody, has to be generated for each different antigen that is to be delivered to an immune cell, such as a dendritic cell (DC).
  • DC dendritic cell
  • the present invention at least addresses this issue and provides an off-the-shelf polypeptide that can be used to deliver a range of antigens to a particular target cell population.
  • the present invention comprises a polypeptide that in some instances is capable of both optimizing neoantigen uptake by an immune cell and/or cross-presentation of the antigen to T cells.
  • the tumor of a patient is sequenced to identify suitable personalized neoantigens.
  • These neoantigens may then be produced and attached to, for example, a DC-activating antibody of one embodiment of the invention that promotes uptake of the neoantigen by the DC and at the same time mediates a superior activation of the DC, which in turn allows the DC to cross-present the neoantigens and activate a strong T cell response towards the tumor, and cure the patient.
  • polypeptides and complexes described herein can be used in contexts other than the delivery of antigens to immune cells.
  • the polypeptides and complexes described herein are able to deliver a range of different cargo to a range of different target sites.
  • Antibodies may be selective for DCs and/or other antigen-presenting cells (APC). Antigen fused to a DC-targeting antibody is more efficiently taken up by the DC and subsequently presented to T cells in the context of MHC class I or II compared to free antigen. Consequently, DC-targeted antigen elicits stronger T cell responses compared to free antigen or antigen coupled to an isotype control antibody, and induces superior anti-tumor responses in mouse models [2]. An additional benefit can be gained by incorporating an immune-activating stimulus in the same molecule, for example by targeting an activating DC receptor with an agonistic antibody, since antigen-adjuvant complexes induce superior anti-tumor responses compared to a corresponding mix of antigen and adjuvant [3].
  • APC antigen-presenting cells
  • Efficient priming of CD8+ CTL responses is important for generating a productive anti-tumor response, and cDCl play a prominent role in priming CTL responses for anti-tumor immunity [4].
  • a productive immune response against a tumor involves both CD8+ and CD4+ T cells, however inducing CD8+ rather than CD4+ T cell responses remains a challenge for cancer vaccines, including neoantigen vaccines. Delivering antigen to a DC population that includes cDCl is likely to promote the induction of antigen-specific CTL.
  • DC target determines which DC population that is targeted, and how much of the antigen that is taken up and how much is presented on MHC II versus MHC I, the latter being critical for presentation to CD8+ T cells. Further, the choice of DC target also affects the level of DC activation following antigen uptake, which determines if antigen-specific T cells are activated or suppressed.
  • a number of different DC targets have been evaluated for antibody-targeted vaccination, including e.g. CR- 1, CLEC9A, DEC-205, CDlc, Dec-1, CDllb, CDllc and CD40 [5-7].
  • CD40 stimulation activates DC and induces cross-presentation.
  • targeted antigens i.e. an antibody fused to an antigenic peptide binding to CD40 induce superior CD8+ T cell responses compared to e.g. DEC-205 [6].
  • CD40 was superior to nine different lectins and scavenger receptors (LOX-1, DC-ASGPR, DCIR, Dectin-1, DEC205, Langerin, MARCO, CLEC6, and DC- SIGN/L) with respect to generating a CD8+ T cell response, using primary human cells in vitro [8].
  • CD40 primarily mediated internalization into early endosomes, which favors antigen processing and cross-presentation to CD8+ T cells.
  • the goal with the present invention is to provide a drug candidate capable of delivering patient-specific tumor neoantigen to DC, and simultaneously activating the DC, thereby leading to superior priming and activation of neoantigen-specific T cells.
  • the activated T cells will then mediate a superior anti-tumor effect against the neoantigen-expressing tumors.
  • the molecule should mediate DC targeting, activation and antigen internalization, all of which could be achieved using CD40 as DC target.
  • the molecule comprising a CD40-binding domain and a tag-binding domain (for example a peptide- tag binding domain i.e.
  • CD40-neoantigen complexes a domain that binds to a peptide tag
  • neoantigens for example neoantigenic peptides fused to a tag (for example a peptide tag)
  • a tag for example a peptide tag
  • CD40-neoantigen complexes should be superior to a formulation comprising an anti-CD40 antibody and neoantigen peptide, in terms of expansion and/or function of neoantigen-specific T cells and/or anti-tumor effect.
  • CD40-neoantigen complex we mean a complex that forms between 1) the molecule that comprises the CD40-binding domain and the tag-binding domain; and 2) the neoantigen fused to the tag - i.e. it is a complex that targets the neoantigen to cells expressing CD40.
  • CD40-tag binding antibodies i.e. antibody that binds to both CD40 and to a tag
  • neoantigen-tag entities i.e. a neoantigen fused to a tag
  • CD40-tag antibodies i.e. capable of binding to CD40 and to the tag
  • the CD40-tag antibodies would be available as an off-the-shelf product, ready to be mixed with the tagged personal neoantigens (for example antigenic peptides) as soon as they have been synthetized.
  • the CD40-tag antibodies could be mixed with tagged antigens that encompass several neoantigen epitopes.
  • Vaccinating against one neoantigen epitope is generally not sufficient to induce a long-lasting anti-tumor effect, and several neoantigen epitopes will likely be needed to elicit a strong anti-tumor response. In this fashion, the invention addresses limitations with the currently available antibody-antigen fusion proteins.
  • the invention provides a polypeptide molecule that in some embodiments is considered to be a cargo-targeting polypeptide or a cargo-delivery polypeptide.
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is able to bind to at least one target antigen, for example a target antigen on an immune cell such as a dendritic cell, and is also able to bind to a tag, for example a peptide tag.
  • the tag for example the peptide tag in preferred embodiments is a coiled-coil oligomerization domain that is attached to a cargo.
  • the cargo may be any cargo that is desired to be targeted to or delivered to a particular site, for example a particular cell type, or tissue, and is further defined elsewhere herein.
  • a tag that is a coiled-coil oligomerisation domain that is attached to a cargo can be referred to as a cargo coiled-coil oligomerisation domain to distinguish it from a corresponding coiled- coil oligomerisation domain present on the polypeptide of the invention, for example on the cargo-targeting or cargo-delivery polypeptide of the invention.
  • a single polypeptide of the invention for example a single cargo-targeting or a cargo-delivery polypeptide of the invention can be used to target a diverse array of cargo a particular cellular targets.
  • the present invention provides a polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprising at least a first target antigen binding domain and at least a first tag binding domain, wherein the first target antigen binding domain is capable of specifically binding to a first target antigen.
  • the first tag binding domain comprises a first coiled-coil oligomerisation domain.
  • the invention provides a polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprising at least a first target antigen binding domain and at least a first tag binding domain wherein the first target antigen binding domain is capable of specifically binding to a first target antigen; and wherein the first tag binding domain comprises a first coiled- coil oligomerisation domain.
  • a polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprising at least a first target antigen binding domain and at least a first tag binding domain wherein the first target antigen binding domain is capable of specifically binding to a first target antigen; and wherein the first tag binding domain comprises a first coiled- coil oligomerisation domain.
  • the target antigen binding domain can be considered to be an immune cell binding domain that is capable of specifically binding to a target present on an immune cell.
  • the tag binding domain does not bind to a peptide tag that is derived from tetanus toxin.
  • binding domain we include the meaning of any peptide sequence that is capable of binding or associating with the relevant second entity.
  • the domain is able to bind specifically to a target antigen.
  • the first target antigen is a target antigen on an immune cell and so the target antigen binding domain can be considered to be an immune cell binding domain.
  • the domain is able to bind specifically to a target on an immune cell.
  • the target is CD40, and the target binding domain comprise an immunoglobulin or fragment thereof that binds to CD40. Exemplary antibodies are described elsewhere herein.
  • the tag binding domain is able to specifically bind to a tag, for example a peptide tag.
  • the tag binding domain comprises a coiled-coil oligomerisation domain that can form a multimer, for example a dimer for example a heterodimer with a corresponding coiled-coil oligomerisation domain.
  • a coiled-coil domain of the heterodimer is present on the polypeptide of the invention, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention, and the corresponding coiled-coil domain of the heterodimer is connected to or attached to the cargo.
  • the interaction between the two coiled-coil oligomerisation domains results in the formation of a complex and the cargo being associated with the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention- in this way the cargo is targeted to the target antigen (for example immune cell) by virtue of the antigen binding domain of the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention.
  • bind specifically we include the meaning that the domain binds to its target in a manner that can be distinguished from binding to non-target domains (i.e. off-targets).
  • a domain that binds specifically may refer to a domain that binds with higher specificity for the intended target compared with that of a non-intended target. Specificity can be determined based on dissociation constant through routine experiments.
  • a domain being "specific for" a target is intended to be synonymous with a domain "directed against” said target.
  • the binding domains will bind only to its respective target, i.e. an antigen, for example an antigen present on an immune cell target, or the relevant tag, for example the peptide tag, for example a coiled-coil oligomerisation tag, and will not bind to any other molecule in the environment, for example in the human body.
  • an antigen for example an antigen present on an immune cell target
  • the relevant tag for example the peptide tag, for example a coiled-coil oligomerisation tag
  • the target antigen binding domain for example the immune cell binding domain is the portion of the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention that directs or targets the polypeptide of the invention to the desired target, for example to the desired immune cell, and requires the target antigen binding domain, for example the immune cell binding domain to be able to bind specifically to a particular target antigen, for example to a particular target antigen on the desired immune cell.
  • the polypeptide of the invention may comprise a single target antigen binding domain, for example a single immune cell binding domain, or may comprise more than one target antigen binding domain, for example more than one immune cell binding domain.
  • a typical monospecific antibody comprises two variable regions, each capable of binding to the same specific target. In this case, where the target antigen is present on an immune cell, the antibody would comprise two immune cell binding domains.
  • the skilled person will be aware of different antibody formats, including those described herein, and will understand that antibody engineering can result in a wide variety of different molecules, with different valences and different specificities.
  • the first target antigen binding domain comprises a first immunoglobulin light chain and a first immunoglobulin heavy chain.
  • the tag binding domain comprises a first coiled-coil oligomerisation domain
  • the first coiled-coil oligomerisation domain is connected to any one or more of: a) the C-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence; b) the C-terminus of the first immunoglobulin heavy chain, optionally connected via a linker sequence; c) the N-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence; and/or d) the N-terminus of the first immunoglobulin heavy chain, optionally connected via a linker sequence.
  • the C-terminus of the first immunoglobulin light chain for example connected via a linker sequence.
  • the linker that connects the light or heavy chain to the coiled- coil oligomerisation domain is a linker of SEQ ID NO: 56.
  • the first immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR region, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and b) the first immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region.
  • the polypeptide of the invention for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises a heavy chain that comprises more than one constant region.
  • the first immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and b) the first immunoglobulin heavy chain comprises or consists of:
  • variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three constant regions.
  • the polypeptide of the invention for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise or consist of one target antigen binding domain, or may comprise more than one target antigen binding domain. Accordingly in some embodiments the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises a second target antigen binding domain, for example wherein the second target antigen binding domain comprises a second immunoglobulin light chain and a second immunoglobulin heavy chain.
  • the heavy chain of the second target antigen binding domain may also comprise one or more than one constant region.
  • the second immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and b) the second immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region; or a) the second immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; and ii) one constant region; and b) the second immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three
  • the tag binding domain (e.g. the coiled-coil oligomerisation domain) may be connected to the polypeptide of the invention at any position.
  • the tag binding domain is a coiled-coil oligomerization domain and the target binding domain comprises an immunoglobulin light chain
  • the coiled-coil oligomerization domain is connected to the C-terminus of the immunoglobulin light chain, and in some instances is connected via a linker.
  • the first target antigen binding domain comprises a first immunoglobulin light chain and a first immunoglobulin heavy chain
  • the first coiled-coil oligomerisation domain is connected to the C-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence.
  • the polypeptide comprises a second target antigen binding domain
  • the second target antigen binding domain comprises a second immunoglobulin light chain and a second immunoglobulin heavy chain
  • the C-terminus of the second immunoglobulin light chain is connected to a tag binding domain, for example to a second coiled-coil oligomerisation domain, for example connected via a linker.
  • one or more coiled coil oligomerisation domains may be connected to any one or more ends of any one or more of the light or heavy chains, for example to the N-terminus and/or C-terminus of the first and/or second light chain and/or the N-terminus and/or C-terminus of the first and/or second heavy chain, for example: a) the C-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence; b) the C-terminus of the first immunoglobulin heavy chain, optionally connected via a linker sequence; c) the N-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence; d) the N-terminus of the first immunoglobulin heavy chain, optionally connected via a linker sequence, e) the C-terminus of the second immunoglobulin light chain, is connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via
  • the polypeptide of the invention for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises a second antigen target binding domain but does not comprise a second tag binding domain.
  • the second antigen target binding domain comprises an immunoglobulin light chain
  • the light chain is not connected to a second tag binding domain, for example is not connected to a second coiled-coil oligomerization domain.
  • the polypeptide of the invention for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention comprises a third target antigen binding domain, and in other embodiments also comprises a fourth target antigen binding domain.
  • the third target antigen binding domain comprises a third immunoglobulin light chain and a third immunoglobulin heavy chain
  • the optional fourth target antigen binding domain comprises a fourth immunoglobulin light chain and a fourth immunoglobulin heavy chain.
  • the third and fourth target antigen binding domains may comprise an immunoglobulin heavy chain that comprises one or more than one constant region.
  • an immunoglobulin heavy chain that comprises one or more than one constant region.
  • the third immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and the third immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region; or
  • the third immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and the third immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three constant regions; and for example when present:
  • the fourth immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3CDR regions, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and the fourth immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region; or
  • the fourth immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and the fourth immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three constant regions.
  • the N and/or C-terminus of the third and/or fourth immunoglobulin light chain is connected to a third and/or a fourth tag binding domain, for example to a third and/or fourth coiled coil oligomerisation domain, for example connected via a linker.
  • the N and/or C-terminus of the third and/or fourth immunoglobulin heavy chain is connected to a third and/or a fourth tag binding domain, for example to a third and/or fourth coiled coil oligomerisation domain, for example connected via a linker.
  • the C-terminus of the third and/or fourth immunoglobulin light chain is not connected to a third and/or fourth tag binding domain, optionally not connected to a third and/or fourth coiled coil oligomerisation domain.
  • polypeptide of the invention for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention may comprise at least 1, 2, 3, 4, 5, 6 or more target antigen binding domains, for example invention may comprise at least 1, 2, 3, 4, 5, 6 immune cell binding domains.
  • the polypeptide of the invention only comprises one Fc region.
  • an Fc region is a portion of the polypeptide where constant regions of the heavy chain from two separate heavy chains interact with one another, typically by forming disulphide bonds between the two heavy chains polypeptides.
  • each of the target antigen binding domains may bind to the same target antigen or feature or molecule on the immune cell.
  • each binding domain may bind to the same feature of molecule on the immune cell. This does not necessarily mean however that each of the immune cell binding domains binds to the same epitope. It is possible for a single target, for example an immune cell surface polypeptide, to comprise a number of epitopes. Polypeptides that comprise at least two immune cell binding domains, for example, wherein each binding domain binds to a different epitope of the same target molecule, are therefore encompassed within the invention.
  • the first and second target antigens are the same.
  • the polypeptide does not comprise a third and fourth target antigen binding domain, and in some instances of this embodiment the polypeptide does comprise a third and fourth target antigen binding domain.
  • the first and second target antigens are different.
  • the polypeptide does not comprise a third and fourth target antigen binding domain, and in some instances of this embodiment the polypeptide does comprise a third and fourth target antigen binding domain.
  • the first and second target antigens are the same and the third target antigen is a different antigen to the first and second target antigen.
  • the polypeptide does not comprise a fourth target antigen binding domain and in some embodiments the polypeptide does comprise a fourth target antigen binding domain.
  • the first and third target antigens are the same and the second antigen is different to the first and third target antigen.
  • the polypeptide does not comprise a fourth target antigen binding domain and in some embodiments the polypeptide does comprise a fourth target antigen binding domain.
  • the first target antigen and the second target antigen and the third target antigen are all different to one another.
  • the polypeptide does not comprise a fourth target antigen binding domain and in some embodiments the polypeptide does comprise a fourth target antigen binding domain.
  • first and second target antigens are the same as each other and wherein the third and fourth antigens are the same as each other, but wherein the first and second target antigens are different to the third and fourth target antigens; or the first and third target antigens are the same as each other and wherein the second and fourth target antigens are the same as each other, but wherein the first and third target antigens are different to the second and fourth target antigen.
  • the first target antigen and the second target antigen and the third target antigen and the fourth target antigen are all different to one another.
  • the first target antigen and the second target antigen and the third target antigen and the fourth target antigen are all the same.
  • Preferred embodiments of the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, have the following features:
  • the polypeptide does not comprise a fourth target antigen binding domain and in some embodiments the polypeptide does comprise a fourth target antigen binding domain .
  • the polypeptide comprises target binding domains that bind to a first, a second, a third and a fourth antigen, and wherein the first and second antigens are the same, and the third and fourth antigens are the same, but where the first and second antigens are different to the third and fourth antigens; B) the first and second target antigens are the same as each other. In some instances of this embodiment the polypeptide does not comprise a third and fourth target antigen binding domain, and in some instances of this embodiment the polypeptide does comprise a third and fourth target antigen binding domain.
  • Fab-arm-exchange uses two parental polypeptides or antibodies that differ in sequence in the Fc region. The sequences differ so that it is energetically more favorable to form heterodimers between the two different parental Fc regions than it is to form homodimers within each Fc region of each parental antibody. In this the exchange of the fab arms is promoted and the resultant antibody will comprise an Fc region that comprises one heavy chain region having a first sequence and one heavy chain region having a second sequence.
  • the polypeptide of the invention for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention has been produced via fab-arm-exchange and so in some embodiments the polypeptide comprises at least a first target binding domain and a second target binding domain, and wherein the first target binding domain comprises a first immunoglobulin heavy chain that comprises three constant regions and wherein the second target binding domain comprises a second immunoglobulin heavy chain that comprises three constant regions, and wherein the first immunoglobin heavy chain sequence and the second immunoglobin heavy chain sequence are different to one another, and wherein the different amino acid sequences promote the formation of a heterodimer between said first and second immunoglobulin heavy chain polypeptides rather than the formation of a homodimer of the first immunoglobulin heavy chain polypeptide and a homodimer of the second immunoglobulin heavy chain polypeptide.
  • the polypeptide of the invention may also comprise a third and possibly a fourth target antigen binding domain, as described elsewhere herein, for example may comprise: a) a third target antigen binding domain, wherein the third target antigen binding domain comprises a third immunoglobulin light chain that comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; optionally comprises ii) one constant region; and a third immunoglobulin heavy chain that comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region; optionally wherein the N-terminus of the third immunoglobulin light chain polypeptide is attached to the C-terminus of the first or second immunoglobulin heavy chain polypeptide; and optionally
  • any one or more of the tag binding domains present in the polypeptide of the invention is preferably a coiled-coil oligomerization domain, that is capable of forming a heterodimer with a corresponding coiled-coil oligomerization domain. Preferences for the position of the coiled-coil oligomerisation domain are described elsewhere herein.
  • polypeptide of the invention for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention can comprise any number of tag binding domains, for example 1, 2, 3, 4, 5, 6, 7 or 8, and can comprise any combination of types of tag binding domains.
  • any light chain and any heavy chain may be attached or connected to a coiled-coil oligomerisation domain.
  • the polypeptide of the invention comprises at least one tag binding domain that is a coiled-coil oligomerisation domain at any one of the N or C terminus of any of the light or heavy chains, the remaining N or C termini of any of the light or heavy chains may not be attached to or connected to a coiled-coil oligomerisation domain.
  • the C-terminus or N-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, but, where present, the second, third and fourth immunoglobulin light chain is not connected to a coiled-coil oligomerisation domain; b) the C-terminus or N-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, and where present the C- terminus of the second immunoglobulin light chain is connected to a second coiled-coil oligomerisation domain, but, where present, the third and fourth immunoglobulin light chain is not connected to a coiled-coil oligomerisation domain; c) the C-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, and where present the C-terminus of the second immunoglobulin light chain is connected to a second coiled-coilisation domain;
  • tag binding domain that is a coiled- coil oligomerisation domain can be attached to either a light or heavy chain, and can be attached to the light or heavy chain at the C and/or N terminus, a range of different iterations is possible and are disclosed herein.
  • the N-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain
  • the N-terminus of the second immunoglobulin light chain is connected to a second coiled-coil oligomerisation domain
  • the C-terminus of the third immunoglobulin light chain is connected to a third coiled-coil oligomerisation domain
  • the C-terminus of the fourth immunoglobulin light chain is connected to a fourth coiled-coil oligomerisation domain
  • the C-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain
  • the N-terminus of the third immunoglobulin light chain is connected to a third coiled-coil oligomerisation domain
  • the N-terminus of the fourth immunoglobulin light chain is connected to a third coiled-coil oligomerisation domain
  • the N-terminus of the fourth immunoglobulin light chain is connected to
  • the invention provides a cargo -targeting polypeptide of the invention that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 coiled-coil oligomerisation domains.
  • coiled-coil oligomerisation domains are in some instances able to form heterodimers, and are in some instances able to form multimers, that comprise more than 2 monomers of the coiled-coil domains.
  • the first coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding first cargo coiled- coil oligomerisation domain, and optionally: when present the second coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding second cargo coiled-coil oligomerisation domain; when present the third coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding third cargo coiled-coil oligomerisation domain; and when present the fourth coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding fourth cargo coiled-coil oligomerisation domain; when present the fifth coiled-coil oligomerisation domain is able to form a multimer, optionally a multimer, optionally
  • coiled-coil oligomerisation domains comprise a peptide that itself comprises repeated units of an amino acid sequence that comprises periodically interspersed hydrophobic residues (it will also be clear that the sequence of the repeated units does not necessarily have to be identical between repeats in a given coiled-coil oligomerisation domain). This results in a tendency for the peptide to form either parallel or anti-parallel, multimeric, heterodimeric or dimeric helix-helix interactions.
  • the coiled-coil oligomerisation domains referred to herein comprises a peptide that comprises repeated unites of an amino acid sequence that comprises a hydrophobic residue at every third or fourth amino acid - for example wherein the hydrophobic residues generate surface(s) on the helix that are hydrophobic and tend to form either parallel or anti-parallel, multimeric, heterodimeric or dimeric helix-helix interactions.
  • the coiled-coil oligomerisation domain comprises:
  • a and d are hydrophobic amino acids, optionally wherein e and g are polar or charged amino acids;
  • the coiled-coil oligomerisation domain comprises:
  • the coiled-coil oligomerisation domain has 3 or 4 repeat units, optionally no more than 4 repeat units.
  • hydrophobic residues the peptide will likely form a coiled-coil, and the other residues of the heptad, b, c, e, f and g can be generally be any residue.
  • Hydrophobic resides are considered to comprise Leu, He, Vai, Phe, Pro, Met and Ala.
  • e and g are polar residues or charged residues, for example Gin, Asn, His, Ser, Thr, Tyr, Cys, Arg, Kys, Asp or Glu.
  • b is leucine or isoleucine; e is Glu or Lys or Arg; and/or g is Glu or Lys or Arg.
  • the coiled-coil oligomerisation domain is not E3 [SEQ ID NO: 71].
  • the coiled-coil oligomerisation domain is not K3 [SEQ ID NO: 75] .
  • the coiled-coil oligomerisation domain is not K4 [SEQ ID NO: 76.]
  • sequence of each repeat sequence within a coiled-coil oligomerisation domain is identical.
  • At least two repeat sequences within the coiled-coil oligomerisation domain are different.
  • Specific examples of coiled-coil oligomerization domains that may be used in the invention as the first coiled-coil oligomerization domain are as follows:
  • GCN4-pI [SEQ ID NO: 123] RMKQLEDKVEELLSKNYHLENEVARLKKLVGER
  • GNC4-PH [SEQ ID NO: 124] RMKQIEDKLEEILSKLYHIENELARIKKIVGER
  • JR2KC [SEQ ID NO: 125]
  • JR2K [SEQ ID NO: 126]
  • JR2E [SEQ ID NO: 127]
  • coiled-coil oligomerisation domains are: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the E5 coiled-coil oligomerisation domain [SEQ ID NO: 73]; the E6 coiled-coil oligomerisation domain [SEQ ID NO: 74]; the K3 coiled-coil oligomerisation domain [SEQ ID NO: 75]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; the leucine zipper domain of [SEQ ID NO: 79] the HAP1 coiled-coil oligomerisation domain [SEQ ID NO: 71]
  • coiled-coil oligomerisation domains are: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; optionally selected from any one of: the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; or optionally selected
  • any one or more of the coiled-coil oligomerisation domains that are part of the polypeptide comprises a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences, or optionally a coiled-coil oligomerisation domain with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences and that
  • the second, third and/or fourth coiled-coil oligomerization domain where present may comprise or consist of the above coiled- coiled oligomerization domains, for example may comprise of consist of any of:
  • Leucine zipper 1 [SEQ ID NO: 119] EYQALEKEVAQLEAENQALEKEVAQLEHE Leucine zipper 2 [SEQ ID NO: 120] EYQALKKKVAQLKAKNQALKKKVAQLKHK rGCN4-pI [SEQ ID NO: 121] REGVLKKLRAVENELHYNKSLLEEVKDLQKMRQL rGCN4-pI' [SEQ ID NO: 122] CGGREGVLKKLRAVENELHYNKSLLEEVKDLQKMRQL
  • GCN4-pI [SEQ ID NO: 123] RMKQLEDKVEELLSKNYHLENEVARLKKLVGER
  • GNC4-PH [SEQ ID NO: 124] RMKQIEDKLEEILSKLYHIENELARIKKIVGER
  • JR2KC [SEQ ID NO: 125]
  • JR2K [SEQ ID NO: 126]
  • JR2E [SEQ ID NO: 127]
  • sequence of the coiled-coil oligomerization domains can vary depending on what cargo is to be delivered.
  • the polypeptide of the invention for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise more than one coiled-coil oligomerization domain but where each coiled-coil domain has the same sequence, the intention being that multiple copies of the same cargo become associated with the polypeptide, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention.
  • the polypeptide for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention may comprise two or more coiled-coil oligomerization domains that have different sequences and so can form a multimer, for example a dimer for example a heterodimer with different coiled-coil domains that are attached or connected to the cargo, in which case a single polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention can be used to deliver two or more different cargo.
  • the first and second coiled-coil oligomerisation domain are the same, for example wherein when present the first and second and third; or the first and second and third and fourth coiled-coil oligomerisation domains are the same and form a multimer, for example a dimer for example a heterodimer with the same partner coiled-coil oligomerisation domain.
  • the polypeptide of the invention for example the cargotargeting polypeptide or cargo-delivery polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 different coiled-coil oligomerisation domains that each forms a multimer, optionally a dimer, optionally a heterodimer with a different partner coiled- coil oligomerisation domain, for example in some embodiments each coiled-coil oligomerisation domain present is different to one another.
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises at least two different coiled- coil oligomerisation domains that each forms a heterodimer with a different partner coiled-coil oligomerisation domain.
  • the first target antigen is an antigen that is present on an immune ceil.
  • the immune cell is an antigen presenting cell, optionally a dendritic cell (DC), B cell and/or macrophage (preferably DC).
  • the polypeptide of the invention may be bi-specific with respect to the target antigen binding domain, for example the immune cell target binding domain, i.e. the polypeptide may comprise at least two different immune cell target binding domains, each capable of binding to a different immune cell target - the different immune cell targets may be present on the same immune cell, or may be targets associated with different immune cells.
  • the polypeptide can comprise more than 2, for example 3 or 4 or more different immune cell targeting domains, each binding to a different immune cell target - which may be on the same or different immune cells.
  • the tag binding domain is preferably a coiled-coil oligomerisation domain.
  • the tag binding domain of the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is the domain that specifically binds to a tag.
  • the tag itself is in preferred embodiments a corresponding coiled-coil oligomerisation domain which is connected to a cargo.
  • the cargo is in some instances a protein or peptide. Where the cargo is a protein or peptide it can be any protein or peptide. In some embodiments where the cargo is a protein peptide the protein or peptide comprises for example an antigen or neoantigen, which it is desirable to target to the immune cell.
  • the cargo is a protein or peptide
  • the cargo protein or cargo peptide and the peptide tag domain are expressed as a single transcript and translated accordingly to produce a single protein or peptide that comprises both the cargo peptide or cargo protein and the peptide tag domain.
  • a linker is present between cargo protein or cargo peptide and the tag domain, for example between the cargo protein or cargo peptide and the coiled coil oligomerisation domain.
  • the linker may be any linker, and may for example be a GS linker as described herein and as will be apparent to the skilled person.
  • the peptide tag domain that is connected to the cargo is a coiled-coil oligomerisation domain, preferably that is capable of forming a multimer for example a dimer for example a heterodimer with the corresponding coiled-coil oligomerisation domain present in the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention.
  • the tag for example a peptide tag may be conjugated to the cargo protein or peptide following expression of the cargo peptide or cargo protein.
  • the peptide tag may be conjugated to the cargo peptide or cargo protein (e.g. a cargo peptide or cargo protein that comprises an antigen or neoantigen) through e.g. a GSSSS linker or metalloprotease cleavable linker or a cieavable valine-citruiline linker.
  • the tag is not a peptide tag that is derived from tetanus toxin.
  • a tag derived from the tetanus toxin such as MTTE [SEQ ID NO: 68] is not encompassed within the meaning of "tag” or "peptide tag” as used herein.
  • tags are well known in the molecular biology field, where it is routine to express a peptide or polypeptide sequence of interest wherein the sequence has been extended to include a relatively short additional sequence, encoding the tag.
  • Applications using tagged peptides typically employ an antibody or antibody derived fragment, capable of specifically binding to the tag.
  • the tag binding domain is an antibody or antibody derived fragment, and binds specifically to the tag.
  • any peptide sequence capable of being specifically recognized by an antibody i.e. capable of inducing the production of antibodies
  • suitable peptide tags include the FLAG-tag comprising the amino acid sequence DYKDDDDK [SEQ ID NO: 69], and the skilled person will be aware of other suitable peptide tags such as V5-tag, Myc- tag, HA-tag, Spot-tag, T7-tag and NE-tag. See for example Mishra 2020 Current Protein and Peptide Science 21 : 821 - 830.
  • the peptide tag is a 33 amino acid sequence as follows:
  • Z33 FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD [SEQ ID NO: 51] or a sequence with at least 80%, 85%, 90%, 95%, 98% or 100% sequence identity to SEQ ID NO: 51 and wherein the tag retains the ability to bind to the Fc region.
  • the peptide tag is not the 33 amino acid sequence of [SEQ ID NO: 51], or is not a sequence with at least 80%, 85%, 90%, 95%, 98% or 100% sequence identity to SEQ ID NO: 51 and wherein the tag retains the ability to bind to the Fc region.
  • the tag binding domain of the polypeptide of the invention for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is not an Fc region; or has an additional tag binding domain other than the Fc region, for example comprises an Fc region and a tag binding domain that is a coiled-coil oligomerisation domain.
  • the cargo for example a cargo protein or cargo peptide, that in some instances comprises an antigen or neoantigen, may be tagged with a non-peptide tag, for example any moiety that acts as a binding partner for a second specific binding domain.
  • a non-peptide tag can be any chemical entity with which a second or further entity has affinity.
  • the tag can be selected from, for example, any organic molecule, a small molecule, or a hapten.
  • Tags can for example take the form of nucleic acids, for example aptamers.
  • Tags as described herein are typically short peptide sequences (i.e. sequences of amino acids). In preferred embodiments the tags described herein are peptide or protein tags, for example short sequences of amino acids.
  • the tag can be of any sequence provided it is able to be bound, preferably specifically bound by the tag binding domain of the polypeptide of the invention. Accordingly, in preferred embodiments the tag is a peptide tag, and the corresponding binding domain is a peptide tag binding domain. Most preferred is that the cargo, for example a cargo protein or cargo peptide comprises a peptide tag that is a coiled-coil oligomerisation domain, such as those specific examples described elsewhere herein.
  • a single polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise a number of different tag binding domains.
  • the cargo targeting polypeptide of the invention comprises a first tag binding domain that is a coiled-coil oligomerisation domain, for example connected to the C-terminus of an immunoglobulin light chain; and may also comprise a tag binding domain that can bind to the FLAG-tag.
  • Other combinations of tag binding domains are also contemplated by the invention.
  • the cargo targeting polypeptide of the invention comprises a first tag binding domain that is a coiled-coil oligomerisation domain, and also comprises at least a second tag binding domain that is not a coiled-coil oligomerisation domain.
  • the tag binding domain is not the Fc region.
  • a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention comprises an Fc region addition to a tag binding domain as described herein, for example comprises an Fc domain and a coiled-coil oligomerisation domain.
  • the cargo is a cargo protein or cargo peptide that comprises an antigen or neoantigen that can be any antigen or neoantigen.
  • the tag and the antigen are both peptides, allowing the expression of a tagged antigen as a single peptide, for example expression of a cargo protein or peptide that comprises an antigen and wherein the cargo protein or peptide is tagged with a tag domain that is a coiled-coil oligomerisation domain.
  • a linker may be present between the sequence encoding the antigen to be delivered (for example to be delivered to the immune cell) and the sequence encoding the tag (for example encoding the coiled-coil oligomerisation domain). Preferences for linkers are as described elsewhere herein.
  • peptide tags the term "peptide” is not construed as limited to a certain size or length of amino acids.
  • a peptide tag could be considered a protein, polypeptide, or multiple subunits thereof and simply indicates that the tag comprises a series of amino acids.
  • a peptide tag also includes within the meaning a coiled-coil oligomerization domain, such as those described herein.
  • the tag binding domain of the polypeptide of the invention binds to a tag that is less than 100 amino acids in length, for example less than 90, 80, 70, 60, 50, 40, 30, 20 or less than 10 amino acids in length, for example binds to a tag that is a coiled-coil oligomerisation domain that is less than 100 amino acids in length, for example less than 90, 80, 70, 60, 50, 40, 30, 20 or less than 10 amino acids in length.
  • the tag that is used to tag the cargo, and the tag binding domain present on the polypeptide can be considered to be binding partners and can essentially comprise any pair of entities, for example a pair of peptides, that are capable of specifically interacting with one another.
  • coiled-coil oligomerization domains as both the tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, and the tag that is used to tag the cargo, for example to tag a cargo protein or cargo peptide is a preferred embodiment.
  • the pair of peptides K3 ((KIAALKE)x3) [SEQ ID NO: 52] (KIAALKEKIAALKEKIAALKE) or K4 ((KIAALKE)x4) [SEQ ID NO: 53] (KIAALKEKIAALKEKIAALKEKIAALKE) are considered to bind to the coiled-coil peptide tag E3 ((EIAALEK)x3) [SEQ ID NO: 54] (EIAALEKEIAALEKEIAALEK).
  • the peptide tag that is used to tag the cargo for example to tag the cargo protein or cargo peptide comprises E3 ((EIAALEK)x3)
  • the tag binding domain of the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises K3 ((KIAALKE)x3) or K4 ((KIAALKE)x4).
  • the peptide tag binding domain of the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises K3 ((KIAALKE)x3) or K4 ((KIAALKE)x4), and consequently has the ability to bind to a cargo, for example an antigen peptide or neoantigen peptide, that has been tagged with the E3 ((EIAALEK)x3) peptide; in other embodiments the tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises E3 ((EIAALEK)x3), and consequently has the ability to bind to a cargo protein or cargo peptide, for example an antigen peptide or neoantigen peptide, that has been tagged with the peptide K3 ((KIAALKE)x3) or K4 ((KIAALKE)x4).
  • K3 (KIAALKE)x3)
  • coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO:
  • More preferred corresponding pairs of coiled-coil oligomerisation domains are: where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77] or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77] or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo
  • Even more preferred corresponding pairs of coiled-coil oligomerisation domains are: where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K4 [SEQ ID NO: 76] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E4 [SEQ ID NO: 72]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is
  • the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76] or K6 [SEQ ID NO: 78].
  • Allowing the polypeptide of the invention for example the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention to contact the tagged cargo in vitro or ex vivo, i.e prior to administration to a subject, and so form a complex outside of the body, has various advantages over administering both the polypeptide of the invention for example the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention and the tagged cargo separately.
  • tag binding domain and the tag of the tagged cargo interaction are less of a requirement for the tag binding domain and the tag of the tagged cargo interaction to be specific to reduce risk of off-target effects provided they bind with strong enough affinity that upon in vivo administration the tag and tag binding domains does not dissociate, thereby leaving both components open to binding with alternative targets.
  • suitable tag/tag binding domain partners will have specificity and/or bind with sufficient affinity to maintain complexes in vivo.
  • administration of the two components that ultimately form the complex of the invention separately but simultaneously, or sequentially has advantages.
  • the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention and the tagged cargo are administered separately.
  • the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention and the tagged cargo are administered separately and at the same time.
  • the cargo-targeting polypeptide or cargodelivery polypeptide of the invention and the tagged cargo are administered separately and at the same time and at the same site or location in the body.
  • the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention and the tagged cargo are administered separately and at different time periods.
  • the cargo-targeting polypeptide or cargo- delivery polypeptide of the invention is administered prior to the tagged cargo.
  • the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention is administered after the tagged cargo.
  • the tag for example the peptide tag used to tag the cargo for example the cargo protein or cargo peptide and towards which the tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is directed is a non-human peptide, to avoid unwanted immune responses.
  • the peptide tag and/or the cargo for example the cargo protein or cargo peptide comprises a biotin tag.
  • the cargo for example the cargo peptide or cargo protein comprises a biotin tag, it also comprises a peptide tag as described herein, for example comprises a coiled- coil oligomerisation domain.
  • biotin label can be used to attach the tagged cargo to a further entity via the interaction between biotin and streptavidin.
  • the tagged cargo is attached to a streptavidin bead via a biotin tag.
  • a complex comprising a polypeptide of the invention, for example the cargo-targeting polypeptide or the cargodelivery polypeptide of the invention, as described herein, and a tagged cargo, for example a tagged cargo protein or peptide (for example a peptide tagged antigen), wherein the tagged cargo (for example the peptide tagged antigen) comprises the tag to which the tag binding domain of the polypeptide binds, also comprises a bead via an interaction between a biotin tag located on the tagged cargo and a streptavidin tag located on the bead. Examples of such complexes are given in the Examples.
  • the tagged cargo may comprise the tag at either end of the cargo.
  • the tag for example the coiled-coil oligomerisation domain may be located at the N-terminus or the C-terminus.
  • the tag is located internally within the cargo protein or peptide.
  • the cargo protein or peptide comprises a tag at the N-terminus and the C- terminus - in some embodiments the tag at the N-terminus is the same as the tag at the C-terminus, but in other embodiments the tag at the N-terminus is different to the tag at the C-terminus.
  • the cargo protein or peptide comprises only one tag and the tag is located at the N-terminus.
  • the cargo protein or peptide comprises only one tag and the tag is located at the C-terminus.
  • the tagged cargo protein or peptide comprises an N-terminal K4 coiled-coil domain followed by an OVA peptide as described in SEQ ID NO: 128.
  • the tagged cargo protein or peptide comprises an OVA peptide followed by a C-terminal K4 coiled-coil domain as described in SEQ ID NO: 129 and 130.
  • the tag binding domains for example the peptide tag binding domains, all have specificity for the same tag (for example specificity for the same peptide tag - for example all of the tag binding domains comprise the same coiled-coil oligomerisation domain which can form a heterodimer with a further coiled-coil oligomerisation domain).
  • the polypeptide of the invention for the example the cargotargeting polypeptide or cargo-delivery polypeptide of the invention with multiple copies of the tagged cargo (for example to load with multiple copies of a tagged antigen) to be targeted and delivered to the specific target site or cell or tissue.
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention to be "loaded” with multiple different cargo, for example multiple different cargo peptides or cargo proteins comprising multiple different antigens.
  • the tag binding domains for example the peptide tag binding domains
  • a different tag for example a peptide tag
  • the polypeptide of the invention can be "loaded” with a range of tagged cargo, for example a range of tagged proteins for example a range of tagged antigens to be delivered.
  • the cargo comprises several different neoantigens (for example different neoantigen peptides) to be present sequentially in one long peptide that is tagged with a single tag for example a single peptide tag.
  • the polypeptide of the invention comprises at least 1, 2, 3, 4 or more tag binding domains (for example coiled-coil oligomerisation domains), wherein each of the tag binding domains (for example each coiled-coil oligomerisation domains) has specificity for the same tag (for example coiled-coil oligomerisation domain).
  • tag binding domains for example coiled-coil oligomerisation domains
  • the polypeptide of the invention comprises at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a first tag (for example a first coiled-coil oligomerisation tag domain ), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a second tag (for example a second coiled-coil oligomerisation tag domain).
  • tag binding domain for example coiled-coil oligomerisation domain
  • a first tag for example a first coiled-coil oligomerisation tag domain
  • tag binding domain for example coiled-coil oligomerisation domain
  • second tag for example a second coiled-coil oligomerisation tag domain
  • the polypeptide of the invention comprises at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a first tag (for example a first coiled-coil oligomerisation tag domain), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a second tag (for example second coiled-coil oligomerisation tag domain), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a third tag (for example a third coiled-coil oligomerisation tag domain).
  • the polypeptide of the invention comprises at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a first tag (for example a first coiled-coil oligomerisation tag domain), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a second tag (for example a second coiled-coil oligomerisation tag domain), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a third tag (for example a third coiled-coil oligomerisation tag domain), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a fourth tag (for example a fourth coiled-coil oligomerisation tag domain).
  • a first tag for example a first coiled-coil oligomerisation tag domain
  • tag binding domain for example coiled-coil oligomerisation domain
  • the polypeptides and methods described herein allow particular tagged cargo to be targeted or directed towards a specific target.
  • the target may be a specific cell, tissue or other structure which can comprise antigens, for example in some embodiments the cargo are cargo proteins or cargo peptides and are targeted to a particular set of immune cells.
  • various immune cells express different polypeptides and other ceil surface molecules and it is routine in the field to classify the different populations of immune cells into subpopulations, based on the expression of one or more polypeptides.
  • the polypeptides of interest are often located on the immune cell surface.
  • the target antigen binding domain is an immune cell binding domain that binds to a molecule, for example a polypeptide, that is located on the surface of the target immune cell.
  • Some cell surface molecules are receptor molecules, which once ligated by, for example, a polypeptide or antibody directed against said receptor molecules, results in a particular function.
  • the ligation of receptor molecules may be by an agonist or antagonist of said receptor.
  • the CD40 receptor which can be found on DC
  • an agonist for example an anti-CD40 antibody
  • the DC is activated.
  • the choice of immune cell target can not only ensure efficient delivery of the tagged cargo peptide or cargo protein, for example tagged antigen, to the immune cell, but can enable other advantageous functions to be activated, enhanced and/or performed, such as activation of the immune cell, enhancement of cytokine release (or enhancement of other ongoing immune cell functions) and/or internalisation of the tagged antigen.
  • APC antigen presenting cells
  • DC dendritic cells
  • B cells B cells
  • macrophages are able to internalise components, such as polypeptides, and present fragments of the molecule on the cell surface, in complex with MHCI or MHCII molecules.
  • the presentation of antigenic fragments on MHCI and MHCII molecules activates the innate and adaptive immune responses.
  • the present invention has particular utility in activating the innate and adaptive immune response through the targeted delivery of antigens to immune cells, and in particular APC such as those described here, including DC.
  • DC are professional APC that play a central role in the induction and regulation of adaptive immune responses, including the induction of cytotoxic T lymphocyte (CTL) responses.
  • CTL cytotoxic T lymphocyte
  • DC can be either plasmacytoid DC (pDC), which mainly reside in the blood and lymphoid organs and are capable of secreting large amounts of cytokines, such as type I interferon, upon activation, and conventional DC (cDC).
  • pDC plasmacytoid DC
  • cDC conventional DC
  • dendritic cell we include both conventional dendritic cells (eDCs, also known as classical dendritic cells) and plasmacytoid dendritic cells (pDCs). eDCs include both cDCl and cDC2. By “dendritic cells”, we also include both immature dendritic cells and mature, activated, dendritic cells. As discussed above, the selection of DC target determines which DC population that is targeted, how much of the antigen that is taken up and how much is presented on MHC II or MHC I, the latter being critical for cross-presentation to CD8+ T cells.
  • CD40 is a cell-surface expressed glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) superfamily and plays a central role in the immune system. It is expressed on a variety of immune cells, such as B cells, DC, monocytes and macrophages, but also on other normal tissues including epithelial cells, endothelial cells and fibroblasts, as well as several tumor types, e.g. on B cell lymphomas and carcinomas. Activation of CD40 on DC results in an anti-tumor immune response via tumor-specific T effector cells. CD40 agonists trigger effective anti-tumor responses in pre-clinical models.
  • TNFR tumor necrosis factor receptor
  • agonistic anti-CD40 antibody treatment of several cancer types, including lymphomas, melanoma, hepatoma, osteosarcoma, renal cell carcinoma, breast cancer and bladder cancer.
  • humanized or human anti-CD40 antibodies have been evaluated in a number of pre-clinical models and consistently demonstrated anti-tumor effects.
  • SGN- 40 a humanized CD40 monoclonal antibody with partial agonistic effects was evaluated using B cell lymphoma models (Raji and Ramos) in severe combined immunodeficiency (SCID) mice, and demonstrated effects on tumor growth and survival with complete response in approximately 50% of treated mice.
  • CP-870,893 a human agonistic anti-CD40 antibody, showed anti-tumor effects against B cell lymphoma, breast, colon, prostate, and pancreatic cancer in SCID mice. Efficacy was observed in CD40 positive as well as in CD40 negative tumors, thus demonstrating the ability of CP-870,893 to enhance anti-tumor immunity.
  • TLR3 is part of the Toll-like receptor family and acts as a receptor for danger signals in the form of double-stranded RNA that may be a form of genetic information derived from viruses. Ligation of TLR3 on a DC initiates inflammatory signaling via IRF3 and NF-KB, which activates the DC. Polyinosinic-polycytidylic acid (poly I : C) is a commonly used ligand of TLR3.
  • Both B cells and DC express high levels of CD40 and may also function as APC. However, it has been demonstrated that DC rather than B cells, macrophages or monocytes are important for generation of antigen-specific T cell responses. These cell populations will, however, act as a sink and may affect the biodistribution.
  • Other additional DC-markers include: XCR-1, CLEC9A, DEC-205, CDlc, Dec-1. Targeting CLEC9A would confer potential advantages since it is a death cell marker and antigen taken up by this receptor ends up in early endosomes and is more likely to result in cross-presentation to CD8+ T cells.
  • CLEC9A is selectively expressed on crosspresenting DC and may be superior to the more widely tested DEC-205 when it comes to inducing CD8+ T cell activation.
  • the target antigen is present on an immune cell that is an APC.
  • the immune cell is an APC and is selected from the group comprising or consisting of a DC, B cell or a macrophage.
  • the immune cell is a DC; even more preferred is cDCl.
  • Cargo-targeting or cargo-delivery polypeptides of the present invention may, in some embodiments, be directed to an immune cell target that is capable of mediating any one or more of: (a) activation of the immune cell (including enhancement of an immune cell activity or function); (b) internalisation of the polypeptide; and/or (c) recruitment of DCs, in particular recruitment of conventional type I dendritic cells (cDCl).
  • an immune cell target that is capable of DC activation includes, but is not limited to, CD40 and TLR3.
  • the immune cell target may facilitate internalization of the ligand.
  • the antigen of interest is capable of being processed by the immune cell in order to be presented on MHC.
  • immune cell targets that facilitate internalization include, but are not limited to, CLEC9A and DEC-205.
  • CLEC9A is a C-type lectin receptor involved in sensing necrotic cells
  • DEC-205 is a type I cell surface protein expressed primarily by DC.
  • the immune cell target may facilitate recruitment of DC, preferably recruitment of cDCl.
  • the immune cell target may be XCR1.
  • eDC can be found in tissues throughout the body, and in lymphoid organs. eDC in tissues capture antigen, transport it through the lymphatic system into draining lymph nodes, and present it to T cells. eDC that reside in lymphoid organs can also capture antigen, which may have diffused to the organ through the lymphatics, and present this to T cells. eDC can be further divided into two subsets, sometimes termed cDCl and cDC2. Transcriptional profiling has shown these subsets to be conserved between mice and humans.
  • cDCl express the chemokine receptor XCR1, which allows them to localize close to XCLl-producing CD8+ T cells in lymphoid tissues, and the dead cell receptor CLEC9A. They are specialized at cross-presenting antigen to CD8+ T cells on MHC I and are required for the priming of CTL responses against TAA in mice. Human cDCl display superior cross-presenting abilities in some, but not all, in vitro settings compared to other human DC subsets. Uptake of exogenous antigen, such as TAA, in DC is primarily accomplished by receptor-mediated endocytosis. Cross-presentation of exogenous antigen is favored by a relatively high endosomal pH, and routing of antigen to early rather than late endosomes. While high endosomal pH is a characteristic of cDCl, which endosomal compartment the antigen is targeted to depend on the endocytic receptor engaged.
  • pDC are generally not very efficient at presenting antigen to T cells
  • targeting antigen to uptake receptors on pDC can lead to effective cross-presentation to CD8+ T cells.
  • the combination of the polypeptide, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention comprising at least a first target antigen binding domain that is an immune cell binding domain and at least one tag binding domain
  • the tagged cargo for example the tagged cargo protein or tagged cargo peptide, for example that comprises an antigen (for example a peptide comprising both the tag (preferably a coiled-coil oligomerisation domain) and the antigen or neoantigen)
  • an antigen for example a peptide comprising both the tag (preferably a coiled-coil oligomerisation domain) and the antigen or neoantigen
  • an antigen for example a peptide comprising both the tag (preferably a coiled-coil oligomerisation domain) and the antigen or neoantigen
  • CD8+ T cells results in a superior expansion of antigen/neoantigen-specific immune cells, for example CD8+ T cells, compared
  • the antibody and antigen is unable to form a complex
  • this results in at least a 1.5 fold increase in expansion of antigen/neoantigen- specific immune cells such as CD8+ T cells, or at least a 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9 or 10 fold increase, for example when administered to a mammal such as a mouse or a human, for example when the agents (as a complex, or administered separately in the absence of the ability to form a complex, i.e.
  • a particular polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is able to target the tagged cargo, for example the tagged cargo protein or cargo peptide, for example where the cargo is an antigen, to a relevant cell population
  • an in vitro experiment can be performed where the tagged cargo protein or peptide, for example tagged antigen is fluorescently labelled.
  • the labelled cargo protein or peptide (for example antigen) is mixed with the cargo-targeting or cargodelivery polypeptide, followed by incubation with relevant cell population.
  • the amount of fluorescently labelled antigen bound to the cells could then be detected by, for example, flow cytometry or fluorescence microscopy.
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention results in the localization of a fluorescently labelled cargo, for example a fluorescently labelled cargo protein or peptides, for example labelled antigen, tagged with the corresponding tag (i.e. the tag to which the tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention binds - preferably a coiled- coil oligomerization domain) to the surface of the appropriate cell population (e.g.
  • the corresponding immune cells to which the target antigen binding domain that is an immune cell binding domain binds for example when the polypeptide of the invention is first incubated with the fluorescently labelled tagged cargo (e.g. fluorescently labelled tagged antigen) followed by incubation with the immune cell.
  • the fluorescently labelled tagged cargo e.g. fluorescently labelled tagged antigen
  • Standard antibodies typically comprise a single, constant region (Fc), and two variable regions which provide target specificity.
  • Fc constant region
  • each of the two variable regions are identical and directed towards the same epitope on the same target, i.e. they are bivalent for the same target, i.e. monospecific.
  • bispecific as used herein means the polypeptide is capable of specifically binding at least two different target entities.
  • Bispecific polypeptides e.g. antibodies, targeting two targets, have the potential to induce specific activation of the immune system in locations where both targets are over expressed.
  • the number of formats engineered is vast and the formats can be grouped according to their general architecture. Brinkmann and Kontermann have proposed a classification of 19 groups (see Brinkmann & Kontermann, 2017, mAbs. 9 : 182-212). The major differences between the groups are in regard to their symmetry, their target valency, their components and the position of these.
  • Fragment-based formats that lack an Fc part have limited half-life and cannot mediate Fc effector functions.
  • IgG-like formats display only monovalent binding to each target.
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise a target antigen binding domain that is an immune cell binding domain that binds to at least one immune ceil receptor, optionally wherein the immune cell receptor is CD40, CLEC9A, DEC-205, XCR1 or TLR3.
  • the target antigen binding domain of the polypeptide of the present invention may bind to CD40.
  • Antibody 1132E3 [Also referred to as A_1132E3, A_1132E3-IgGl, 1132E3- IgGl, IgGl-1132E3]
  • Antibody 1132E4 [Also referred to as A_1132E4, A_1132E4-IgGl, 1132E4- IgGl, IgGl-1132E4]
  • Antibody 1132E5 [Also referred to as A_1132E5, A_1132E5-IgGl, 1132E5- IgGl, IgGl-1132E5]
  • Antibody 1132E6 [Also referred to as A_1132E6, A_1132E6-IgGl, 1132E6-
  • Antibody 1132K3 Heavy chain [SEQ ID NO: 88]
  • Antibody 1132E3-IgG2 [Also referred to as A_1132E3-IgG2, 1132E3IgG2, IgG2-1132E3]
  • Antibody 1132E4-IgG2 [Also referred to as A_1132E4-IgG2, 1132E4IgG2,
  • Bispecific antibodies the bispecific antibodies used in the Examples comprise an IgG light chain as described in the table below and which comprises the sequence of the relevant coiled-coil oligomerisation domain, and the polypeptide also comprises an IgG heavy chain - Fab light chain of [SEQ ID NO: 117] and a Fab heavy chain of SEQ ID NO: 118.
  • the polypeptide of the invention for example the cargo-targeting or cargo-delivery polypeptide of the invention comprises, in some embodiments: a heavy chain of SEQ NO: 88 and light chain selected from the group comprising or consisting of SEQ ID NO: 89-SEQ ID NO: 105; a heavy chain of SEQ ID NO: 106 and a light chain of SEQ ID NO: 89 or of SEQ ID NO: 90; or an IgG heavy chain-Fab light chain comprising or consisting of SEQ ID NO: 117, a Fab heavy chain comprising or consisting of SEQ ID NO: 118, and an IgG light chain that comprises a coiled-coil oligomerisation domain selected from any of [SEQ ID NO: 107-116] or comprises a sequence that has a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or
  • the polypeptide may be an antibody that is selected from ADC-1013; clones 1132/1133, 1140/1135, 1150/1151 and 1107/1108 from WO 2015/091853; CP-870,893, APX005M, ChiLob 7/4, and SEA-CD40.
  • polypeptide may be an antibody that comprises or consists of any one or more of the following sequences: AntibodY 132Z1133
  • VH amino acid sequence (VH) amino acid sequence:
  • VL Variable light chain amino acid sequence
  • VH CDRs CDR1 : GFTFSSYA (SEQ ID NO: 3)
  • CDR2 IGSYGGGT (SEQ ID NO: 4)
  • VL CDRs CDR1 : QSISSY (SEQ ID NO: 6)
  • CDR2 AAS (SEQ ID NO: 7)
  • VH Variable heavy chain
  • VL Variable light chain
  • VH amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGS
  • VL Variable light chain amino acid sequence
  • VH CDRs CDR1 : GFTFSSYA (SEQ ID NO: 13)
  • CDR2 ISGSGGST (SEQ ID NO: 14)
  • VL CDRs CDR1 : QSISSY (SEQ ID NO: 16)
  • VH Variable heavy chain
  • VL Variable light chain
  • VH amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIGGSSSY
  • VL Variable light chain amino acid sequence
  • VH CDRs CDR1 : GFTFSSYA (SEQ ID NO: 23)
  • VL CDRs CDR1 : QSISSY (SEQ ID NO: 26)
  • VH Variable heavy chain
  • VL Variable light chain
  • VH amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGS
  • VL Variable light chain amino acid sequence
  • VH CDRs CDR1 : GFTFSSYA (SEQ ID NO: 33)
  • CDR2 ISGSGGST (SEQ ID NO: 34)
  • VL CDRs CDR1 : QSISSY (SEQ ID NO: 36)
  • VH Variable heavy chain
  • VL Variable light chain
  • VH amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWLSYISGGSSYI
  • VSS (SEQ ID NO: 41)
  • VL Variable light chain amino acid sequence
  • VH CDRs CDR1 : GFTFSTYGMH (SEQ ID NO: 43)
  • CDR2 GKGLEWLSYISGGSSYIFYADSVRGR (SEQ ID NO: 44)
  • VL CDRs CDR1 : CTGSSSNIGAGYNVY (SEQ ID NO: 46)
  • CDR3 CAAWDKSISGLV (SEQ ID NO: 48)
  • VH Variable heavy chain
  • VL Variable light chain
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is selected from the group consisting of: antibodies or antigen binding fragments thereof.
  • an antibody or an antigen-binding fragment thereof we include substantially intact antibody molecules, as well as chimeric antibodies, humanised antibodies, isolated human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen-binding fragments and derivatives of the same.
  • Suitable antigen-binding fragments and derivatives include Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g.
  • Fab fragments fragments, Fab' fragments and F(ab)2 fragments
  • single variable domains e.g. VH and VL domains
  • dAbs single domain antibodies
  • the potential advantages of using antibody fragments, rather than whole antibodies, are several-fold.
  • the smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue.
  • antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • the antigen-binding fragment is selected from the group consisting of: Fv fragments (such as a single chain Fv fragment, or a disulphide-bonded Fv fragment), Fab-like fragments (such as a Fab fragment; a Fab' fragment or a F(ab)2 fragment) and single domain antibodies.
  • Fv fragments such as a single chain Fv fragment, or a disulphide-bonded Fv fragment
  • Fab-like fragments such as a Fab fragment; a Fab' fragment or a F(ab)2 fragment
  • single domain antibodies single domain antibodies
  • an antibody or an antigen-binding fragment thereof is also intended to encompass antibody mimics (for example, non-antibody scaffold structures that have a high degree of stability yet allow variability to be introduced at certain positions).
  • antibody mimics include: affibodies (also called Trinectins; Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also called Tetranectins; Innovations Pharmac. Technol. (2006), 27-30); adnectins (also called monobodies; Meth. Mol.
  • chimeric T cell receptors also known as chimeric T cell receptors, chimeric immunoreceptors, and chimeric antigen receptors or CARs
  • CARs engineered receptors, which graft an arbitrary specificity onto an immune effector cell.
  • CARs are used to graft the specificity of a monoclonal antibody onto a T cell; with transfer of their coding sequence facilitated by retroviral vectors.
  • fusions comprising a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to CD3-zeta transmembrane and endodomain.
  • scFv single-chain variable fragment
  • the invention also encompasses modified versions of antibodies and antigen-binding fragments thereof, whether existing now or in the future, e.g. modified by the covalent attachment of polyethylene glycol or another suitable polymer (see below).
  • antibodies may be generated via any one of several methods which employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi. et al, 1989; Winter et al., 1991, the disclosures of which are incorporated herein by reference) or generation of monoclonal antibody molecules by cell lines in culture.
  • hybridoma technique examples include, but are not limited to, the hybridoma technique, the human B cell hybridoma technique, and the Epstein-Barr virus (EBV)- hybridoma technique (Kohler et al., 1975,Kozbor et al., 1985; Cote et al., 1983; Cole et al., 1984., the disclosures of which are incorporated herein by reference).
  • EBV Epstein-Barr virus
  • antibody fragments can be obtained using methods well known in the art (see, for example, Harlow & Lane, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, New York, the disclosures of which are incorporated herein by reference).
  • antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • human or humanised antibodies are preferably used.
  • Humanised forms of non-human (e.g. murine) antibodies are genetically engineered chimeric antibodies or antibody fragments having preferably minimal-portions derived from non-human antibodies.
  • Humanised antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementary determining region of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired functionality.
  • donor antibody such as mouse, rat or rabbit having the desired functionality.
  • Fv framework residues of the human antibody are replaced by corresponding non-human residues.
  • Humanised antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences.
  • the humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially ail of the complementarity determining regions correspond to those of a non-human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence.
  • Humanised antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986, Riechmann et al., 1988, Presta, 1992, the disclosures of which are incorporated herein by reference).
  • the humanised antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. Humanisation can be essentially performed as described (see, for example, Jones et al., 1986, Reichmann et al., 1988, Verhoeyen et al., 1988, US 4,816,567, the disclosures of which are incorporated herein by reference) by substituting human complementarity determining regions with corresponding rodent complementarity determining regions.
  • humanised antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanised antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be identified using various techniques known in the art, including phage display libraries (see, for example, Hoogenboom & Winter, 1991, Marks et al., 1991, Cole et al., 1985, Boerner et al., 1991, the disclosures of which are incorporated herein by reference).
  • bispecific polypeptides e.g. antibodies
  • the bispecific polypeptides may be of any suitable structural format.
  • the polypeptide is an antigen-binding fragment selected from the group consisting of: an Fv fragment (such as a single chain Fv fragment, or a disulphide-bonded Fv fragment); a Fab-like fragment (such as a Fab fragment; a Fab' fragment; or a F(ab)2 fragment); and domain antibodies.
  • an Fv fragment such as a single chain Fv fragment, or a disulphide-bonded Fv fragment
  • a Fab-like fragment such as a Fab fragment; a Fab' fragment; or a F(ab)2 fragment
  • the tag binding domain of the polypeptide is an Fc region of an antibody or antibody fragment having the ability to specifically bind to the tag, for example to the peptide tag.
  • the tag for example the peptide tag may be a molecule with affinity to the Fc portion of an antibody, such as an antibody or fragment thereof that is specific to Fc (an anti-Fc antibody or fragment thereof); and Protein A, which is a protein derived from Staphylococcus aureus that binds strongly to IgGs.
  • the peptide tag is a 33 amino acid sequence as follows:
  • the tag binding domain of the polypeptide of the invention is an Fc region of an antibody or antigen binding fragment thereof that is capable of binding to the Z33 peptide sequence of SEQ ID NO: 51.
  • the peptide tag is linked to a larger peptide or polypeptide, for example that comprises an antigen or neoantigen, through a linker, for example a GSSSS linker [SEQ ID NO: 55] or a cleavable valine-citrulline linker.
  • a linker for example a GSSSS linker [SEQ ID NO: 55] or a cleavable valine-citrulline linker.
  • the binding domain localized to the Fc region can be engineered by introduction of mutations to enhance the affinity to the Fc-binding peptide tag.
  • mutations would improve the stability of the DC-targeting antibody-neoantigen complex and prevent swapping of the Fc in an in vivo setting.
  • the tag binding domain is a coiled-coil oligomerization domain and the tag that is used to tag the cargo, for example a cargo protein or peptide is also a coiled-coil oligomerization domain, such that the two coiled- coil oligomerization domains interact when contacted to form a muitimer, for example a dimer, for example a heterodimer.
  • a single polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may have multiple tag binding domains of different formats, so for example may have a tag binding domain that is a coiled-coil oligomerization domain, and may also have a tag binding domain that is an Fc region.
  • the Fc region is not considered to be a tag binding domain.
  • the tag that is used to tag the cargo for example the cargo protein or cargo peptide may be linked to the cargo via a peptide linker, for example a GS linker or a cleavable valine-citrulline linker or a meta I loprotease cleavable linker as described elsewhere herein.
  • a peptide linker for example a GS linker or a cleavable valine-citrulline linker or a meta I loprotease cleavable linker as described elsewhere herein.
  • the polypeptide for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention comprises at least 2 tag binding domains (for example at least 2 peptide tag binding domains), for example 3, 4, 5, 6 or more peptide tag binding domains (for example at least 3, 4, 5, 6 peptide tag binding domains).
  • at least 2 tag binding domains are present, in some embodiments at least one is selected from the group consisting of: a coiled-coil oligomerisation domain, and at least a second tag binding domain is selected from antibodies or antigen binding fragments thereof; or an Fc region of an antibody having the ability to specifically bind to the peptide tag.
  • the tag binding domain (for example the peptide tag binding domain) is a domain other than an Fc region.
  • the polypeptide may comprise an Fc region, but the Fc region is not the domain that binds to the tagged antigen/neoantigen.
  • the polypeptide may comprise a number of tag binding domains (for example a number of peptide tag binding domains) as described herein, and none of those are the Fc region, i.e. the Fc region is not used to bind to the tagged antigen/neoantigen.
  • the Fc region is used to bind to the tagged cargo or the Fc region is one of a number of binding domains used to bind to the tagged cargo.
  • the polypeptide comprises: a) at least a first target antigen binding domain that is an immune cell binding domain that comprises or consists of an IgG antibody; b) at least a first target antigen binding domain that is an immune cell binding domain that comprises or consists of an Fv fragment; c) at least a first target antigen binding domain that comprises or consists of an IgG antibody and at least one tag binding domain that comprises or consists of a Fab- like fragment; d) at least a first target antigen binding domain that comprises or consists of an IgG antibody and at least one tag binding domain that comprises or consists of a domain antibody; e) at least a first target antigen binding domain that comprises or consists of an IgG antibody and at least one tag binding domain that comprises or consists of a coiled-coil oligomerisation domain; f) at least a first target antigen binding domain that comprises or consists of an Fv fragment and at least one tag
  • the antibody may be an IgG antibody, for example an IgGl, IgG2, IgG3 or IgG4 antibody.
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is a bispecific polypeptide, and optionally comprises or consists of a format selected from the group consisting of (wherein the bispecific is comprised of a first target antigen binding domain 1 (Bl) and a second target antigen binding domain 2 (B2) : a) IgG-scFv bispecific antibodies, optionally wherein Bl is an intact IgG and B2 is an scFv attached to Bl at the N-terminus of a light chain; at the C-terminus of a light chain; at the N-terminus of a heavy chain; and/or at the C-terminus of a heavy chain of the IgG.
  • Bl target antigen binding domain 1
  • B2 second target antigen binding domain 2
  • the scFv could be attached to the N-terminus and C- terminus of the light chain; attached to the N-terminus of both the light and heavy chains, etc.
  • monovalent bispecific antibodies such as a DuoBody® (Genmab AS, Copenhagen, Denmark) or 'knob-in-hole' bispecific antibody (for example, an scFv- KIH, scFv-KIHr, a BiTE-KIH or a BiTE-KIHr (see Xu et al., 2015, mAbs 7(l) :231-242);
  • c) scFv2-Fc bispecific antibodies (such as ADAPTIRTM bispecific antibodies from Emergent Biosolutions Inc); d) BiTE/scFv2 bispecific antibodies; e) DVD-Ig bispecific antibodies; f) DART-based bispecific antibodies (for example, DART2-Fc or DART); g) DNL-Fab3 bispecific antibodies; and h)
  • the bispecific polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention antibody may be an IgG-scFv antibody.
  • the IgG-scFv antibody may be in either VH-VL or VL-VH orientation.
  • the scFv may be stabilised by a S-S bridge between VH and VL.
  • binding domain Bl and binding domain B2 are fused directly to each other.
  • Bl is the immune cell binding domain and B2 is the tag binding domain. In other embodiments Bl is the tag binding domain and B2 is the immune cell binding domain.
  • binding domain Bl and binding domain B2 are joined via a polypeptide linker.
  • a polypeptide linker may be a short linker peptide between about 10 to about 25 amino acids.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N- terminus of the VH with the C-terminus of the VL, or vice versa.
  • the linker may be selected from the group consisting of the amino acid sequence SGGGGSGGGGS [SEQ ID NO: 56], SGGGGSGGGGSAP [SEQ ID NO: 57], NFSQP [SEQ ID NO: 58], KRTVA [SEQ ID NO: 59], GGGSGGGG [SEQ ID NO: 60], GGGGSGGGGS [SEQ ID NO: 61], GGGGSGGGGSGGGGS [SEQ ID NO: 62], GSTSGSGKPGSGEGSTKG [SEQ ID NO: 63] (Whitlow et al.
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises an Fc region or a variant of said region, optionally wherein the region is an IgGl, IgG2, IgG3 or IgG4 region, preferably IgGl or IgG2.
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is a monospecific antibody directed towards an immune cell target.
  • the polypeptide comprises a tag binding domain that is a coiled coil oligomerisation domain that can bind to a first tag that is also a coiled-coil oligomerisation domain, and may also comprise an Fc region that has been modified to be capable of binding to a second tag that is not a coiled coil oligomerisation domain; or a bispecific antibody wherein one paratope comprises the immune cell binding domain and the second paratope comprises the tag binding domain (for example the tag binding domain).
  • the tag binding domain is a coiled coil oligomerisation domain that can bind to a first tag that is also a coiled-coil oligomerisation domain, and may also comprise an Fc region that has been modified to be capable of binding to a second tag that is not a coiled coil oligomerisation domain.
  • the invention in addition to providing a polypeptide, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention capable of targeting a cargo for example a cargo protein or cargo peptide, such as an antigenic peptide or a neoantigen to a particular target site of cell type, for example to an immune cell or population of immune cells, the invention also provides a complex comprising a polypeptide of the invention, for example the cargo-targeting polypeptide or the cargo-delivery polypeptide of the invention, as described herein, and a tagged cargo, for example a tagged cargo protein or peptide (for example a peptide tagged antigen), wherein the tagged cargo (for example the peptide tagged antigen) comprises the tag to which the tag binding domain of the polypeptide binds.
  • a polypeptide of the invention for example the cargo-targeting polypeptide or the cargo-delivery polypeptide of the invention, as described herein, and a tagged cargo, for example
  • Preferences for features of the complex of the invention for example in relation to the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, and the tagged cargo for example tagged cargo protein or peptides (for example tagged peptides that comprise the one or more antigens) are as described herein in relation to other aspects of the invention.
  • the complex of the invention comprises the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and the tagged cargo for example tagged protein or peptide (for example a tagged peptide antigen).
  • the preferred tag, and tag binding domain are complementary coiled-coil oligomerisation domains, that can form multimers for example dimers for example heterodimers.
  • the invention provides a complex comprising a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention and a tagged cargo, for example a tagged cargo protein or cargo peptide, wherein the tagged cargo comprises a tag that is a cargo coiled-coil oligomerisation domain that is an interaction partner for the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention.
  • the complex does not comprise: a polypeptide according the invention, for example a cargo-targeting or cargodelivery polypeptide of the invention that comprises a tag binding domain that is a coiled-coil oligomerisation domain that is E3 [SEQ ID NO: 71] and a cargo that is attached to a tag that is a coiled-coil oligomerisation domain that is K3 [SEQ ID NO:
  • a polypeptide according the invention for example a cargo-targeting or cargodelivery polypeptide of the invention that comprises a tag binding domain that is a coiled-coil oligomerisation domain that is E3 [SEQ ID NO: 71] and a cargo that is attached to a tag that is a coiled-coil oligomerisation domain that is K4 [SEQ ID NO:
  • a polypeptide according the invention for example a cargo-targeting or cargodelivery polypeptide of the invention that comprises a tag binding domain that is a coiled-coil oligomerisation domain that is K3 [SEQ ID NO: 75] and a cargo that is attached to a tag that is a coiled-coil oligomerisation domain that is E3 [SEQ ID NO: 71]; a polypeptide according the invention, for example a cargo-targeting or cargodelivery polypeptide of the invention that comprises a tag binding domain that is a coiled-coil oligomerisation domain that is K4 [SEQ ID NO: 76] and a cargo that is attached to a tag that is a coiled-coil oligomerisation domain that is E3 [SEQ ID NO: 71],
  • the coiled-coil oligomerization domain of the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and the tagged cargo protein or peptide is selected from any of the coiled coil oligomerisation domains described by SEQ ID NO: 71-87 or 119-127, or select from any of the coiled coil oligomerisation domains described by SEQ ID NO: 71-87, or a coiled-coil oligomerisation domain with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences, or wherein any of the coiled-coil oligomerisation domains has a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • Certain coiled-coil oligomerization domains interact with certain other coiled-coil oligomerization domains.
  • the skilled person will readily be able to determine which coiled-coil oligomerization domains are suitable to be used as a corresponding pair - one on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, and one used as the tag on the tagged cargo protein or peptide.
  • the coiled-coil oligomerisation domain present on the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77] or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO:
  • the corresponding pairs of coiled-coil oligomerisation domains are as follows: where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the
  • the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76] or K6 [SEQ ID NO: 78]; and where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76] or K6 [SEQ ID NO: 78].
  • the cargo coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76]; and where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K4 [SEQ ID NO: 76] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E4 [SEQ ID NO: 72].
  • the complex may comprise a single polypeptide of the invention, for example a single cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and two cargo molecules.
  • the invention provides a complex of the invention wherein:
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises: at least a first tag binding domain that is a coiled-coil oligomerisation domain and a second tag binding domain that is a coiled-coil oligomerisation domain; and at least a first cargo comprising a first cargo coiled-coil oligomerisation domain and a second cargo comprising a second cargo coiled-coil oligomerisation domain; wherein the first tag binding domain interacts with the first cargo coiled-coil oligomerisation domain; and wherein the second tag binding domain interacts with the second cargo coiled-coil oligomerisation domain.
  • the first cargo is different to the second cargo.
  • the cargo can be any compound, agent, moiety or molecule to which it is possible to attach or connect or otherwise incorporate a tag as described herein.
  • the cargo is selected from any one or more of: a) a protein or a peptide; b) a nucleic acid; c) a toxin; d) a small molecule, for example a small molecule drug, imaging agent, toxin, radionucleotide labelled molecule; e) a virus particle; f) a viral vector; g) crispr editing components; h) lipid vesicles, optionally liposomes and/or exosomes; i) a PAMP or a DAMP; j) bacterial cellular fragments and structures; and/or k) a nanoparticle.
  • Any of these entities may be conjugated or otherwise connected to a tag as described herein, for example connected to a coiled-coil oligomerisation domain.
  • the protein or peptide can be an antigenic protein or peptide.
  • the antigenic protein or peptide has been identified as a neoantigen that has arisen in a tumour or a cell.
  • the cargo is a neoantigen.
  • the antigenic protein or peptide is or comprises or consists of: a cancer antigen, for example a HPV-associated cancer antigen; an antigenic peptide or protein sequence that is derived from a pathogen, optionally wherein the pathogen is a bacteria, fungus or virus, optionally wherein the virus is Human Papillomavirus (HPV).
  • HPV Human Papillomavirus
  • the cargo is: a) A peptide or protein that is, or comprises, an antigen, optionally wherein: i) the antigen has been identified as a neoantigen that has arisen in a tumour or cell; ii) the antigen is a cancer antigen, optionally wherein the cancer antigen is a Human Papillomavirus (HPV)-associated cancer antigen; iii) the antigen is derived from a pathogen, optionally wherein the pathogen is a bacteria, fungus or virus, optionally wherein the virus is Human Papillomavirus (HPV); or b) a nanoparticle that comprises an antigen, optionally comprises an antigen: i) that has been identified as a neoantigen that has arisen in a tumour or cell; ii) that is a cancer antigen, optionally wherein the cancer antigen is a Human Papillomavirus (HPV)-associated cancer antigen; iii) that
  • the nanoparticle is a protein nanoparticle.
  • the cargo is a toxin, optionally wherein the cargo is a toxic protein, toxic peptide or toxic nanoparticle, for example wherein the toxic protein, toxic peptide or toxic nanoparticle is a drug-conjugated protein, peptide or nanoparticle.
  • the cargo is a viral particle that has been engineered to express the relevant tag, for example the relevant coiled-coil oligomerisation domain.
  • the cargo is a protein or peptide
  • the tag is also a protein or peptide
  • the cargo and the tag may be expressed as one transcript and translated into a single amino acid chain. Generating such constructs is routine for the skilled person.
  • a linker As described elsewhere herein, between the cargo and the tag it is possible to incorporate a linker.
  • Various linkers known to the skilled person are considered to be useful for example GS linkers and metalloprotease cleavage linkers.
  • Cleavable linkers are considered to be advantageous in some uses since it is possible to produce a construct that is cleaved when in an appropriate environment, for example a tumour microenvironment where the appropriate meta I loproteases are present.
  • the tag may be attached to the cargo by chemical conjugation.
  • the cargo and the tag are both proteins or peptides, but is particularly suited to situations where the cargo is not proteinaceous and so cannot be generated from a single mRNA transcript that also encodes the tag.
  • the tag (for example the peptide tag for example the coiled- coil oligomerization domain) may also be adapted to be multifunctional.
  • the tag may facilitate the binding of an antigen or epitope of interest to a polypeptide of the present invention, in addition to acting as an adjuvant.
  • adjuvant means anything that can elicit or enhance an immune response, for example an immune response directed towards an antigen.
  • Tags that also act as adjuvants may be tags that are epitopes for other immune cells, such as a T cell epitope or B cell epitope.
  • a tag that comprises a sequence that is also a T cell epitope means that there may exist T cells in the subject that are specific to the sequence of the tag, and their interaction with said tag may boost or provoke their activity.
  • a B cell epitope means that the tag may have antibodies directed against it in situ.
  • a tag derived from the tetanus toxin (TTx) such as MTTE
  • TTx tetanus toxin
  • MTTE tetanus toxin
  • universal B cell epitope means that the majority of individuals in a given population have antibodies specific to that particular epitope. This may occur due to herd vaccination (i.e. the vaccination or the majority of a population against a particular disease, such as tetanus, measles, mumps, rubella etc).
  • Tags comprising particular universal B cell epitopes may be a detriment to the purpose of the present invention.
  • use of the MTTE peptide as a tag may be unworkable for large portions of the population due to patients having anti-MTTE antibodies.
  • a polypeptide of the present invention that is specific to the MTTE peptide (being used as a tag attached to an antigen or epitope of interest) may have patient antibodies directed against it that prevent their intended function.
  • polypeptides of the present invention are not intended to comprise a second binding domain specific for TTx or the MTTE peptide derived therefrom.
  • the peptide tag is not a tetanus derived tag.
  • a tag for example a peptide tag
  • a human peptide tag that is a natural human peptide is less likely to provoke an immune response against it (due to it not being considered "foreign" by the immune system).
  • the tag for example a peptide tag
  • the tag could be derived from a non-human source, such as bacteria (i.e. a bacterial protein).
  • the structure of the peptide tag is another consideration, such as the secondary and/or tertiary structures. Regarding secondary structures, it may be preferable that the peptide tag comprises largely of a-helices and few to no p-sheets.
  • a peptide tag may comprise or consist of an a-helical structure.
  • Peptide secondary structure can be predicted using software that is publicly available, such as Jpred4 (http://www.compbio.dundee.ac.uk/jpred/, Drozdetskiy et al., Nucl. Acids Res. 43(W1) : W389-W394, 2015) and PASTA 2.0 (http://protein.bio.unipd.it/pasta2/, Walsh et al., Nucl. Acids Res. 42(W) : W301-W307, 2014), or can be determined experimentally using routine techniques such as circular dichroism spectroscopy (see e.g. Greenfield, N., Nat 5 Protoc. 1(6): 2876-2890, 2006).
  • the tag is a non-human tag, for example where the tag is a peptide tag, the peptide tag is a non-human peptide.
  • the complex of the invention comprises a polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention as described herein wherein the polypeptide of the invention comprises an Fc region, and a tagged cargo, for example a tagged cargo protein or peptide (for example a tagged antigen), wherein the tag portion of the tagged cargo (for example a peptide tagged antigen) binds to the Fc region of the polypeptide of the invention.
  • a polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention as described herein wherein the polypeptide of the invention comprises an Fc region, and a tagged cargo, for example a tagged cargo protein or peptide (for example a tagged antigen), wherein the tag portion of the tagged cargo (for example a peptide tagged antigen) binds to the Fc region of the polypeptide of the invention.
  • the tag has the sequence of [SEQ ID NO: 51] or a sequence with at least 80%, 85%, 90%, 95%, 98% or 100% sequence identity to SEQ ID NO: 51 and wherein the tag retains the ability to bind to the Fc region.
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise more than one tag binding domain (for example more than one peptide tag binding domain), and may be capable of binding to more than one copy of the same tag, or to multiple different tags (for example multiple different peptide tags).
  • the complex comprises a polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, wherein the polypeptide comprises at least two tag binding domains (for example two tag binding domains that are coiled coil oligomerisation domains) capable of binding to at least two different tags (for example at least two different coiled coil oligomerisation domains), and wherein the complex comprises at least two tagged cargos, for example at least two tagged proteins or peptides (for example at least two peptide tagged antigens).
  • the polypeptide comprises at least two tag binding domains (for example two tag binding domains that are coiled coil oligomerisation domains) capable of binding to at least two different tags (for example at least two different coiled coil oligomerisation domains)
  • the complex comprises at least two tagged cargos, for example at least two tagged proteins or peptides (for example at least two peptide tagged antigens).
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise at least 2, 3 or 4 tag binding domains (for example at least 2, 3 or 4 peptide tag binding domains), and the complex may comprise at least 2, 3 or 4 tagged antigens (for example peptide tagged antigens), for example tagged neoantigens.
  • a single complex may comprise multiple copies of a first tagged cargo for example a first tagged cargo protein or peptide, (for example a first peptide tagged antigen), for example a first tagged neoantigen; or may comprise at least 2, 3, or 4 different tagged cargo for example at least 2, 3 or 4 different tagged cargo proteins or peptides (for example may comprise at least 2, 3, or 4 different peptide tagged antigens), for example at least 2, 3 or 4 different tagged neoantigens or peptide tagged neoantigens.
  • Neoantigen neoantigen
  • the skilled person will understand what is meant by the term neoantigen, and we include the meaning of newly formed antigens that have not been previously recognized by the immune system in an individual.
  • the genetic instability of tumor cells often leads to the occurrence of a large number of mutations, and expression of non-synonymous mutations can produce neoantigens, i.e. antigens that are specific to that tumor and to that individual.
  • Neoantigens are highly immunogenic as they are not expressed in normal tissues. They can activate CD4+ and CD8+ T cells to generate an immune response and have the potential to become new targets of tumor immunotherapy. Neoantigens can arise from altered tumor proteins formed as a result of tumor mutations or from viral proteins.
  • tumour cell we also include the meaning of antigens to which the individual has already been exposed, but which are more highly expressed by a tumour cell.
  • stimulation of the immune system by the polypeptide of the invention would be expected to raise an immune response that would be directed primarily to areas in which the antigen is most highly expressed - i.e. the tumour.
  • the antigen is a peptide, protein or fragment thereof that comprises an antigenic peptide or protein sequence. Accordingly, it is considered to be advantageous if the complex of the invention comprises a neoantigen that has arisen in a particular tumour, since administration of the complex will result in the neoantigen being targeted to the desired immune cell, for example an APC such as a DC, where the immune cell can be activated, the neoantigen can be internalised and subsequently presented to other relevant cells of the immune system, ultimately directing the immune system against the tumour.
  • the desired immune cell for example an APC such as a DC
  • the antigen is a neoantigen (for example a peptide neoantigen), and the complex of the invention comprises a polypeptide of the invention, and at least one tagged neoantigen such as at least one tagged peptide neoantigen.
  • the complex of the invention comprises a polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and 2, 3, 4 or more different tagged cargo that are or comprise or consist of antigens, for example peptide tagged antigens such as peptide tagged neoantigens.
  • the peptide tagged neoantigens may be tagged with the same peptide tag, or the neoantigens may be tagged with different peptide antigens.
  • the antigen for example the peptide tagged antigen
  • the skilled person is readily able to sequence the genomic material of a given tumour to identify suitable neoantigens.
  • the tagged cargo protein or peptide that comprises or consists of a tagged antigen for example the peptide tagged antigen comprises an antigenic peptide that is a cancer antigen.
  • the cancer antigen is an HPV-associated cancer antigen.
  • the neoantigen is a personalized neoantigen and requires the identification of suitable neoantigens in the individual tumour(s).
  • the polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, complex and methods described herein are useful for targeting any cargo, for example a cargo protein or peptide, for example an antigenic peptide to any cell type, for example to immune cells, such as APC, for example DC.
  • the polypeptide, for example a cargo- targeting polypeptide or a cargo-delivery polypeptide of the invention, complex and methods described herein may be ultimately aimed at the treatment or prevention of cancer and may comprise cancer antigens or cancer neoantigens, as described herein.
  • the present invention is also useful for the treatment or prevention of pathogenic infections, for example infection with a bacteria, fungus or virus.
  • suitable antigens such as antigenic peptides from the pathogen should be tagged with the appropriate tag such as a peptide tag, to which the polypeptide of the invention binds.
  • the necessary antigens are targeted to immune cells, such as APC, for example DC, initiating the appropriate immune response.
  • the tagged cargo comprises or consists of a tagged antigen, and wherein the antigen is a peptide that comprises an antigenic amino acid sequence that is derived from a pathogen, for example wherein the pathogen is a bacteria, fungus or virus, for example wherein the virus is HPV.
  • the present invention also provides a method of forming the complex of the invention, wherein the complex is formed by contacting a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide according to the invention (i.e. a polypeptide capable of binding to a target antigen for example to an immune cell and capable of binding to a tagged cargo for example to a tagged antigen) with a tagged cargo (for example a tagged antigen) in vitro, wherein the tagged cargo (for example tagged antigen) comprises the corresponding tag (for example a coiled-coil oligomerisation domain) to which the tag binding domain of the polypeptide binds.
  • a polypeptide of the invention for example a cargo-targeting polypeptide or a cargo-delivery polypeptide according to the invention
  • a tagged cargo for example a tagged antigen
  • the tagged cargo comprises the corresponding tag (for example a coiled-coil oligomerisation
  • Preferences for features of the method of forming the complex of the invention for example in relation to the tagged cargo (for example tagged antigen) and to the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention are as described herein in relation to other aspects of the invention.
  • both the tag binding domain of the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and the tag that is used to tag the cargo are both complementary coiled-coil oligomerisation domains that are capable of interacting with one another and forming a multimer for example a dimer for example a heterodimer.
  • the method can also be used to prepare a complex that comprises more than one cargo for example more than one cargo antigen (for example more than one antigenic peptide) as will be apparent to the skilled person, for example by contacting the polypeptide of the invention with a number of tagged cargo (for example a number of tagged antigens).
  • the tagged cargo for example tagged antigens, e.g. antigens tagged with a coiled-coil oligomerisation domain tag
  • polypeptide for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises the appropriate corresponding tag binding domain(s) so that the polypeptide can bind to the tag(s).
  • the contacting may be performed simultaneously, i.e.
  • the polypeptide of the invention for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention can be contacted to a mixture of different tagged cargo, such as a mixture of different tagged peptide antigens; or the contacting can be performed sequentially, wherein the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is contacted to a first tagged cargo (for example a first tagged antigen), and is subsequently contacted to a second tagged cargo, and a third and fourth as appropriate (.e.g. a second, third and fourth tagged antigen).
  • a first tagged cargo for example a first tagged antigen
  • the invention provides a pharmaceutical composition comprising a complex of the invention.
  • Preferences for features of the pharmaceutical composition for example in relation to the tagged antigen (for example peptide tagged antigen) and to the polypeptide of the invention are as described herein in relation to other aspects of the invention.
  • the pharmaceutical composition will comprise the necessary excipients and carriers to ensure that the active agent, i.e. the complex, is maintained and supported and delivered in a functional state.
  • more than one cargo for example more than one antigen (for example more than one peptide tagged antigen, for example peptide tagged neoantigen), is administered to a patient.
  • this could be achieved by preparing a complex wherein the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention binds to a number of different tagged cargo, for example a number of different peptide antigens or neoantigens - i.e. each individual complex of polypeptide/antigen comprises more than one antigen.
  • an alternative means of delivering multiple cargo proteins or peptides for example delivering multiple antigens or neoantigens is to prepare a composition or pharmaceutical composition that comprises a number of different complexes of the invention, for example the pharmaceutical composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different complexes, wherein each complex is associated with a different cargo (for example different antigen or neoantigen), i.e. each individual complex only comprises one cargo (e.g. one antigen), but the pharmaceutical composition comprises a number of different complexes.
  • the invention provides a pharmaceutical composition wherein the composition comprises more than one different complex according to the invention, and wherein the cargo , for example tagged antigen(s) of each complex are different, for example wherein the sequence of the tag (for example the coiled-coil oligomerisation domain) is the same and the sequence of the cargo protein or cargo peptide (for example antigen) is different; or wherein the sequence of the tag (for example the coiled-coil oligomerisation domains) is different and the sequence of the peptide is different.
  • the cargo for example tagged antigen(s) of each complex are different, for example wherein the sequence of the tag (for example the coiled-coil oligomerisation domain) is the same and the sequence of the cargo protein or cargo peptide (for example antigen) is different; or wherein the sequence of the tag (for example the coiled-coil oligomerisation domains) is different and the sequence of the peptide is different.
  • the invention also provides a nucleic acid encoding the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention.
  • the invention also provides a nucleic acid encoding a tagged cargo protein or peptide of the invention.
  • the invention also provides a tagged cargo protein or peptide wherein the cargo protein or peptide comprises a first domain that is a coiled-coil oligomerisation domain and a second domain.
  • the invention provides a nucleic acid that encodes the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and which also encodes the tagged cargo protein or peptide of the invention.
  • the invention also provides a vector comprising any one or more the nucleic acids of the invention.
  • the invention provides a cell comprising any one or more of the nucleic acids of the invention or the vectors of the invention. Preferences for the nucleic acids, vectors and cells of the invention are as described herein in relation to other aspects of the invention.
  • the invention provides a number of medical uses relating to the administration of the complex or pharmaceutical composition of the invention or nucleic acid of the invention or vectors of the invention or cells of the invention . Preferences for features of the medical uses, for example in relation to the tagged peptide and to the polypeptide of the invention are as described herein in relation to other aspects of the invention.
  • the invention provides a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in medicine.
  • the invention also provides a method of treatment or method of preventing a disease, wherein the method comprises the administration of a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or ceil of the invention.
  • the invention also provides the use of a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in a method of manufacture of a medicament for the treatment or prevention of disease.
  • Therapeutic agents of the present invention are intended to be pre-formed as complexes prior to administration, with the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and the tagged cargo forming a complex with one another via the tag binding domain prior to administration.
  • the target antigen binding portion of the complex will direct the cargo (for example antigen) to the same target (for example immune cells).
  • the first component is a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, i.e. comprising a target antigen binding domain (e.g.
  • a tagged antigen that is not bound to a polypeptide of the invention for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention, i.e. with a domain specific to a target antigen (for example specific to an immune cell target), such as a DC target (e.g.
  • CD40 may fail to traffic to the DC and thus not be processed and presented to generate an adaptive immune response.
  • a polypeptide e.g. anti-CD40 polypeptide
  • tagged cargo for example tagged antigen
  • the present invention requires that the complexes are preformed prior to administration, to ensure the cargo (e.g. antigen or neoantigen) reaches the intended target for example target cells based on the target antigen binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention.
  • the cargo e.g. antigen or neoantigen
  • the complex and pharmaceutical compositions of the invention have a particular use in the treatment or prevention of cancer.
  • the cargo is a neoantigen, identified from a patient's tumour
  • the complex and pharmaceutical composition are particularly useful in the treatment of cancer.
  • the invention provides a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in a method of treating or preventing cancer, for example wherein the tagged cargo (for example a tagged antigen) comprises an antigenic sequence that has been identified as a neoantigen that has arisen in a tumour or cell; or a cancer antigen.
  • the tagged cargo for example a tagged antigen
  • the tagged cargo comprises an antigenic sequence that has been identified as a neoantigen that has arisen in a tumour or cell; or a cancer antigen.
  • the invention provides a method of treating or preventing cancer, wherein the method comprises administering a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention.
  • the invention also provides the use of a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention in the manufacture of a medicament for the treatment of cancer.
  • polypeptide and complexes, nucleic acid of the invention, or vector of the invention or cell of the invention thereof may also be used as a therapeutic or prophylactic vaccine.
  • the invention also provides a vaccine comprising any one or more of the polypeptide and complexes, nucleic acid of the invention, or vector of the invention or cell of the invention.
  • the vaccination is a cancer vaccine.
  • the vaccine is a vaccine against a contagious disease.
  • the invention also provides a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in a method of treating or preventing a pathogenic infection, for example for treating or preventing a bacterial, fungal or viral infection, wherein the tagged cargo comprises an antigenic peptide sequence derived from a bacteria, fungus or virus, for example from HPV.
  • the invention provides a method of treating or preventing a pathogenic infection, for example for treating or preventing a bacterial, fungal or viral infection, wherein the method comprises administering a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention wherein the cargo comprises an antigenic peptide sequence derived from a bacteria, fungus or virus, for example from HPV.
  • the invention also provides the use of a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention in the manufacture of a medicament for the treatment or prevention of a pathogenic infection, for example for treating or preventing a bacterial, fungal or viral infection, wherein the cargo comprises an antigenic peptide sequence derived from a bacteria, fungus or virus, for example from HPV.
  • the present invention lends itself to be used in methods of personalised therapy, particularly for the treatment of cancer.
  • a tissue sample from a tumour or other sample for example a blood sample where the cancer is a blood cancer, or a sample of exosomes, can be analysed to identify neoantigens that have arisen specifically in the cancer cell or tissue, and not in other healthy tissues.
  • Such analysis needs to be performed for each individual patient, producing a truly tailored therapy.
  • tagged versions of the antigens can easily be produced using, for example, standard laboratory cloning and expression techniques to create a tagged antigen - for example an antigen tagged with a coiled- coil oligomerisation domain.
  • the tagged antigens for example coiled-coil oligomerisation domain tagged antigens
  • they can be contacted with the or a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, producing one or more complexes that can be administered to the subject.
  • the actual polypeptide of the invention for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, for use in the treatment of multiple patients, can be universal.
  • the actual target antigen binding domain for example the immune cell binding domain
  • tag binding domains for example coiled-coil oligomerisation domains
  • the only variable part of the system is the cargo, for example the cargo protein or peptide sequence, for example the neoantigen sequence.
  • the invention provides a complex of the invention or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in a method of personalised therapy, wherein a tumourspecific antigen such as a neoantigen or viral antigen have been identified as having arisen in a patient (i.e. is specific for the tumour, either by virtue of certain mutations occurring in the tumour DNA, or by virally transmitted antigens), and wherein the tagged cargo protein or peptide comprises the neoantigen.
  • a tumourspecific antigen such as a neoantigen or viral antigen
  • the personalised therapy is for the treatment or prevention of cancer.
  • the information regarding suitable neoantigens for use in the personalised treatment of a patient are already available.
  • the step of analysing the sample from the patient and obtaining the necessary information regarding suitable neoantigens is part of the method of the invention.
  • the invention provides a complex of the invention or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in a method of personalised therapy, for example for the personalised treatment of cancer, wherein the method of personalised therapy involves the initial step of identifying a neoantigen that has arisen in a patient.
  • the invention also provides a method of personalised therapy, wherein the method comprises administering a complex of the invention or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for example for the personalised treatment of cancer.
  • the method involves the initial step of identifying a neoantigen that has arisen in a patient.
  • the invention provides the use of a complex of the invention or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention in the manufacture of a medicament for personalised therapy, for example for the personalised treatment of cancer.
  • the therapy involves the initial step of identifying a neoantigen that has arisen in a patient.
  • the invention also provides an in vitro method of producing a complex comprising a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention and a tagged cargo for example a tagged cargo protein or peptide (for example a tagged antigen), wherein the tagged cargo (for example tagged antigen) comprises the corresponding tag (for example peptide tag) to which the tag binding domain of the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention binds, wherein the method comprises contacting the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention with a tagged cargo (for example tagged antigen) in vitro or ex vivo.
  • Preferences for the in vitro or ex vivo method of producing a complex of the invention for example in relation to the tagged peptide and to the polypeptide of the invention, are
  • the complex of the invention is considered useful for in vivo administration.
  • the complex of the invention is also useful in an in vitro or ex vivo method of activating an immune cell, wherein the method comprises contacting the immune cell with a complex of the invention or a pharmaceutical composition of the invention, wherein the immune cell comprises the antigen to which the target antigen binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention binds.
  • the invention provides an in vitro or ex vivo method of activating an immune cell, wherein the method comprises contacting the immune cell with a complex of the invention or a pharmaceutical composition of the invention, wherein the immune cell comprises the antigen to which the target antigen binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention .
  • the method comprises contacting the immune cell with a complex of the invention or a pharmaceutical composition of the invention, wherein the immune cell comprises the antigen to which the target antigen binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention .
  • the tagged peptide and the polypeptide of the invention are as described herein in relation to other aspects of the invention.
  • the invention provides a kit for the in vitro preparation of a complex according to the invention wherein the kit comprises one or more polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the inventions of the invention and one or more tagged cargo, for example one or more tagged antigens; and/or comprises one or more nucleic acids or vectors or cells according to the invention.
  • the kit comprises one or more polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the inventions of the invention and one or more tagged cargo, for example one or more tagged antigens; and/or comprises one or more nucleic acids or vectors or cells according to the invention.
  • Preferences for features of the kits of the invention are as described herein in relation to other aspects of the invention.
  • the kit comprises a buffer suitable for the in vitro formation of the complex.
  • the invention provides a complex that comprises a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention in RUBYTM format, that has 1 target antigen binding domain capable of binding to CD40 and one K3 coiled-coil oligomerisation domain peptide tag binding domain that can form a heterodimer with the E4 coiled-coil oligomerisation domain, and wherein the complex comprises a cargo that is a neoantigen that was identified in a patient, wherein the neoantigen is tagged with the E4 coiled-coil oligomerisation domain.
  • a polypeptide of the invention for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention in RUBYTM format
  • the complex comprises a cargo that is a neoantigen that was identified in a patient, wherein the neoantigen is tagged with the E4 coiled-coil
  • the invention also provides a complex formed by contacting a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention that comprises a first and a second tag binding domain, wherein the first and second tag binding domain each binds to a different tag (i.e. a first and second tag), with a mixture of 2 tagged cargo proteins or peptides that are 2 tagged peptide neoantigens, wherein one peptide neoantigen has been tagged with the first tag and the other peptide neoantigen has been tagged with the second tag.
  • a polypeptide of the invention for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention that comprises a first and a second tag binding domain, wherein the first and second tag binding domain each binds to a different tag (i.e. a first and second tag), with a mixture of 2 tagged cargo proteins or peptides that are 2
  • FIG. 1 Human CD40 transgenic (hCD40tg) mice were given either anti-CD40- Fc(OVA) antibody or anti-CD40 antibody and the OVA peptide separately. Control mice received vehicle only. The treatments were given on two occasions, 7 days between. Seven days after the second treatment, inguinal lymph nodes were collected for flow cytometry analysis of viable CD45+ CD3+ CD8+ OVA-MHCI tetramer+ T cells. The graphs show frequency ( ⁇ SEM) of OVA (SIINFEKL)-MHCI tetramer+ among CD8+ T cells.
  • FIG. 1 Human CD40 transgenic (hCD40tg) mice were given OVA peptide-conjugated Fc-binding peptide (Z33) or free OVA peptide (SIINFEKL) in combination with anti- CD40 antibody, isotype control antibody, or Poly I:C adjuvant. Control mice received vehicle only. The treatments were given on two occasions, 7 days between. Seven days after the second treatment, inguinal lymph nodes were collected for flow cytometry analysis of viable CD45+ CD3+ CD8+ OVA-MHCI tetramer+ T cells. The graphs show frequency ( ⁇ SEM) of OVA (SIINFEKL)-MHCI tetramer+ among CD8+ T cells.
  • FIG. 3 Human CD40 transgenic (hCD40tg) mice were inoculated with EG7-OVA cells s.c. on the right flank on day 0, followed by s.c. treatment with either anti-CD40- Fc(OVA) antibody or vehicle control on the left flank on day 1 and 8.
  • the graphs show the mean ( ⁇ SEM) tumor volume.
  • FIG. 4 Human CD40 transgenic (hCD40tg) mice were inoculated with EG7-OVA cells s.c. on the right flank on day 0, followed by s.c. treatment with anti-CD40-Fc(OVA) antibody, or anti-CD40 antibody and OVA peptide given separately, or vehicle control, on the left flank on day 1 and 8.
  • the graphs show the mean ( ⁇ SEM) tumor volume.
  • Figure 5 Binding of the different peptides to antibodies fused with coiled coil peptides, A) binding of K3 and K4 to monoclonal antibodies fused with E3, B) binding of HAP2 to monoclonal antibodies fused with HAP1, HAP2,HAP3, HAP5 and HAP6, C) binding of K4 to IgGl monoclonal antibodies fused with E3, E4, E5 and E6, D) binding of K4 to IgG2 monoclonal antibodies fused with E3, E4, E5 and E6, E) binding of K6 to IgGl monoclonal antibodies fused with E3, E4, E5 or E6, F) binding of K6 to IgG2 monoclonal antibodies fused with E3, E4, E5 or E6.
  • Figure 6 Representative results of binding kinetics of the different peptides to antibodies fused with coiled coil peptides, A) binding of K3 to a monoclonal antibody fused with E3, B) binding of K4 to monoclonal antibody fused with E3, C) binding of K4 to monoclonal antibody fused with E4, D) binding of K6 to monoclonal antibody fused with E3, E) binding of K6 to monoclonal antibody fused with E4.
  • biotinylated peptides were coupled to streptavidin sensors and assayed against antibody in solution whereas antibodies were coupled to FAB2G sensors and assayed against peptides in solution (B-E).
  • FIG. 8 CD40 activation by A) monoclonal antibodies and B) bispecific antibodies fused with E3 and complexed with K4, CHO cells expressing Fc gamma receptor I (CHO- CD64), CHO cells expressing EpCAM (CHO-EpCAM) and CHO control cells were used.
  • Figure 9 Shows ELISA results for monoclonal antibodies fused with different peptides binding to antigen 1.
  • Figure 10 Binding kinetics of mAbs fused with coiled peptides against human Fey receptors.
  • Figure 14 A (parts A-F) Sensorgrams illustrating binding kinetics of antibodies with coiled coils to human FcRn. A and D) mAbE4, B and E) mAbE4K46 and C and F) mAb control without any peptide. Dissociation was performed at pH 6.0 (A-C) and 7.4 (D- F). B (parts G-L) Sensorgrams illustrating binding kinetics of antibodies with coiled coils to mouse FcRn. G and J) mAbE4, H and K) mAbE4K46 and I and L) mAb control without any peptide. Dissociation was performed at pH 6.0 (G-I) and 7.4 (J-L).
  • FIG. 18 ELISA results for monoclonal antibodies fused with different peptides binding after 7 days incubation in human serum or BSA.
  • Figure 19 Exemplary schematics showing the relative arrangements of the light and heavy chains, and coiled-coil oligomerisation domains of some potential formats. It will be clear that other arrangements are also possible.
  • Figure 20 FACS analysis of antibody-peptide complexes binding to cells.
  • K4-Ova Biotin peptides were used in these experiments. Cells only A), Cells + 0.5pg K4 Ova-biotin B), Cells + 5pg 1132E4 only C), Cells+5ugll32E4+0.5 pg K4-Ova-biotin D), Cells+ lugll32E4+0.1 pg K4-Ova-biotin E) and Cells+0.2ugll32E4+0.02 pg K4-Ova- biotin F).
  • FIG. 21 FACS analysis of antibody-peptide complexes binding to cells. K6-Ova Biotin peptides were used in these experiments. Cells only A), Cells + 0.5pg K6 Ova-biotin
  • FIG. 22 FACS analysis of Antibody-peptide-bead complexes binding to cells. Antibody-peptide-bead complexes were prepared first before addition to cells. Unstained A), Cells+ beads-anti SA PE stained B), Cells+ beads-anti Fc APC stained
  • Figure 24 A - Tumor growth over time. B - Survival over time.
  • Figure 25 Results of binding kinetics of the different peptides to antibodies fused with coiled coil peptide E4, A) binding of K4-Ova to an IgGl monoclonal antibody fused with E4, B) binding of K4-Ova to an IgG2 monoclonal antibody fused with E4, C) binding of K4-Ova to an IgGl monoclonal antibody control without E4, D) binding of Ova-K4-1 to an IgGl monoclonal antibody fused with E4, E) binding of Ova-K4-1 to an IgG2 monoclonal antibody fused with E4, F) binding of Ova-K4-1 to an IgGl monoclonal antibody control without E4, G) binding of Ova-K4-2 to an IgGl monoclonal antibody fused with E4, H) binding of Ova-K4-2 to an IgG2 monoclonal antibody fused with E4, I) binding of Ova-K4-2 to an IgGl monoclonal antibody control
  • a polypeptide comprising at least one immune cell binding domain and at least one tag binding domain, wherein the at least one immune cell binding domain is capable of specifically binding to an immune cell target; and wherein the at least one tag binding domain is capable of specifically binding to a peptide tag, wherein the peptide tag is not derived from tetanus toxin.
  • polypeptide according to embodiment 1 wherein the polypeptide comprises more than one immune cell binding domain optionally comprises 2, 3, or 4 immune cell binding domains.
  • polypeptide according to any one of embodiments 1 or 2 wherein the polypeptide comprises more than one peptide tag binding domain, optionally comprises 2, 3, or 4 peptide tag binding domains.
  • polypeptide according to embodiment 3 wherein one or more of the more than one peptide tag binding domains binds to a different peptide tag.
  • the immune cell is an antigen presenting cell, such as a dendritic cell (DC), B cell and/or macrophage (preferably DC).
  • DC dendritic cell
  • B cell preferably DC
  • macrophage preferably DC
  • the immune cell target is capable of mediating: activation of the immune cell; and/or internalisation of the polypeptide; and/or recruitment of conventional type I dendritic cells (cDCl).
  • cDCl conventional type I dendritic cells
  • the immune cell binding domain is an antibody selected from: ADC-1013; clones 1132/1133, 1140/1135, 1150/1151 and 1107/1108 from WO 2015/091853; CP- 870,893, APX005M, ChiLob 7/4, SEA-CD40; wherein:
  • 1150/1151 comprises one or more sequences selected from SEQ ID NO: 21-28;
  • 1107/1108 comprises one or more sequences selected from SEQ ID NO: 31-38.
  • a FLAG tag DYKDDDDK
  • a peptide probe sequence optionally wherein the peptide probe sequence is a coiled-coil peptide tag E3 comprising the amino acid sequence (EIAALEK)x3.
  • polypeptide according to any one of embodiments 1-12 wherein the peptide tag is a non-human peptide.
  • polypeptide according to any one of embodiments 1-13 wherein the immune cell binding domain is selected from the group consisting of: antibodies or antigen binding fragments thereof.
  • polypeptide according to any one of embodiments 1-14 wherein the peptide tag binding domain is: a) selected from group consisting of: antibodies or antigen binding fragments thereof; and/or b) is not an Fc region.
  • the antigen-binding fragment is selected from the group consisting of: an Fv fragment (such as a single chain Fv fragment, or a disulphide-bonded Fv fragment); a Fab-like fragment (such as a Fab fragment; a Fab' fragment; or a F(ab)2 fragment); and domain antibodies.
  • an Fv fragment such as a single chain Fv fragment, or a disulphide-bonded Fv fragment
  • a Fab-like fragment such as a Fab fragment; a Fab' fragment; or a F(ab)2 fragment
  • polypeptide according to any one of embodiments 1-14 and 16 wherein the peptide tag binding domain is an Fc region of an antibody having the ability to specifically bind to the peptide tag.
  • polypeptide according to any one of embodiments 1-17 wherein the polypeptide comprises at least 2 peptide tag binding domains, wherein at least one peptide tag binding domain is selected from the group consisting of: antibodies or antigen binding fragments thereof; and wherein at least one peptide tag binding domain is an Fc region of an antibody having the ability to specifically bind to the peptide tag. 19.
  • polypeptide according to any one of embodiments 1-18 wherein the polypeptide comprises: a) at least one immune cell binding domain that comprises or consists of an IgG antibody and at least one peptide tag binding domain that comprises or consists of an IgG antibody; b) at least one immune cell binding domain that comprises or consists of an IgG antibody and at least one peptide tag binding domain that comprises or consists of an Fv fragment; c) at least one immune cell binding domain that comprises or consists of an IgG antibody and at least one peptide tag binding domain that comprises or consists of a Fab-like fragment; d) at least one immune cell binding domain that comprises or consists of an IgG antibody and at least one peptide tag binding domain that comprises or consists of a domain antibody; e) at least one immune cell binding domain that comprises or consists of an IgG antibody and at least one peptide tag binding domain that comprises or consists of a coiled-coil peptide tag (such as E3 ((EIAALEK)
  • polypeptide according any one of embodiments 1-19 wherein the polypeptide is a bispecific polypeptide, and optionally comprises or consists of a format selected from the group consisting of: a) IgG-scFv bispecific antibodies; b) monovalent bispecific antibodies; c) scFv2-Fc bispecific antibodies; d) BiTE/scFv2 bispecific antibodies; e) DVD-Ig bispecific antibodies; f) DART-based bispecific antibodies; g) DNL-Fab3 bispecific antibodies; and h) scFv-HSA-scFv bispecific antibodies i) RUBYTM format antibodies, wherein the antibody comprises:
  • polypeptide according to any one of embodiments 1-20 wherein the polypeptide comprises an Fc region or a variant of said region, optionally wherein the region is an IgGl, IgG2, IgG3 or IgG4 region, optionally IgGl or IgG2.
  • polypeptide according to any of embodiments 1-21 wherein the polypeptide is a monospecific antibody directed towards the immune cell target, and wherein the Fc region has been modified to be capable of binding to the peptide tag; a bispecific antibody wherein one paratope comprises the immune cell binding domain and the second paratope comprises the peptide tag binding domain, and optionally wherein the Fc region of the antibody has been modified to be capable of binding to a peptide tag.
  • a complex comprising a polypeptide according to any one of embodiments 1- 22 and a tagged peptide antigen, wherein the tagged peptide antigen comprises the peptide tag to which the tag binding domain of the polypeptide binds.
  • the tagged peptide antigen comprises an antigenic peptide sequence that is a cancer antigen, optionally wherein the cancer antigen is a Human Papillomavirus (HPV)- associated cancer antigen.
  • HPV Human Papillomavirus
  • the tagged peptide antigen comprises an antigenic sequence that is derived from a pathogen, optionally wherein the pathogen is a bacteria, fungus or virus, optionally wherein the virus is Human Papillomavirus (HPV).
  • a pathogen optionally wherein the pathogen is a bacteria, fungus or virus, optionally wherein the virus is Human Papillomavirus (HPV).
  • HPV Human Papillomavirus
  • a pharmaceutical composition comprising a complex according to any of embodiments 23 and 28.
  • composition according to embodiment 29 wherein the composition comprises more than one different complex according to any of embodiments 23-28, and wherein the sequence of the tagged peptide antigen(s) of each complex are different, optionally wherein the sequence of the tag is the same and the sequence of the peptide antigen is different; or wherein the sequence of the tag is different and the sequence of the peptide is different.
  • a complex according to any one of embodiments 23-28 or a pharmaceutical composition according to any one of embodiments 29 or 30 for use in a method of treating or preventing cancer optionally wherein the tagged peptide antigen comprises an antigenic sequence that has been identified as a neoantigen that has arisen in a tumour or cell; or a cancer antigen.
  • HPV Human Papillomavirus
  • the complex for use according to any of embodiments 34 or 35 wherein the method of personalised therapy involves the initial step of identifying a neoantigen that has arisen in a patient.
  • a method for the treatment or prevention of a disease wherein the method comprises administering a complex according to any one of embodiments 23-28 or a pharmaceutical composition according to any one of embodiments 29 or 30.
  • a method for the treatment or prevention of cancer comprising administering a complex according to any one of embodiments 23-28 or a pharmaceutical composition according to any one of embodiments 29 or 30, optionally wherein the tagged peptide antigen comprises an antigenic sequence that has been identified as a neoantigen that has arisen in a cell, optionally a tumour cell; or a cancer antigen.
  • a method for the treatment or prevention of a pathogenic infection comprising administering a complex according to any one of embodiments 23- 28 or a pharmaceutical composition according to any one of embodiments 29 or 30, optionally wherein the method is for the treatment of prevention of a bacterial, fungal or viral infection, wherein the tagged peptide antigen comprises an antigenic sequence derived from a bacteria, fungus or virus, optionally from Human Papillomavirus (HPV).
  • HPV Human Papillomavirus
  • a method for the personalised treatment or prevention of a disease in a patient comprising administering a complex according to any one of embodiments 23-28 or a pharmaceutical composition according to any one of embodiments 29 or 30, optionally wherein the tagged peptide antigen comprises an antigenic sequence that has been identified as a neoantigen that has arisen in a cell, optionally a tumour cell; or a cancer antigen.
  • a method for personalised therapy comprising administering a complex according to any of embodiments 23-28 or a pharmaceutical composition according to any of embodiments 29 or 30 to a patient, wherein a neoantigen has been identified as having arisen in the patient, and wherein the tagged antigenic peptide comprises the neoantigen.
  • a method of producing a complex comprising a polypeptide according to any one of embodiments 1-22 and a tagged peptide antigen, wherein the tagged peptide antigen comprises the peptide tag to which the tag binding domain of the polypeptide binds, wherein the method comprises contacting the polypeptide according to any of embodiments 1-22 with a tagged peptide antigen in vitro.
  • An in vitro or ex vivo method of activating an immune cell comprising contacting the immune cell with a complex according to any of embodiments 23-28 or a pharmaceutical composition according to any of embodiments 29 or 30, wherein the immune cell comprises the immune cell target to which the immune cell binding domain of the polypeptide binds.
  • a vector comprising the nucleic acid according to embodiment 46.
  • a cell comprising the nucleic acid according to embodiment 46 or the vector according to embodiment 47.
  • kits for the in vitro preparation of a complex according to any of embodiments 23-28 wherein the kit comprises a polypeptide according to any of embodiments 1-22 and a tagged peptide antigen.
  • kit according to embodiment 49 wherein the kit comprises a buffer suitable for the in vitro formation of the complex.
  • Coiled coil peptide sequences were connected via a peptide linker to antibody sequences.
  • the coiled coil peptides used in the studies were: E3, E4, E5, E6, K3, K4, K5, K6, Leucine zipper, HAP1, HAP2, HAP3, HAP4, HAP5, HAP6, HAP7, HAP8. Their sequences can be found in Table 1.
  • the E3, E4, E5, E6, Leucine zipper, HAP1, HAP2, HAP3, HAP4, HAP5, HAP6, HAP7 and HAP8 coiled coil peptides were connected to the C-terminus of the antibody light chain. Both IgGl and IgG2 isotypes were tested.
  • the antibody used in these studies was 1132, a CD40 agonistic antibody that has been shown to activate antigen presenting cells.
  • Protein aggregation was measured with SE-HPLC in a 1260 Infinity II system (Agilent Technologies) using a TSK gel Super SW mAB HTP 4pm, 4.6x150mm column (TOSOH Bioscience) and 100 mM Sodium Phosphate, pH 6.8, 300mM NaCI as mobile phase at ambient temperature and a flow rate of 0.35 ml/min.
  • the aim of the study was to evaluate the oligomerization capacity between antibodies fused to coiled coil peptides and free coiled coil peptides using ELISA.
  • Plates were coated with 0.5 pg/mL antigen, rh CD40 Fc Chimera (1493-CDB, R&D Systems) in PBS over night at 4°C. After washing in PBS/0.05% Tween 20 (PBST), the plates were blocked with PBS/2% BSA for at least 30 minutes at room temperature before being washed again.
  • PBST PBS/0.05% Tween 20
  • Antibodies fused with peptides IgGl-1132E3 (IgGlmAbE3), IgG2-1132E3 (IgG2mAbE3), IgGl-1132E3 (IgGlmAbE3), IgG2-1132E3 (IgG2mAbE3), IgGl-1132HAPl (IgGlmAbHAPl), IgGl-1132HAP2 (IgGlmAbHAP2), IgGl-1132HAP3 (IgGlmAbHAP3), IgGl-1132HAP5 (IgGlmAbHAP5), IgGl-1132HAP6 (IgGlmAbHAP6), IgGl-1132E4 (IgGlmAbE4), IgG2-1132E4 (IgG2mAbE4), IgGl- 1132E5 (IgGlmAbE5), IgG2-1132E5 (IgG2mAbE5), IgG
  • K4 showed good binding to antibodies coupled with peptides E3 ( Figure 5A) whereas no binding was observed for K3 or HAP2 to antibodies fused to E3 ( Figure 5A) or HAP1, 2, 3, 5 or 6 respectively (Figure 5B).
  • K4 displayed binding to antibodies fused with peptides E4, E5 and E6 both for IgGl and IgG2 subclasses ( Figure 5C and D) although at lower degree compared to antibodies fused to E3.
  • K6 displayed good binding to antibodies fused with peptides E3, E4, E5 and E6 both for IgGl and IgG2 subclasses ( Figure 5E and F).
  • the control antibody 1332 without coiled coil peptide
  • the tested antibodies were (IgGl-1132E3 (IgGlmAbE3), IgG2-1132E3 (IgG2mAbE3), IgGl-1132E3 (IgGlmAbE3), IgG2-1132E3 (IgG2mAbE3), IgGl-1132E4 (IgGlmAbE4), IgG2-1132E4 (IgG2mAbE4), IgGl- 1132HAP3 (IgGlmAbHAP3), IgGl-1132HAP6 (IgGlmAbHAP6), IgGl-1132HAP7 (IgGlmAbHAP7)).
  • the peptides K4 or K6 were diluted 1 /2 in lx Kinetic buffer starting at 0.3 pM. Sensor regeneration using lOmM Glycine pH 1.7 was performed before capture of the next antibody. Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, inter-step correlation by alignment against dissociation was performed and the data were smoothed by a Savitzky-Golay filter in the data analysis software (v.9.0.0.14). The processed data were fitted using a 1: 1 Langmuir binding model with X2 as a measurement of fitting accuracy.
  • K3, K4 and K6 showed good binding to antibodies coupled with peptides E3 or E4. No binding was observed for HAP2 at all.
  • a faster dissociation observed for K3 (Figure 6A) was improved by increasing the length of the peptide to K4 and K6 (
  • Coiled coils were also fused to bispecific antibodies to evaluate compatibility with other antibody modalities than IgG.
  • E3, Leucin zipper, HAP1, HAP2, HAP3, HAP4, HAP5, HAP6, HAP7 and HAP8 coiled coil peptides were fused connected via a peptide linker to the C-terminal IgG light chain of a bispecific antibody called 1132-3174 consisting of a CD40 antibody 1132 and an EpCAM antibody 3174 in the RUBY format
  • RUBYTM bsAb are of the Appended IgG class of bsAb formats with FAb domains linked through their light chains to the C -terminal end of IgG molecules.
  • the bispecific antibody 1132-3174 and the 1132-3174 variants connected with coiled coil peptides were expressed using transient Expi293 HEK (Life technologies) cells in 30 mL according to manufacturer's instructions. Purification of the antibodies from supernatants was made on protein A using the NGC system (BioRad). Cells were transfected with three different vectors encoding separately for each of the three polypeptides chains (LI, Hl and H2).
  • Dual ELISA Plates were coated with 0.5 pg/mL CD40 (recombinant human CD40 Fc Chimera (1493- CDB, R&D Systems)) in PBS over night at 4°C. After washing in PBS/0.05% Tween 20 (PBST), the plates were blocked with PBS/2% BSA for at least 30 minutes at room temperature before being washed again. Samples serially diluted in PBS/0.5% BSA were then added and allowed to bind for at least 1 hour at room temperature. After washing, plates were incubated with 0.5 pg/mL recombinant human EpCAM Fc-Biotin (10694-H02H-B, Sino Biological) for at least 1 hour at room temperature. Dual complexed bsAb with CD40 and EpCAM were detected with HRP-labelled streptavidin. SuperSignal Pico Luminescent was used as substrate and luminescence signals were measured using Fl uostar Optima. H
  • Sensor tips were regenerated using 10 mM glycine, pH 1.7. Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, inter-step correlation by alignment against dissociation was performed and the data were smoothed by a Savitzky-Golay filter in the data analysis software (v.9.0.0.14), The processed data were fitted using a 1: 1 Langmuir binding model with X2 as a measurement of fitting accuracy.
  • the rank threshold is preferred over affinity as different alleles show biases towards high or low binding affinity. Peptides with a percentile rank ⁇ 10 were included in the binding predictions, as recommended by IEDB.
  • AbEpiAnalyser analyzes the potential immunogenicity of antibodies and sequence-modified proteins by first predicting the binding of each 15-mer peptide using the NetMHCII3. 1 algorithm to a large panel of human MHC class II alleles and subsequently calculates a positionspecific risk score. The score reflects the frequency of the HLA-DR, HLA-DP and HLA- DQ binding sub-peptides overlapping a given position in the defined population. The position-specific risk scores are summed over each sequence.
  • a cutoff value of 0.1 is set for the for position-specific risk scores (i.e. all position-specific risk scores ⁇ 0.1 are set to 0).
  • potential T-cell neo-epitopes are identified.
  • the North American population has a weight of 52.7%, European 25.3%, North East Asian 9.2%, South East Asian 5.9%, South Central American 1.7%, Oceanian 1.3%, Western Asian 1.3%, Sub-Saharan African 1.0%, South Asian 0.7%, North African 0.6%, and Australian 0.1%. Results are presented as position-specific risk scores.
  • HAP1 and HAP4 displayed the highest immunogenicity risk scores.
  • the risk scores for the E peptides were zero indicating that these coiled coils are particularly beneficial for fusion to antibodies from an immunogenicity perspective.
  • CD40 bioassay Promega. Briefly, CD40 Effector Cells were incubated overnight at 37°C. Serially diluted mono or bispecific antibody coiled coil fusion samples (1132E3 (mAbE3) and (1132-3174E3 (bsAbE3) respectively) complexed to soluble coiled coil counterparts (K4) were added followed by addition of CHO cells expressing Fc gamma receptor 1 (Fcyl (CD64)) or human EpCAM (for bsAb antibodies) or CHO-control cells. Plates were incubated at 37°C for 6 hours. Bio-GioTM Reagent was added and plates incubated at room temperature for 5- 15 minutes.
  • mAbE3 mono or bispecific antibody coiled coil fusion samples
  • bsAbE3 soluble coiled coil counterparts
  • the samples tested consisted of 1132E3 IgGl (mAbE3) or 1132-3174E3 (bsAbE3) complexed with soluble K4 peptide, mAbE3 and bsAbE3 in the absence of soluble K4 peptide and monoclonal and bispecific antibody controls consisting of 1132 mAb or 1132-3174 bsAb that were not fused to any coiled coil peptides.
  • mAbE3/K4 diluted to 12pg/ml (80nM) was mixed with K4 diluted to 0.64pg/ml (160nM) to a final concentration of mAbE3/K4 40nM to 80nM.
  • the complexes were incubated for 1 hour before serial dilution and addition to cells.
  • mAbE3 and mAb control were diluted to a start concentration of 6pg/ml (40nM).
  • bsAbE3 diluted to 24pg/ml, (96nM) was mixed with K4 diluted to 0.77pg/ml (192nM) to a final concentration of bsAb48nM/K4 96nM.
  • the complexes were incubated for 1 hr before serial dilution and addition to cells.
  • bsAbE3 and bsAb control were diluted to a start concentration of 12pg/ml (48nM).
  • Plates were coated with 0.5 pg/mL of CD40 in PBS over night at 4°C. After washing in PBS/0.05% Tween 20 (PBST), the plates were blocked with PBS/2% BSA for at least 30 minutes at room temperature before being washed again.
  • PBST PBS/0.05% Tween 20
  • Antibodies fused to coiled coil peptides (1132E3 (mAbE3), 1132Leucin zipper (mAbLeucine Zipper), 1132HAP1 (mAbHAPl), 1132HAP3 (IgGlmAbHAP3), 1132HAP4 (ImAbHAP4), 1132HAP5 (mAbHAP5), 1132HAP6 (mAbHAP6), 1132HAP7 (mAbHAP7), 1132HAP8 (mAbHAP8)) or naked antibodies used as control (1132 (mAb)) serially diluted in PBS/0.5% BSA were then added and allowed to bind for at least 1 hour at room temperature. After washing, plates were incubated with a secondary anti kappa antibody for at least 1 hour at room temperature. After washing, SuperSignal Pico Luminescent was used as substrate and luminescence signals were measured using Fluostar Optima.
  • the coiled coil fused antibody (A__1132E4IgGl) was diluted to 400nM, mixed with K6-Ova diluted at 800nM to obtain a complex at a final concentration of 200nM antibody and K6-Ova 400nM.
  • the captured antibodies were assayed against human or mouse Fc gamma receptors for 60 seconds followed by dissociation for 60 seconds.
  • Fc gamma receptors were serially diluted 1 /z in seven dilutions in lx Kinetic buffer (ForteBio) starting at 0.1 pM.
  • sample buffer 100 mM Sodium phophatase, 150 mM NaCI, 0.05 % Tween 20 pH 6
  • the captured antibodies were assayed against serially diluted ( 1 /z in seven dilutions starting at 1.6pm) human FcRn (ITF02-200, Immunitrack) or mouse FcRn (ITF08-200, Immunitrack) for 60 seconds. This was followed by dissociation in sample buffer at pH 6.0 or 7.4 for 60 seconds. Sensor regeneration using lOmM Glycine pH 1.7 was performed before capture of the next antibody.
  • A__1132E4 was mixed with 54nM K6-Ova to obtain a complex at final concentration of 13,5 nM A_1132E4, 27nM K6-Ova.
  • Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, interstep correlation by alignment against dissociation was performed and the data were smoothed by a Savitzky-Golay filter in the data analysis software (v.9.0.0.14). The processed data were fitted using a 1 : 1 Langmuir binding model. For pH 7.4 where a high off rate was obtained, only the initial 5-10 seconds were used for curve fitting and data processing.
  • CD40 agonistic antibody 1132E4 in combination with four different peptides, carrying the peptide antigen sequence for ovalbumin SIINFEKL as is or connected to coiled coil peptides K4 or K6 or the peptide Z33 (which binds to the Fc region of human IgGl antibodies), to induce OVA-specific CD8 + T cell expansion in comparison to the same OVA-peptides used as monotherapies was studied in vivo.
  • human CD40 transgenic mice were subcutaneously treated with either vehicle (PBS) or molar equivalent amounts of the peptides (14.1, 19.3, 18.9 and 3.3 pg of K4-Ova, K6-Ova, Z33-Ova and ovalbumin peptide control (SIINFEKL), respectively), alone or in combination with 33 pg of 1132E4.
  • vehicle PBS
  • molar equivalent amounts of the peptides (14.1, 19.3, 18.9 and 3.3 pg of K4-Ova, K6-Ova, Z33-Ova and ovalbumin peptide control (SIINFEKL), respectively
  • SIINFEKL ovalbumin peptide control
  • Storage stability Storage stability evaluation was performed by freeze-thawing or incubating the samples, of monoclonal antibodies connected to coiled coil peptides (A__1132IgGl, A_1132IgG2, A_1132E4IgGl, A___1132E4IgG2) or control antibody not connected to a coiled coil peptide (A_1132), at room temperature (RT) or 40°C for up to 2 weeks.
  • Freeze-thawing was performed by incubating proteins (the same proteins as listed above) at -80°C and thawing them in a water bath set at 37°C after 24, 48 and 72 hours.
  • SEC-HPLC analysis was performed only on samples thawed after 24 and 72 hours. For proteins incubated at 40°C, SEC-HPLC analysis was performed for samples at weeks 1 and 2. In addition, all samples were visually inspected for any particles at each of the three-time points.
  • PEG polyethylene glycol
  • Protein samples of monoclonal antibodies connected to coiled coil peptides (A_1132IgGl, A__1132IgG2, A__1132E4IgGl, A___1132E4IgG2) or control antibody not connected to a coiled coil peptide (A__1132), were incubated in BSA (PBS + 0.1% BSA) or 50% human serum at 37°C for 7 days. Mono ELISA was performed after seven days to assess binding.
  • Protein solubility can be determined by exposing protein samples to different concentrations of PEG. Addition of PEG to the protein solution leads to precipitation as a result of exclusion volume effects. PEG being a long-chain polymer occupies more space in the solution than a protein of a similar molecular mass and this lowers protein solubility resulting into protein precipitation.
  • the antibodies with coiled coil peptides showed good colloidal stability comparable to the antibody control, not connected to a coiled coil peptide, and performed better than the assay control which as expected had about 50% of the protein precipitated at PEG concentration of approximately 8-9 % ( Figure 17).
  • the serum stability assay showed no loss in binding after seven days incubation in human serum, compared to BSA for any the antibodies connected to coiled coil peptides or the control antibody without coiled coil peptide ( Figure 18)
  • K4-Ova-biotin or K6-Ova-biotin were diluted to lpg/ml from which 1/5 serial dilutions were prepared.
  • 1132E4 was diluted to lOpg/ml from which 1/5 serial dilutions were prepared.
  • K4-Ova-biotin or K6-Ova biotin were mixed with 1132E4 on a 1 : 1 ratio to obtain complex concentrations ranging from 5ug/0.5ug to 0,00032 /0, 000032 1132E4/K4-Ova-Biotin or 1132E4/K6-Ova-Biotin.
  • incubation was performed for Ihour at room temperature with gentle rotation.
  • B16-F10 CD40 expressing cells and B16-F10 wt cells were used. Cells were seeded at 300000 cells/well in 25pl. The cells were incubated with 25pl antibody-peptide complexes. Cells without any treatment, cells treated with only the peptide (K4-Ova- biotin or K6-Ova-biotin) and cells treated with only the antibody were used as controls. Incubation was performed for 30 minutes at 4°C. Staining was performed using PE Streptavidin (BD Pharmingen #554061) followed by FACS analysis. Results
  • Antibody-peptide-beads complexes were prepared by mixing equimolar concentrations of 1132E4IgG2 and K6-Ova-biotin at a ratio of 1 : 1 to obtain a complex at 5ug/0.5ug antibody/K6-peptide complex. This was followed by addition of Streptavidin MicroBeads. To obtain an antibody-peptide-bead mixture, a 12pL beads/500ul antibody-peptide mixture was prepared. An antibody-peptide mixture without beads was prepared as control.
  • Two 1:4 serial dilutions of the antibody-peptide-beads complexes and control mixtures were prepared. The solutions were incubated at room temperature with gentle rotation to allow complex formation. B16-CD40 or B16-wt cells were prepared at 8x10 ⁇ 6 cells/ml for each cell line. Additional controls were prepared at 12 L beads/8xl0 ⁇ 6 B16-CD40 or B16-wt cells. Finally, 300 000 cells/well were plated followed by addition of the antibody K6-peptide-beads complexes or controls and incubation at 4°C for 30 minutes.
  • non-tumor bearing male huCD40tg mice Forty-four non-tumor bearing male huCD40tg mice (ICH) of Alligator's own strain were used in the experiment. On day 0 and day 7, non-tumor bearing male huCD40tg mice were subcutaneously treated with either vehicle (PBS), or molar equivalent amounts of the peptides (14.1, 19.3, 18.9 and 3.3 pg of K4-Ova, K6-Ova, Z33-Ova and ovalbumin peptide control (SIINFEKL) alone or in combination with 33 pg of 1132E4 (huCD40) in IgGl or IgG2 format, or 1132 CD40 mAb alone. Seven days after the second treatment (D14), all mice were sacrificed and their inguinal lymph nodes closest to the treatment site (iLN) were removed for FACS analysis of OVA-specific CD8+ T cells.
  • PBS vehicle
  • SIINFEKL molar equivalent amounts of
  • mice On day 0, 0.2e6 MB49-EpCAM + -ovalbumin+ cells, growing in log phase, were injected subcutaneously in the right outer flank of human CD40 transgenic mice in a volume of 100 pl PBS. On day 10 and day 17, mice were subcutaneously treated in the inside of the left hindleg with either vehicle (PBS), 1.5 pg free OVA-peptide (SIINFEKL), 60 pg of 1132E4-IgG2, 6.5 pg K4-OVA or 60 pg of 1132E4-IgG2 in combination with 6.5 pg K4-OVA. The peptide doses given correspond to equal molar amounts.
  • the tumor growth was observed and measured with a caliper in width (w), length (I) and height (h) of which the tumor volume was calculated (w/2 x 1/2 x h/2 x n x (4/3)). Tumor measurements were performed until the end of the experiment, when the tumor volume of the last mouse was approaching the ethical limit of 2000 mm3.
  • the K4-0va where the ova fused with the ova peptide on the C terminal was synthesized with higher purity (>98%) compared to ova-K4-l(>70%) or Ova-K4-2 (>70%) peptides.
  • the Ova peptide is fused on the N-terminal.
  • the ova peptide is linked to the N-terminal.
  • the ova peptide is surrounded by hydrophobic amino acids Lysine and Tryptophan.
  • a control IgGl antibody (called 1188) and monoclonal antibodies coupled with coiled coil peptides, 1132E4 IgGl and 1132E4 IgG2, were expressed using transient Expi293 HEK (Life technologies) cultures in 30 mL according to manufacturer's instructions. Purification of the antibodies from supernatants was made on protein A using the NGC system (BioRad). Cells were transfected with two different vectors encoding separately for each of the two polypeptides chains (i.e. the immunoglobulin heavy chain and the light chain to which a coiled coil peptide was fused).
  • Protein aggregation was measured with SE-HPLC in a 1260 Infinity II system (Agilent Technologies) using a TSK gel Super SW mAB HTP 4 m, 4.6x150mm column (TOSOH Bioscience) and 100 mM Sodium Phosphate, pH 6.8, 300mM NaCI as mobile phase at ambient temperature and a flow rate of 0.35 ml/min.
  • Example 17 Priming of ovalbumin-specific CD8+ T cells by anti-CD40-
  • the OVA peptide SIINFEKL [SEQ ID NO: 70] was conjugated to the Fc of an anti-CD40 antibody (bivalent, monospecific) by use of Z33, a 33 amino acid long Fc-binding peptide, which binds between the CH2 and CH3 region of the Fc domain.
  • the SIINFEKL peptide was in turn covalently linked to the Z33 peptide via a GSSSS linker.
  • the purpose with this anti-CD40-Fc(OVA) antibody is to evaluate a concept wherein a peptide is conjugated to an anti-CD40 agonist and how such conjugation impacts T cell responses to the peptide.
  • the OVA peptide can be exchanged to one or more tumor antigens to induce an immune response directed against tumor cells expressing such antigen(s).
  • the aim of this experiment was to evaluate the effect of the anti-CD40-Fc(OVA) antibody on the priming of OVA-specific CD8+ T cells, compared to an anti-CD40 antibody and OVA peptide administered separately.
  • mice Human CD40 transgenic (hCD40tg) mice, 8-9 weeks of age, were given a mixture of 33 pg anti-CD40 antibody and 18.9 pg Z33-OVA complex s.c. on two occasions, 7 days between. Additional cohorts of mice were instead given 33 pg anti-CD40 antibody and 3.3 pg OVA peptide separately or vehicle (PBS) control.
  • PBS vehicle
  • mice were sacrificed and inguinal lymph nodes collected.
  • the lymph nodes were mashed through cell strainers to obtain single cell suspensions and the cells were subsequently Fc blocked and stained with an antibody cocktail containing fluorescently-labelled anti-mouse antibodies for CDllb, CD19, MHCII, NK1.1 (dump channel), and CD45, CD3, CD4 and CD8, as well as OVA (SIINFEKL) MHCI tetramer.
  • the cells were also stained with Fixable Viability Stain 780 (BD Biosciences) to assess the cell viability. Samples were analysed by flow cytometry in order to determine the frequency of viable CD45+ CD3+ CD8+ OVA-MHCI tetra mer+ T cells.
  • Example 18 Effect on priming of OVA-specific T cells in vivo
  • the OVA peptide-linked Fc-binding peptide Z33 is capable of binding to antibodies, thereby forming an antibody-antigenic peptide complex.
  • the antigenic peptide is targeted to CD40- expressing antigen-presenting cells such as dendritic cells (DC), which enhances crosspresentation of the antigen and increases priming of antigen-specific T cells.
  • DC dendritic cells
  • the purpose with this Fc-binding peptide is to generate a concept where T cell priming is improved when the Fc-binding peptide is combined with a DC-targeting and activating antibody.
  • the aim of this experiment was to evaluate the effect on T cell priming when Z33-OVA or free OVA peptide is combined with either a CD40 agonistic antibody, an isotype control antibody, or an adjuvant to which Z33 does not bind.
  • Human CD40 transgenic mice 12-14 weeks of age, were given a mixture of 18.9 pg Z33-OVA peptide or a molar equivalent dose of 3.3 pg free OVA peptide (SIINFEKL) and either 33 pg anti-CD40 antibody, 33 pg isotype control antibody, or 50 pg Poly I : C (a TLR3 ligand used as adjuvant) s.c. on two occasions, 7 days between.
  • An additional cohort of mice were instead given vehicle (PBS) control.
  • mice were sacrificed and inguinal lymph nodes collected.
  • the lymph nodes were mashed through cell strainers to obtain single cell suspensions and the cells were subsequently Fc blocked and stained with an antibody cocktail containing fluorescently-labelled anti-mouse antibodies for CDllb, CD19, MHCII, NK1.1 (dump channel), and CD45, CD3, CD4 and CD8, as well as OVA (SIINFEKL) MHCI tetramer.
  • the cells were also stained with Fixable Viability Stain 780 (BD Biosciences) to assess the cell viability. Samples were analysed by flow cytometry in order to determine the frequency of viable CD45+ CD3+ CD8+ OVA-MHCI tetra mer+ T cells.
  • an immunostimulatory signal alone was not sufficient to induce superior T cell expansion with Z33-OVA compared to OVA peptide, further supporting the notion that conjugating the antigenic peptide to a DC-targeting agonistic antibody is important for achieving an improved T cell priming effect.
  • Example 19 Anti-tumor efficacy of an OVA-expressing tumor model, comparing anti-CD40-Fc(OVA) to vehicle control
  • mice subcutaneously with the anti-CD40-Fc(OVA) antibody i.e. anti-CD40 antibody complexed with the Z33-OVA peptide
  • an OVA-specific T cell response was induced in draining lymph nodes.
  • the OVA peptide can be exchanged for tumor antigen peptides to induce a tumor-specific immune response against tumors expressing such antigens. This tumor-targeting immune response would be expected to reduce the growth of an established tumor.
  • the aim of this experiment was to evaluate the effect of the anti-CD40-Fc(OVA) antibody on the growth of the OVA-expressing tumor EG7-OVA in a therapeutic vaccination setting.
  • mice Human CD40 transgenic (hCD40tg) female mice, 10-12 weeks of age, were inoculated with 1.0 x 10 6 EG7-OVA cells s.c. on the right flank on day 0. On day 1 and 8, mice were given either a mixture of 100 pg anti-CD40 antibody and 57 pg Z33-OVA peptide (anti-CD40-Fc(OVA) antibody) (which were mixed together prior to administration to allow complexes between the antibody and tagged antigen to form prior to administration to the mice) or vehicle control (Dextrose) s.c. on the left flank. Tumor volume and survival was monitored.
  • anti-CD40-Fc(OVA) antibody which were mixed together prior to administration to allow complexes between the antibody and tagged antigen to form prior to administration to the mice
  • vehicle control Dextrose
  • Example 20 Comparison of the anti-tumor efficacy of CD40-Fc(OVA) (premixed combination of anti-CD40 and Z33-OVA) and separate administration of anti-CD40 and OVA in an OVA-expressing tumor model
  • the aim of this experiment was to evaluate the effect of the anti-CD40-Fc(OVA) antibody (i.e. the pre-mixed complex of the anti-CD40 antibody and the Z33-OVA peptide) on the growth of the OVA-expressing tumor EG7-OVA in a therapeutic vaccination setting, compared to the separate administration of the anti-CD40 and OVA peptide (SIINFEKL).
  • the anti-CD40-Fc(OVA) antibody i.e. the pre-mixed complex of the anti-CD40 antibody and the Z33-OVA peptide
  • mice Human CD40 transgenic (hCD40tg) female mice, 9-11 weeks of age, were inoculated with 1.0 x 10 6 EG7-OVA cells s.c. on the right flank on day 0. On day 1 and 8, mice were given either a mixture of 100 pg anti-CD40 antibody and 19 pg Z33-OVA peptide (anti-CD40-Fc(OVA) antibody) (which were mixed together prior to administration to allow complexes between the antibody and tagged antigen to form prior to administration to the mice) or anti-CD40 and 3.3 pg OVA peptide (a molar equivalent dose to Z33-OVA peptide) s.c. on the left flank. Tumor volume and survival was monitored.
  • SIINFEKL OVA peptide

Abstract

The present invention provides polypeptides capable of targeting a cargo, such as a neoantigen, to particular sites in the body, and associated therapeutic methods. In particular embodiments, the polypeptides are antibodies or antibody-based polypeptides.

Description

Methods and compositions
Field
The present invention relates to the field of targeted delivery of cargo.
Background
Although immune checkpoint inhibitors have improved the clinical benefit for many cancer patients, there is still a great unmet medical need; especially immunologically "cold" tumors with less T cell infiltration generally do not respond to immune checkpoint inhibitors [ 1]. Therapeutic cancer vaccines have the potential to increase the tumortargeting T cell pool, providing a possible treatment for some of these immunologically cold tumors. In addition, any tumor not responding completely to PD-l-targeting therapies could benefit from cancer vaccines.
Therapeutic cancer vaccines have a poor clinical track record, mainly due to the choice of vaccination antigens, which mostly have been tumor-associated self-antigens that do not generate a strong tumor-specific T cell response. The breakthroughs in immunotherapy have shown that for a vaccine to generate a strong T cell response, it is critical to target tumor-specific antigens, e.g. neoantigens.
There are several lines of evidence that demonstrate that neoantigens are critical for obtaining a successful immune response. First, neoantigen-load (mutational burden) of patients' tumors correlate with favorable clinical outcome. Second, tumor neoantigen-specific T cells are expanded by successful immunotherapies (targeting PD- 1, CTLA-4). Finally, neoantigen-specific T cells are responsible for the anti-tumor effect of successful immunotherapies.
Thus, the main challenges to address when developing immune cell-targeted therapies are to: i) provide the right type of immune activation that generates an effective antitumor T cell response, and ii) target tumor antigens, such as neoantigens, that can induce a strong antitumor response. Brief summary of invention
An issue with current methods of antibody-targeted vaccination is that a new antigenbinding molecule, i.e. antibody, has to be generated for each different antigen that is to be delivered to an immune cell, such as a dendritic cell (DC). The present invention at least addresses this issue and provides an off-the-shelf polypeptide that can be used to deliver a range of antigens to a particular target cell population.
The present invention comprises a polypeptide that in some instances is capable of both optimizing neoantigen uptake by an immune cell and/or cross-presentation of the antigen to T cells. In preferred situations, the tumor of a patient is sequenced to identify suitable personalized neoantigens. These neoantigens may then be produced and attached to, for example, a DC-activating antibody of one embodiment of the invention that promotes uptake of the neoantigen by the DC and at the same time mediates a superior activation of the DC, which in turn allows the DC to cross-present the neoantigens and activate a strong T cell response towards the tumor, and cure the patient.
The polypeptides and complexes described herein can be used in contexts other than the delivery of antigens to immune cells. For example the polypeptides and complexes described herein are able to deliver a range of different cargo to a range of different target sites.
Antibody-targeted vaccination
Antibodies may be selective for DCs and/or other antigen-presenting cells (APC). Antigen fused to a DC-targeting antibody is more efficiently taken up by the DC and subsequently presented to T cells in the context of MHC class I or II compared to free antigen. Consequently, DC-targeted antigen elicits stronger T cell responses compared to free antigen or antigen coupled to an isotype control antibody, and induces superior anti-tumor responses in mouse models [2]. An additional benefit can be gained by incorporating an immune-activating stimulus in the same molecule, for example by targeting an activating DC receptor with an agonistic antibody, since antigen-adjuvant complexes induce superior anti-tumor responses compared to a corresponding mix of antigen and adjuvant [3]. Efficient priming of CD8+ CTL responses is important for generating a productive anti-tumor response, and cDCl play a prominent role in priming CTL responses for anti-tumor immunity [4]. A productive immune response against a tumor involves both CD8+ and CD4+ T cells, however inducing CD8+ rather than CD4+ T cell responses remains a challenge for cancer vaccines, including neoantigen vaccines. Delivering antigen to a DC population that includes cDCl is likely to promote the induction of antigen-specific CTL.
The selection of a DC target determines which DC population that is targeted, and how much of the antigen that is taken up and how much is presented on MHC II versus MHC I, the latter being critical for presentation to CD8+ T cells. Further, the choice of DC target also affects the level of DC activation following antigen uptake, which determines if antigen-specific T cells are activated or suppressed. A number of different DC targets have been evaluated for antibody-targeted vaccination, including e.g. CR- 1, CLEC9A, DEC-205, CDlc, Dec-1, CDllb, CDllc and CD40 [5-7].
The main advantage with targeting CD40 is that CD40 stimulation activates DC and induces cross-presentation. Despite its poor internalization properties, targeted antigens (i.e. an antibody fused to an antigenic peptide) binding to CD40 induce superior CD8+ T cell responses compared to e.g. DEC-205 [6]. In fact, it was recently demonstrated that CD40 was superior to nine different lectins and scavenger receptors (LOX-1, DC-ASGPR, DCIR, Dectin-1, DEC205, Langerin, MARCO, CLEC6, and DC- SIGN/L) with respect to generating a CD8+ T cell response, using primary human cells in vitro [8]. Further, it was shown that CD40 primarily mediated internalization into early endosomes, which favors antigen processing and cross-presentation to CD8+ T cells.
The goal with the present invention is to provide a drug candidate capable of delivering patient-specific tumor neoantigen to DC, and simultaneously activating the DC, thereby leading to superior priming and activation of neoantigen-specific T cells. The activated T cells will then mediate a superior anti-tumor effect against the neoantigen-expressing tumors. The molecule should mediate DC targeting, activation and antigen internalization, all of which could be achieved using CD40 as DC target. The molecule comprising a CD40-binding domain and a tag-binding domain (for example a peptide- tag binding domain i.e. a domain that binds to a peptide tag) will then be mixed with neoantigens, for example neoantigenic peptides fused to a tag (for example a peptide tag), prior to administration to a patient, resulting in the formation of CD40-neoantigen complexes. Antigen-targeting to DC mediates a more efficient vaccination effect compared to non-targeted antigen, therefore CD40-neoantigen complexes should be superior to a formulation comprising an anti-CD40 antibody and neoantigen peptide, in terms of expansion and/or function of neoantigen-specific T cells and/or anti-tumor effect. By "CD40-neoantigen complex" we mean a complex that forms between 1) the molecule that comprises the CD40-binding domain and the tag-binding domain; and 2) the neoantigen fused to the tag - i.e. it is a complex that targets the neoantigen to cells expressing CD40.
The concept of mixing CD40-tag binding antibodies (i.e. antibody that binds to both CD40 and to a tag) with neoantigen-tag entities (i.e. a neoantigen fused to a tag) to generate CD40-neoantigen complexes provides a flexibility that is not available for current CD40-antigen fusion concepts. For a truly personalized neoantigen vaccination approach, a patient's tumor must first be sequenced and neoantigen epitopes identified, followed by production of the neoantigen. This can be completed in a matter of weeks for peptides, whereas generation of a new antibody or antibody-antigen fusion protein would take more than a year, making it impossible to use as a personal neoantigen vaccine. The CD40-tag antibodies (i.e. capable of binding to CD40 and to the tag) on the other hand would be available as an off-the-shelf product, ready to be mixed with the tagged personal neoantigens (for example antigenic peptides) as soon as they have been synthetized. Further, the CD40-tag antibodies could be mixed with tagged antigens that encompass several neoantigen epitopes. Vaccinating against one neoantigen epitope is generally not sufficient to induce a long-lasting anti-tumor effect, and several neoantigen epitopes will likely be needed to elicit a strong anti-tumor response. In this fashion, the invention addresses limitations with the currently available antibody-antigen fusion proteins.
It is not only improved methods of targeted delivery of antigens to immune cells that are needed. There is also a general need for improved and simple methods to target various entities to particular cells, or tissues, or other sites within the body.
Detailed description of the invention
The invention provides a polypeptide molecule that in some embodiments is considered to be a cargo-targeting polypeptide or a cargo-delivery polypeptide. The polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is able to bind to at least one target antigen, for example a target antigen on an immune cell such as a dendritic cell, and is also able to bind to a tag, for example a peptide tag. The tag, for example the peptide tag in preferred embodiments is a coiled-coil oligomerization domain that is attached to a cargo. The cargo may be any cargo that is desired to be targeted to or delivered to a particular site, for example a particular cell type, or tissue, and is further defined elsewhere herein. A tag that is a coiled-coil oligomerisation domain that is attached to a cargo can be referred to as a cargo coiled-coil oligomerisation domain to distinguish it from a corresponding coiled- coil oligomerisation domain present on the polypeptide of the invention, for example on the cargo-targeting or cargo-delivery polypeptide of the invention.
In this way a single polypeptide of the invention, for example a single cargo-targeting or a cargo-delivery polypeptide of the invention can be used to target a diverse array of cargo a particular cellular targets.
The present invention provides a polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprising at least a first target antigen binding domain and at least a first tag binding domain, wherein the first target antigen binding domain is capable of specifically binding to a first target antigen. In some preferred embodiments the first tag binding domain comprises a first coiled-coil oligomerisation domain.
Accordingly in some embodiments the invention provides a polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprising at least a first target antigen binding domain and at least a first tag binding domain wherein the first target antigen binding domain is capable of specifically binding to a first target antigen; and wherein the first tag binding domain comprises a first coiled- coil oligomerisation domain.
In some embodiments the target antigen binding domain can be considered to be an immune cell binding domain that is capable of specifically binding to a target present on an immune cell.
In some preferred embodiments, the tag binding domain does not bind to a peptide tag that is derived from tetanus toxin.
By the term "binding domain" we include the meaning of any peptide sequence that is capable of binding or associating with the relevant second entity. In the case of the first target antigen binding domain, the domain is able to bind specifically to a target antigen. For example in some embodiments the first target antigen is a target antigen on an immune cell and so the target antigen binding domain can be considered to be an immune cell binding domain. In this case the domain is able to bind specifically to a target on an immune cell. In preferred embodiments the target is CD40, and the target binding domain comprise an immunoglobulin or fragment thereof that binds to CD40. Exemplary antibodies are described elsewhere herein.
The tag binding domain is able to specifically bind to a tag, for example a peptide tag.
Preferably the tag binding domain comprises a coiled-coil oligomerisation domain that can form a multimer, for example a dimer for example a heterodimer with a corresponding coiled-coil oligomerisation domain. One coiled-coil domain of the heterodimer is present on the polypeptide of the invention, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention, and the corresponding coiled-coil domain of the heterodimer is connected to or attached to the cargo. The interaction between the two coiled-coil oligomerisation domains results in the formation of a complex and the cargo being associated with the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention- in this way the cargo is targeted to the target antigen (for example immune cell) by virtue of the antigen binding domain of the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention.
By "bind specifically" we include the meaning that the domain binds to its target in a manner that can be distinguished from binding to non-target domains (i.e. off-targets). For example, a domain that binds specifically may refer to a domain that binds with higher specificity for the intended target compared with that of a non-intended target. Specificity can be determined based on dissociation constant through routine experiments. A domain being "specific for" a target is intended to be synonymous with a domain "directed against" said target.
Preferably the binding domains will bind only to its respective target, i.e. an antigen, for example an antigen present on an immune cell target, or the relevant tag, for example the peptide tag, for example a coiled-coil oligomerisation tag, and will not bind to any other molecule in the environment, for example in the human body. However, it will be appreciated that some degree of off-target binding may be tolerated, and the skilled person will understand how to determine whether a particular binding activity is of the required specificity or not.
The target antigen binding domain, for example the immune cell binding domain is the portion of the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention that directs or targets the polypeptide of the invention to the desired target, for example to the desired immune cell, and requires the target antigen binding domain, for example the immune cell binding domain to be able to bind specifically to a particular target antigen, for example to a particular target antigen on the desired immune cell.
The polypeptide of the invention, for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention may comprise a single target antigen binding domain, for example a single immune cell binding domain, or may comprise more than one target antigen binding domain, for example more than one immune cell binding domain. For example, a typical monospecific antibody comprises two variable regions, each capable of binding to the same specific target. In this case, where the target antigen is present on an immune cell, the antibody would comprise two immune cell binding domains. The skilled person will be aware of different antibody formats, including those described herein, and will understand that antibody engineering can result in a wide variety of different molecules, with different valences and different specificities.
Accordingly in some embodiments the first target antigen binding domain comprises a first immunoglobulin light chain and a first immunoglobulin heavy chain. In some embodiments where the tag binding domain comprises a first coiled-coil oligomerisation domain, the first coiled-coil oligomerisation domain is connected to any one or more of: a) the C-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence; b) the C-terminus of the first immunoglobulin heavy chain, optionally connected via a linker sequence; c) the N-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence; and/or d) the N-terminus of the first immunoglobulin heavy chain, optionally connected via a linker sequence. the C-terminus of the first immunoglobulin light chain, for example connected via a linker sequence.
In some embodiments the linker that connects the light or heavy chain to the coiled- coil oligomerisation domain is a linker of SEQ ID NO: 56. In some embodiments of the polypeptide of the invention, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention; a) the first immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR region, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and b) the first immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region.
In some embodiments the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises a heavy chain that comprises more than one constant region. For example in some embodiments: a) the first immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and b) the first immunoglobulin heavy chain comprises or consists of:
I) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three constant regions.
As described above, the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise or consist of one target antigen binding domain, or may comprise more than one target antigen binding domain. Accordingly in some embodiments the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises a second target antigen binding domain, for example wherein the second target antigen binding domain comprises a second immunoglobulin light chain and a second immunoglobulin heavy chain.
As for the first target antigen binding domain, the heavy chain of the second target antigen binding domain may also comprise one or more than one constant region. Accordingly in some embodiments: a) the second immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and b) the second immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region; or a) the second immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; and ii) one constant region; and b) the second immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three constant regions.
The tag binding domain (e.g. the coiled-coil oligomerisation domain) may be connected to the polypeptide of the invention at any position.
Where the tag binding domain is a coiled-coil oligomerization domain and the target binding domain comprises an immunoglobulin light chain, in some preferred embodiments the coiled-coil oligomerization domain is connected to the C-terminus of the immunoglobulin light chain, and in some instances is connected via a linker.
Accordingly in some embodiments wherein the first target antigen binding domain comprises a first immunoglobulin light chain and a first immunoglobulin heavy chain, and wherein the first coiled-coil oligomerisation domain is connected to the C-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence.
In some embodiments wherein the polypeptide comprises a second target antigen binding domain, wherein the second target antigen binding domain comprises a second immunoglobulin light chain and a second immunoglobulin heavy chain, in some embodiments the C-terminus of the second immunoglobulin light chain is connected to a tag binding domain, for example to a second coiled-coil oligomerisation domain, for example connected via a linker. However, as described above, one or more coiled coil oligomerisation domains may be connected to any one or more ends of any one or more of the light or heavy chains, for example to the N-terminus and/or C-terminus of the first and/or second light chain and/or the N-terminus and/or C-terminus of the first and/or second heavy chain, for example: a) the C-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence; b) the C-terminus of the first immunoglobulin heavy chain, optionally connected via a linker sequence; c) the N-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence; d) the N-terminus of the first immunoglobulin heavy chain, optionally connected via a linker sequence, e) the C-terminus of the second immunoglobulin light chain, is connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence; f) the C-terminus of the second immunoglobulin heavy chain, is connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence; g) the N-terminus of the second immunoglobulin light chain, is connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence; and/or h) the N-terminus of the second immunoglobulin heavy chain, is connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence.
In some embodiments the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises a second antigen target binding domain but does not comprise a second tag binding domain. For example where the second antigen target binding domain comprises an immunoglobulin light chain, the light chain is not connected to a second tag binding domain, for example is not connected to a second coiled-coil oligomerization domain.
In some embodiments wherein the polypeptide of the invention, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention comprises a third target antigen binding domain, and in other embodiments also comprises a fourth target antigen binding domain. In some embodiments the third target antigen binding domain comprises a third immunoglobulin light chain and a third immunoglobulin heavy chain, and the optional fourth target antigen binding domain comprises a fourth immunoglobulin light chain and a fourth immunoglobulin heavy chain.
As mentioned in relation to the first and second target antigen binding domain, the third and fourth target antigen binding domains may comprise an immunoglobulin heavy chain that comprises one or more than one constant region. For example in some embodiments:
A) the third immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and the third immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region; or
B) the third immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and the third immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three constant regions; and for example when present:
C) the fourth immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3CDR regions, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and the fourth immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region; or
D) the fourth immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and the fourth immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three constant regions.
In some embodiments the N and/or C-terminus of the third and/or fourth immunoglobulin light chain is connected to a third and/or a fourth tag binding domain, for example to a third and/or fourth coiled coil oligomerisation domain, for example connected via a linker.
In some embodiments the N and/or C-terminus of the third and/or fourth immunoglobulin heavy chain is connected to a third and/or a fourth tag binding domain, for example to a third and/or fourth coiled coil oligomerisation domain, for example connected via a linker.
In some embodiments the C-terminus of the third and/or fourth immunoglobulin light chain is not connected to a third and/or fourth tag binding domain, optionally not connected to a third and/or fourth coiled coil oligomerisation domain.
In one embodiment therefore the polypeptide of the invention, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention may comprise at least 1, 2, 3, 4, 5, 6 or more target antigen binding domains, for example invention may comprise at least 1, 2, 3, 4, 5, 6 immune cell binding domains.
It will be appreciated that in preferred embodiments, the polypeptide of the invention only comprises one Fc region. The skilled person will appreciate that an Fc region is a portion of the polypeptide where constant regions of the heavy chain from two separate heavy chains interact with one another, typically by forming disulphide bonds between the two heavy chains polypeptides.
In some embodiments of the present invention, where the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises more than one target antigen binding domain, for example more than one immune cell binding domain, each of the target antigen binding domains may bind to the same target antigen or feature or molecule on the immune cell. For example, where the polypeptide comprises two immune cell binding domains, each binding domain may bind to the same feature of molecule on the immune cell. This does not necessarily mean however that each of the immune cell binding domains binds to the same epitope. It is possible for a single target, for example an immune cell surface polypeptide, to comprise a number of epitopes. Polypeptides that comprise at least two immune cell binding domains, for example, wherein each binding domain binds to a different epitope of the same target molecule, are therefore encompassed within the invention.
Accordingly, in some embodiments the first and second target antigens are the same. In some instances of this embodiment the polypeptide does not comprise a third and fourth target antigen binding domain, and in some instances of this embodiment the polypeptide does comprise a third and fourth target antigen binding domain.
In some embodiments the first and second target antigens are different. In some instances of this embodiment the polypeptide does not comprise a third and fourth target antigen binding domain, and in some instances of this embodiment the polypeptide does comprise a third and fourth target antigen binding domain.
In some embodiments of the polypeptide of the invention, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention the first and second target antigens are the same and the third target antigen is a different antigen to the first and second target antigen. In some of these embodiments the polypeptide does not comprise a fourth target antigen binding domain and in some embodiments the polypeptide does comprise a fourth target antigen binding domain.
In some embodiments of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention the first and third target antigens are the same and the second antigen is different to the first and third target antigen. In some of these embodiments the polypeptide does not comprise a fourth target antigen binding domain and in some embodiments the polypeptide does comprise a fourth target antigen binding domain.
In some embodiments the first target antigen and the second target antigen and the third target antigen are all different to one another. In some of these embodiments the polypeptide does not comprise a fourth target antigen binding domain and in some embodiments the polypeptide does comprise a fourth target antigen binding domain.
In some embodiments the first and second target antigens are the same as each other and wherein the third and fourth antigens are the same as each other, but wherein the first and second target antigens are different to the third and fourth target antigens; or the first and third target antigens are the same as each other and wherein the second and fourth target antigens are the same as each other, but wherein the first and third target antigens are different to the second and fourth target antigen.
In some embodiments the first target antigen and the second target antigen and the third target antigen and the fourth target antigen are all different to one another.
In some embodiments the first target antigen and the second target antigen and the third target antigen and the fourth target antigen are all the same.
Preferred embodiments of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, have the following features:
A) the first and third target antigens are the same and the second antigen is different to the first and third target antigen. In some of these embodiments the polypeptide does not comprise a fourth target antigen binding domain and in some embodiments the polypeptide does comprise a fourth target antigen binding domain . In preferred embodiments the polypeptide comprises target binding domains that bind to a first, a second, a third and a fourth antigen, and wherein the first and second antigens are the same, and the third and fourth antigens are the same, but where the first and second antigens are different to the third and fourth antigens; B) the first and second target antigens are the same as each other. In some instances of this embodiment the polypeptide does not comprise a third and fourth target antigen binding domain, and in some instances of this embodiment the polypeptide does comprise a third and fourth target antigen binding domain.
It is possible to produce polypeptides that comprise different target binding domains using methods to obtain heterodimerization of different heavy chains, for example using fab-arm-exchange, knob-in-hole, ionic electrostatic pairing, LUZ-Y or SEEDbody technologies. Fab-arm-exchange uses two parental polypeptides or antibodies that differ in sequence in the Fc region. The sequences differ so that it is energetically more favorable to form heterodimers between the two different parental Fc regions than it is to form homodimers within each Fc region of each parental antibody. In this the exchange of the fab arms is promoted and the resultant antibody will comprise an Fc region that comprises one heavy chain region having a first sequence and one heavy chain region having a second sequence.
In some embodiments then the polypeptide of the invention, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention has been produced via fab-arm-exchange and so in some embodiments the polypeptide comprises at least a first target binding domain and a second target binding domain, and wherein the first target binding domain comprises a first immunoglobulin heavy chain that comprises three constant regions and wherein the second target binding domain comprises a second immunoglobulin heavy chain that comprises three constant regions, and wherein the first immunoglobin heavy chain sequence and the second immunoglobin heavy chain sequence are different to one another, and wherein the different amino acid sequences promote the formation of a heterodimer between said first and second immunoglobulin heavy chain polypeptides rather than the formation of a homodimer of the first immunoglobulin heavy chain polypeptide and a homodimer of the second immunoglobulin heavy chain polypeptide.
It will be appreciated that where the polypeptide of the invention, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention has been produced via fab-arm-exchange, it may also comprise a third and possibly a fourth target antigen binding domain, as described elsewhere herein, for example may comprise: a) a third target antigen binding domain, wherein the third target antigen binding domain comprises a third immunoglobulin light chain that comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; optionally comprises ii) one constant region; and a third immunoglobulin heavy chain that comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region; optionally wherein the N-terminus of the third immunoglobulin light chain polypeptide is attached to the C-terminus of the first or second immunoglobulin heavy chain polypeptide; and optionally wherein the polypeptide further comprises: b) a fourth target antigen binding domain, wherein the fourth target antigen binding domain comprises a fourth immunoglobulin light chain that comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and a fourth immunoglobulin heavy chain that comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region; optionally wherein the N-terminus of the fourth immunoglobulin light chain polypeptide is attached to the C-terminus of the first or second immunoglobulin heavy chain polypeptide.
As described elsewhere herein, any one or more of the tag binding domains present in the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention, is preferably a coiled-coil oligomerization domain, that is capable of forming a heterodimer with a corresponding coiled-coil oligomerization domain. Preferences for the position of the coiled-coil oligomerisation domain are described elsewhere herein.
It will also be appreciated that the polypeptide of the invention, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention can comprise any number of tag binding domains, for example 1, 2, 3, 4, 5, 6, 7 or 8, and can comprise any combination of types of tag binding domains.
The N or C terminus of any light chain and any heavy chain may be attached or connected to a coiled-coil oligomerisation domain. Likewise, provided the polypeptide of the invention comprises at least one tag binding domain that is a coiled-coil oligomerisation domain at any one of the N or C terminus of any of the light or heavy chains, the remaining N or C termini of any of the light or heavy chains may not be attached to or connected to a coiled-coil oligomerisation domain.
In some embodiments then : a) the C-terminus or N-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, but, where present, the second, third and fourth immunoglobulin light chain is not connected to a coiled-coil oligomerisation domain; b) the C-terminus or N-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, and where present the C- terminus of the second immunoglobulin light chain is connected to a second coiled-coil oligomerisation domain, but, where present, the third and fourth immunoglobulin light chain is not connected to a coiled-coil oligomerisation domain; c) the C-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, and where present the C-terminus of the second immunoglobulin light chain is connected to a second coiled-coil oligomerisation domain, and the C-terminus of the third immunoglobulin light chain is connected to a third coiled-coil oligomerisation domain but, where present, the fourth immunoglobulin light chain is not connected to a coiled-coil oligomerisation domain; d) the C-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, and where present the C-terminus of the second immunoglobulin light chain is connected to a second coiled-coil oligomerisation domain, and the C-terminus of the third immunoglobulin light chain is connected to a third coiled-coil oligomerisation domain, and the C-terminus of the fourth immunoglobulin light chain is connected to a fourth coiled-coil oligomerisation domain.
It will be dear to the skilled person that since the tag binding domain that is a coiled- coil oligomerisation domain can be attached to either a light or heavy chain, and can be attached to the light or heavy chain at the C and/or N terminus, a range of different iterations is possible and are disclosed herein. For example in some embodiments: the N-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, and where present the N-terminus of the second immunoglobulin light chain is connected to a second coiled-coil oligomerisation domain, and the C-terminus of the third immunoglobulin light chain is connected to a third coiled-coil oligomerisation domain, and the C-terminus of the fourth immunoglobulin light chain is connected to a fourth coiled-coil oligomerisation domain; or the C-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, and where present the C-terminus of the second immunoglobulin light chain is connected to a second coiled-coil oligomerisation domain, and the N-terminus of the third immunoglobulin light chain is connected to a third coiled-coil oligomerisation domain, and the N-terminus of the fourth immunoglobulin light chain is connected to a fourth coiled-coil oligomerisation domain; or the C-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain and the N -terminus of the first immunoglobin light chain is connected to a second coiled-coil oligomerisation domain, and where present the C-terminus of the second immunoglobulin light chain is not connected to a second coiled-coil oligomerisation domain, and the C-terminus of the third immunoglobulin light chain is connected to a third coiled-coil oligomerisation domain, and the C- terminus of the fourth immunoglobulin light chain is not connected to a fourth coiled- coil oligomerisation domain, and the N-terminus of the fourth immunoglobulin light chain is not connected to a fourth coiled-coil oligomerisation domain.
In this way it is possible to produce a cargo-targeting polypeptide of the invention that has between 1 and 12 tag binding domains that are oligomerisation domains. Accordingly, in some embodiments the invention provides a cargo -targeting polypeptide of the invention that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 coiled-coil oligomerisation domains.
The skilled person will appreciate that coiled-coil oligomerisation domains are in some instances able to form heterodimers, and are in some instances able to form multimers, that comprise more than 2 monomers of the coiled-coil domains.
Accordingly, in some embodiments the first coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding first cargo coiled- coil oligomerisation domain, and optionally: when present the second coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding second cargo coiled-coil oligomerisation domain; when present the third coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding third cargo coiled-coil oligomerisation domain; and when present the fourth coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding fourth cargo coiled-coil oligomerisation domain; when present the fifth coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding fifth cargo coiled-coil oligomerisation domain; when present the sixth coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding sixth cargo coiled-coil oligomerisation domain; and when present the seventh coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding seventh cargo coiled-coil oligomerisation domain when present the eighth coiled-coil oligomerisation domain is able to form a muitimer, optionally a heterodimer with a corresponding eighth cargo coiled-coil oligomerisation domain; when present the ninth coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding ninth cargo coiled-coil oligomerisation domain; and when present the tenth coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding tenth cargo coiled-coil oligomerisation domain; when present the eleventh coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding eleventh cargo coiled-coil oligomerisation domain; and when present the twelfth coiled-coil oligomerisation domain is able to form a multimer, optionally a heterodimer with a corresponding twelfth cargo coiled-coil oligomerisation domain. The skilled person will be able to identify suitable coiled-coil oligomerisation domains to use in the present invention. A key feature of coiled-coil oligomerisation domains is that they comprise a peptide that itself comprises repeated units of an amino acid sequence that comprises periodically interspersed hydrophobic residues (it will also be clear that the sequence of the repeated units does not necessarily have to be identical between repeats in a given coiled-coil oligomerisation domain). This results in a tendency for the peptide to form either parallel or anti-parallel, multimeric, heterodimeric or dimeric helix-helix interactions.
In some embodiments then the coiled-coil oligomerisation domains referred to herein comprises a peptide that comprises repeated unites of an amino acid sequence that comprises a hydrophobic residue at every third or fourth amino acid - for example wherein the hydrophobic residues generate surface(s) on the helix that are hydrophobic and tend to form either parallel or anti-parallel, multimeric, heterodimeric or dimeric helix-helix interactions.
For example, in some embodiments the coiled-coil oligomerisation domain comprises:
A) repeating units of a heptad sequence which follows the consensus of any one or more of: i) abcdefg wherein a and d are hydrophobic amino acids, optionally wherein e and g are polar or charged amino acids; ii) Hpphppp where h is a hydrophobic residue and p is a polar and/or charged residue; iii) Hpphpph where h is a hydrophobic residue and p is a polar and/or charged residue; iv) Hpphhpp where h is a hydrophobic residue and p is a polar and/or charged residue; v) Hpphhph where h is a hydrophobic residue and p is a polar and/or charged residue; vi) Hphhphp where h is a hydrophobic residue and p is a polar and/or charged residue; and/or vii) Hhphphp where h is a hydrophobic residue and p is a polar and/or charged residue;
B) a leucine zipper; and/or C) repeating units of a non-heptad sequence, optionally with a periodicity of 11/3, 15/4, 18/5 or 25/7, optionally wherein the repeat sequence comprises any one or more of the following consensus sequences: viii) abbcdefg ix) abcabcdefg x) abcdabcdefg xi) abcPPPefg xii) abcdefghijk xiii) abcdefghijklmo. wherein a and d are hydrophobic amino acids, optionally wherein e and g are polar or charged amino acids;
In some embodiments, the coiled-coil oligomerisation domain comprises:
At least 2, 3, 4, 5, 6, 7, 8 or more of the repeat units;
Less than 8, 7, 6, 5, 4 or less than 3 of the repeat units; and/or
Between 2 and 8 of the repeat units, optionally between 3 and 7, or 4 and 6 of the repeat units.
In some preferred embodiments, the coiled-coil oligomerisation domain has 3 or 4 repeat units, optionally no more than 4 repeat units.
The skilled person will appreciate that provided a and d are hydrophobic residues, the peptide will likely form a coiled-coil, and the other residues of the heptad, b, c, e, f and g can be generally be any residue. Hydrophobic resides are considered to comprise Leu, He, Vai, Phe, Pro, Met and Ala. In some instances e and g are polar residues or charged residues, for example Gin, Asn, His, Ser, Thr, Tyr, Cys, Arg, Kys, Asp or Glu. In some instances b is leucine or isoleucine; e is Glu or Lys or Arg; and/or g is Glu or Lys or Arg.
In one embodiment the coiled-coil oligomerisation domain is not E3 [SEQ ID NO: 71].
In one embodiment the coiled-coil oligomerisation domain is not K3 [SEQ ID NO: 75] .
In one embodiment the coiled-coil oligomerisation domain is not K4 [SEQ ID NO: 76.]
In some embodiments, the sequence of each repeat sequence within a coiled-coil oligomerisation domain is identical.
In some embodiments, at least two repeat sequences within the coiled-coil oligomerisation domain are different. Specific examples of coiled-coil oligomerization domains that may be used in the invention as the first coiled-coil oligomerization domain are as follows:
Leucine zipper 1 [SEQ ID NO: 119] EYQALEKEVAQLEAENQALEKEVAQLEHE
Leucine zipper 2 [SEQ ID NO: 120] EYQALKKKVAQLKAKNQALKKKVAQLKHK rGCN4-pI [SEQ ID NO: 121] REGVLKKLRAVENELHYNKSLLEEVKDLQKMRQL rGCN4-pI' [SEQ ID NO: 122] CGGREGVLKKLRAVENELHYNKSLLEEVKDLQKMRQL
GCN4-pI [SEQ ID NO: 123] RMKQLEDKVEELLSKNYHLENEVARLKKLVGER
GNC4-PH [SEQ ID NO: 124] RMKQIEDKLEEILSKLYHIENELARIKKIVGER
JR2KC [SEQ ID NO: 125]
AADLKKAIKALKKHLKAKGPCDAAQLKKQLKQAFKAFKRAG
JR2K [SEQ ID NO: 126]
NAADLKKAIKALKKHLKAKGPVDAAQLKKQLKQAFKAFKRAG
JR2E [SEQ ID NO: 127]
NAADLEKAIEALEKHLEAKGPCDAAQLEKQLEQAFEAFERAG the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the E5 coiled-coil oligomerisation domain [SEQ ID NO: 73]; the E6 coiled-coil oligomerisation domain [SEQ ID NO: 74]; the K3 coiled-coil oligomerisation domain [SEQ ID NO: 75]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; the leucine zipper domain of [SEQ ID NO: 79] the HAP1 coiled-coil oligomerisation domain [SEQ ID NO: 80]; the HAP2 coiled-coil oligomerisation domain [SEQ ID NO: 81]; the HAP3 coiled-coil oligomerisation domain [SEQ ID NO: 82]; the HAP4 coiled-coil oligomerisation domain [SEQ ID NO: 83]; the HAP5 coiled-coil oligomerisation domain [SEQ ID NO: 84]; the HAP6 coiled-coil oligomerisation domain [SEQ ID NO: 85]; the HAP7 coiled-coil oligomerisation domain [SEQ ID NO: 86]; or the HAP8 coiled-coil oligomerisation domain [SEQ ID NO: 87].
Preferred examples of coiled-coil oligomerisation domains are: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the E5 coiled-coil oligomerisation domain [SEQ ID NO: 73]; the E6 coiled-coil oligomerisation domain [SEQ ID NO: 74]; the K3 coiled-coil oligomerisation domain [SEQ ID NO: 75]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; the leucine zipper domain of [SEQ ID NO: 79] the HAP1 coiled-coil oligomerisation domain [SEQ ID NO: 80]; the HAP2 coiled-coil oligomerisation domain [SEQ ID NO: 81]; the HAP3 coiled-coil oligomerisation domain [SEQ ID NO: 82]; the HAP4 coiled-coil oligomerisation domain [SEQ ID NO: 83]; the HAP5 coiled-coil oligomerisation domain [SEQ ID NO: 84]; the HAP6 coiled-coil oligomerisation domain [SEQ ID NO: 85]; the HAP7 coiled-coil oligomerisation domain [SEQ ID NO: 86]; or the HAP8 coiled-coil oligomerisation domain [SEQ ID NO: 87]. More preferred specific examples of coiled-coil oligomerisation domains are: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; optionally selected from any one of: the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; or optionally selected from any one of: the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71].
It will be appreciated that minor divergence from the above sequences will still be expected to result in a functional coiled-coil oligomerization domain, and so in some embodiments any one or more of the coiled-coil oligomerisation domains that are part of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences, or optionally a coiled-coil oligomerisation domain with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences and that is able to form a heterodimer with a corresponding coiled-coil oligomerisation domain.
It will also be appreciated that the second, third and/or fourth coiled-coil oligomerization domain where present may comprise or consist of the above coiled- coiled oligomerization domains, for example may comprise of consist of any of:
Leucine zipper 1 [SEQ ID NO: 119] EYQALEKEVAQLEAENQALEKEVAQLEHE Leucine zipper 2 [SEQ ID NO: 120] EYQALKKKVAQLKAKNQALKKKVAQLKHK rGCN4-pI [SEQ ID NO: 121] REGVLKKLRAVENELHYNKSLLEEVKDLQKMRQL rGCN4-pI' [SEQ ID NO: 122] CGGREGVLKKLRAVENELHYNKSLLEEVKDLQKMRQL
GCN4-pI [SEQ ID NO: 123] RMKQLEDKVEELLSKNYHLENEVARLKKLVGER
GNC4-PH [SEQ ID NO: 124] RMKQIEDKLEEILSKLYHIENELARIKKIVGER
JR2KC [SEQ ID NO: 125]
AADLKKAIKALKKHLKAKGPCDAAQLKKQLKQAFKAFKRAG
JR2K [SEQ ID NO: 126]
NAADLKKAIKALKKHLKAKGPVDAAQLKKQLKQAFKAFKRAG
JR2E [SEQ ID NO: 127]
NAADLEKAIEALEKHLEAKGPCDAAQLEKQLEQAFEAFERAG the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the E5 coiled-coil oligomerisation domain [SEQ ID NO: 73]; the E6 coiled-coil oligomerisation domain [SEQ ID NO: 74]; the K3 coiled-coil oligomerisation domain [SEQ ID NO: 75]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; the leucine zipper domain of [SEQ ID NO: 79] the HAP1 coiled-coil oligomerisation domain [SEQ ID NO: 80]; the HAP2 coiled-coil oligomerisation domain [SEQ ID NO: 81]; the HAP3 coiled-coil oligomerisation domain [SEQ ID NO: 82]; the HAP4 coiled-coil oligomerisation domain [SEQ ID NO: 83]; the HAP5 coiled-coil oligomerisation domain [SEQ ID NO: 84]; the HAP6 coiled-coil oligomerisation domain [SEQ ID NO: 85]; the HAP7 coiled-coil oligomerisation domain [SEQ ID NO: 86]; or the HAP8 coiled-coil oligomerisation domain [SEQ ID NO: 87], or may comprise or consist of any of: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the E5 coiled-coil oligomerisation domain [SEQ ID NO: 73]; the E6 coiled-coil oligomerisation domain [SEQ ID NO: 74]; the K3 coiled-coil oligomerisation domain [SEQ ID NO: 75]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; the leucine zipper domain of [SEQ ID NO: 79] the HAP1 coiled-coil oligomerisation domain [SEQ ID NO: 80]; the HAP2 coiled-coil oligomerisation domain [SEQ ID NO: 81]; the HAP3 coiled-coil oligomerisation domain [SEQ ID NO: 82]; the HAP4 coiled-coil oligomerisation domain [SEQ ID NO: 83]; the HAP5 coiled-coil oligomerisation domain [SEQ ID NO: 84]; the HAP6 coiled-coil oligomerisation domain [SEQ ID NO: 85]; the HAP7 coiled-coil oligomerisation domain [SEQ ID NO: 86]; or the HAP8 coiled-coil oligomerisation domain [SEQ ID NO: 87], or may comprise of consist of any of: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; optionally selected from any one of: the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72] ; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; or optionally selected from any one of: the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; or comprises a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences, or optionally a coiled-coil oligomerisation domain with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences and that is able to form a heterodimer with a corresponding coiled-coil oligomerisation domain.
It will appreciated that the sequence of the coiled-coil oligomerization domains can vary depending on what cargo is to be delivered. For example, the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise more than one coiled-coil oligomerization domain but where each coiled-coil domain has the same sequence, the intention being that multiple copies of the same cargo become associated with the polypeptide, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention. In other instances, the polypeptide, for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention may comprise two or more coiled-coil oligomerization domains that have different sequences and so can form a multimer, for example a dimer for example a heterodimer with different coiled-coil domains that are attached or connected to the cargo, in which case a single polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention can be used to deliver two or more different cargo.
Accordingly, in some embodiments the first and second coiled-coil oligomerisation domain are the same, for example wherein when present the first and second and third; or the first and second and third and fourth coiled-coil oligomerisation domains are the same and form a multimer, for example a dimer for example a heterodimer with the same partner coiled-coil oligomerisation domain.
In some embodiments, the polypeptide of the invention, for example the cargotargeting polypeptide or cargo-delivery polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 different coiled-coil oligomerisation domains that each forms a multimer, optionally a dimer, optionally a heterodimer with a different partner coiled- coil oligomerisation domain, for example in some embodiments each coiled-coil oligomerisation domain present is different to one another.
In some embodiments the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises at least two different coiled- coil oligomerisation domains that each forms a heterodimer with a different partner coiled-coil oligomerisation domain.
As described above, in some instances the first target antigen is an antigen that is present on an immune ceil. In some embodiments the immune cell is an antigen presenting cell, optionally a dendritic cell (DC), B cell and/or macrophage (preferably DC).
In some embodiments, the polypeptide of the invention may be bi-specific with respect to the target antigen binding domain, for example the immune cell target binding domain, i.e. the polypeptide may comprise at least two different immune cell target binding domains, each capable of binding to a different immune cell target - the different immune cell targets may be present on the same immune cell, or may be targets associated with different immune cells. Similarly, the polypeptide can comprise more than 2, for example 3 or 4 or more different immune cell targeting domains, each binding to a different immune cell target - which may be on the same or different immune cells.
As described elsewhere herein the tag binding domain is preferably a coiled-coil oligomerisation domain. The tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is the domain that specifically binds to a tag. The tag itself is in preferred embodiments a corresponding coiled-coil oligomerisation domain which is connected to a cargo. The cargo is in some instances a protein or peptide. Where the cargo is a protein or peptide it can be any protein or peptide. In some embodiments where the cargo is a protein peptide the protein or peptide comprises for example an antigen or neoantigen, which it is desirable to target to the immune cell. Where the cargo is a protein or peptide, preferably the cargo protein or cargo peptide and the peptide tag domain, for example the coiled-coil oligomerisation domain, are expressed as a single transcript and translated accordingly to produce a single protein or peptide that comprises both the cargo peptide or cargo protein and the peptide tag domain. In some instances, a linker is present between cargo protein or cargo peptide and the tag domain, for example between the cargo protein or cargo peptide and the coiled coil oligomerisation domain. The linker may be any linker, and may for example be a GS linker as described herein and as will be apparent to the skilled person.
As will be apparent throughout, preferably the peptide tag domain that is connected to the cargo is a coiled-coil oligomerisation domain, preferably that is capable of forming a multimer for example a dimer for example a heterodimer with the corresponding coiled-coil oligomerisation domain present in the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention.
In other embodiments where the cargo is a protein or peptide, the tag, for example a peptide tag may be conjugated to the cargo protein or peptide following expression of the cargo peptide or cargo protein. For example, the peptide tag may be conjugated to the cargo peptide or cargo protein (e.g. a cargo peptide or cargo protein that comprises an antigen or neoantigen) through e.g. a GSSSS linker or metalloprotease cleavable linker or a cieavable valine-citruiline linker.
In ail embodiments, the tag is not a peptide tag that is derived from tetanus toxin. For example, a tag derived from the tetanus toxin (TTx), such as MTTE [SEQ ID NO: 68], is not encompassed within the meaning of "tag" or "peptide tag" as used herein.
The concept of "tags" is well known in the molecular biology field, where it is routine to express a peptide or polypeptide sequence of interest wherein the sequence has been extended to include a relatively short additional sequence, encoding the tag. Applications using tagged peptides typically employ an antibody or antibody derived fragment, capable of specifically binding to the tag. Similarly, in some embodiments of the invention, the tag binding domain is an antibody or antibody derived fragment, and binds specifically to the tag.
It will be clear to the skilled person that any peptide sequence capable of being specifically recognized by an antibody, i.e. capable of inducing the production of antibodies, can be considered a peptide tag. Examples of suitable peptide tags include the FLAG-tag comprising the amino acid sequence DYKDDDDK [SEQ ID NO: 69], and the skilled person will be aware of other suitable peptide tags such as V5-tag, Myc- tag, HA-tag, Spot-tag, T7-tag and NE-tag. See for example Mishra 2020 Current Protein and Peptide Science 21 : 821 - 830.
In one embodiment, the peptide tag is a 33 amino acid sequence as follows:
Z33: FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD [SEQ ID NO: 51] or a sequence with at least 80%, 85%, 90%, 95%, 98% or 100% sequence identity to SEQ ID NO: 51 and wherein the tag retains the ability to bind to the Fc region.
In some other embodiments, the peptide tag is not the 33 amino acid sequence of [SEQ ID NO: 51], or is not a sequence with at least 80%, 85%, 90%, 95%, 98% or 100% sequence identity to SEQ ID NO: 51 and wherein the tag retains the ability to bind to the Fc region.
In some embodiments the tag binding domain of the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is not an Fc region; or has an additional tag binding domain other than the Fc region, for example comprises an Fc region and a tag binding domain that is a coiled-coil oligomerisation domain.
The cargo, for example a cargo protein or cargo peptide, that in some instances comprises an antigen or neoantigen, may be tagged with a non-peptide tag, for example any moiety that acts as a binding partner for a second specific binding domain. Thus, a non-peptide tag can be any chemical entity with which a second or further entity has affinity. The tag can be selected from, for example, any organic molecule, a small molecule, or a hapten. Tags can for example take the form of nucleic acids, for example aptamers. Tags as described herein are typically short peptide sequences (i.e. sequences of amino acids). In preferred embodiments the tags described herein are peptide or protein tags, for example short sequences of amino acids. The tag can be of any sequence provided it is able to be bound, preferably specifically bound by the tag binding domain of the polypeptide of the invention. Accordingly, in preferred embodiments the tag is a peptide tag, and the corresponding binding domain is a peptide tag binding domain. Most preferred is that the cargo, for example a cargo protein or cargo peptide comprises a peptide tag that is a coiled-coil oligomerisation domain, such as those specific examples described elsewhere herein.
It will be apparent from the disclosure herein that a single polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise a number of different tag binding domains. For example in some embodiments the cargo targeting polypeptide of the invention comprises a first tag binding domain that is a coiled-coil oligomerisation domain, for example connected to the C-terminus of an immunoglobulin light chain; and may also comprise a tag binding domain that can bind to the FLAG-tag. Other combinations of tag binding domains are also contemplated by the invention.
For example in one embodiment the cargo targeting polypeptide of the invention comprises a first tag binding domain that is a coiled-coil oligomerisation domain, and also comprises at least a second tag binding domain that is not a coiled-coil oligomerisation domain.
In some embodiments described throughout, the tag binding domain is not the Fc region. In some embodiments throughout the polypeptide, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention comprises an Fc region addition to a tag binding domain as described herein, for example comprises an Fc domain and a coiled-coil oligomerisation domain.
In some embodiments the cargo is a cargo protein or cargo peptide that comprises an antigen or neoantigen that can be any antigen or neoantigen. In further preferred embodiments, the tag and the antigen are both peptides, allowing the expression of a tagged antigen as a single peptide, for example expression of a cargo protein or peptide that comprises an antigen and wherein the cargo protein or peptide is tagged with a tag domain that is a coiled-coil oligomerisation domain. In some embodiments a linker may be present between the sequence encoding the antigen to be delivered (for example to be delivered to the immune cell) and the sequence encoding the tag (for example encoding the coiled-coil oligomerisation domain). Preferences for linkers are as described elsewhere herein.
For peptide tags, the term "peptide" is not construed as limited to a certain size or length of amino acids. For example, a peptide tag could be considered a protein, polypeptide, or multiple subunits thereof and simply indicates that the tag comprises a series of amino acids. A peptide tag also includes within the meaning a coiled-coil oligomerization domain, such as those described herein.
In some embodiments the tag binding domain of the polypeptide of the invention binds to a tag that is less than 100 amino acids in length, for example less than 90, 80, 70, 60, 50, 40, 30, 20 or less than 10 amino acids in length, for example binds to a tag that is a coiled-coil oligomerisation domain that is less than 100 amino acids in length, for example less than 90, 80, 70, 60, 50, 40, 30, 20 or less than 10 amino acids in length.
The tag that is used to tag the cargo, and the tag binding domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, for example a coiled-coil oligomerization domain, can be considered to be binding partners and can essentially comprise any pair of entities, for example a pair of peptides, that are capable of specifically interacting with one another.
As described above, the use of coiled-coil oligomerization domains as both the tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, and the tag that is used to tag the cargo, for example to tag a cargo protein or cargo peptide is a preferred embodiment.
For example, the pair of peptides K3 ((KIAALKE)x3) [SEQ ID NO: 52] (KIAALKEKIAALKEKIAALKE) or K4 ((KIAALKE)x4) [SEQ ID NO: 53] (KIAALKEKIAALKEKIAALKEKIAALKE) are considered to bind to the coiled-coil peptide tag E3 ((EIAALEK)x3) [SEQ ID NO: 54] (EIAALEKEIAALEKEIAALEK).
Accordingly, in some embodiments, the peptide tag that is used to tag the cargo, for example to tag the cargo protein or cargo peptide comprises E3 ((EIAALEK)x3), and the tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises K3 ((KIAALKE)x3) or K4 ((KIAALKE)x4). It will be clear that in this instance, and since the E3 ((EIAALEK)x3), K3 ((KIAALKE)x3) and K4 ((KIAALKE)x4) sequences are relatively small, the location of K3 ((KIAALKE)x3) or K4 ((KIAALKE)x4) and E3 ((EIAALEK)x3) is interchangeable. For instance, in one embodiment, the peptide tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises K3 ((KIAALKE)x3) or K4 ((KIAALKE)x4), and consequently has the ability to bind to a cargo, for example an antigen peptide or neoantigen peptide, that has been tagged with the E3 ((EIAALEK)x3) peptide; in other embodiments the tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises E3 ((EIAALEK)x3), and consequently has the ability to bind to a cargo protein or cargo peptide, for example an antigen peptide or neoantigen peptide, that has been tagged with the peptide K3 ((KIAALKE)x3) or K4 ((KIAALKE)x4).
Other particular corresponding pairs of coiled-coil oligomerisation domain that may be used with one another to form a multimer, for example a dimer for example a heterodimer are: where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E5 [SEQ ID NO: 73] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E6 [SEQ ID NO: 74] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K3 [SEQ ID NO: 75] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73]or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K4 [SEQ ID NO: 76] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73]or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K5 [SEQ ID NO: 77] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73]or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K6 [SEQ ID NO: 78] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73]or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is HAP1 [SEQ ID NO: 80] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is HAP2 [SEQ ID NO: 81] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP1 [SEQ ID NO: 80], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is HAP3 [SEQ ID NO: 82] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP1 [SEQ ID NO: 80], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is HAP4 [SEQ ID NO: 83] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP1 [SEQ ID NO: 80], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is HAP5 [SEQ ID NO: 84] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP1 [SEQ ID NO: 80], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is HAP6 [SEQ ID NO: 85] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP1 [SEQ ID NO: 80], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is HAP7 [SEQ ID NO: 86] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP1 [SEQ ID NO: 80] or HAP8 [SEQ ID NO: 87]; or where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is HAP8 [SEQ ID NO: 87] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP1 [SEQ ID NO: 81]; or wherein any of the coiled-coil oligomerisation domains has a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences, or wherein any of the coiled-coil oligomerisation domains has a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences and that is able to form a heterodimer with a corresponding coiled- coil oligomerisation domain. More preferred corresponding pairs of coiled-coil oligomerisation domains are: where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77] or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77] or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K4 [SEQ ID NO: 76] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K5 [SEQ ID NO: 77] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], and where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K6 [SEQ ID NO: 78] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72],
Even more preferred corresponding pairs of coiled-coil oligomerisation domains are: where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K4 [SEQ ID NO: 76] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E4 [SEQ ID NO: 72]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76] or K6 [SEQ. ID NO: 78]; and where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76] or K6 [SEQ ID NO: 78].
Allowing the polypeptide of the invention for example the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention to contact the tagged cargo in vitro or ex vivo, i.e prior to administration to a subject, and so form a complex outside of the body, has various advantages over administering both the polypeptide of the invention for example the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention and the tagged cargo separately. For example the formation of such complexes ex vivo means there is less of a requirement for the tag binding domain and the tag of the tagged cargo interaction to be specific to reduce risk of off-target effects provided they bind with strong enough affinity that upon in vivo administration the tag and tag binding domains does not dissociate, thereby leaving both components open to binding with alternative targets. Thus, suitable tag/tag binding domain partners will have specificity and/or bind with sufficient affinity to maintain complexes in vivo.
Again, such an approach allows for the production of an off-the-shelf product that can be mixed with one or more tagged cargo that are antigens, for example one or more tagged personalized neoantigen peptides and administered to patients to induce a personal neoantigen-specific T cell response.
In other embodiments administration of the two components that ultimately form the complex of the invention separately but simultaneously, or sequentially, has advantages. For example in some embodiments the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention and the tagged cargo are administered separately. In some embodiments the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention and the tagged cargo are administered separately and at the same time. In some embodiments the cargo-targeting polypeptide or cargodelivery polypeptide of the invention and the tagged cargo are administered separately and at the same time and at the same site or location in the body.
In other embodiments the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention and the tagged cargo are administered separately and at different time periods. For example in one embodiment the cargo-targeting polypeptide or cargo- delivery polypeptide of the invention is administered prior to the tagged cargo. In other embodiments the cargo-targeting polypeptide or cargo-delivery polypeptide of the invention is administered after the tagged cargo.
Preferably the tag, for example the peptide tag used to tag the cargo for example the cargo protein or cargo peptide and towards which the tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is directed is a non-human peptide, to avoid unwanted immune responses.
In some embodiments, the peptide tag and/or the cargo for example the cargo protein or cargo peptide comprises a biotin tag.
Where the cargo for example the cargo peptide or cargo protein comprises a biotin tag, it also comprises a peptide tag as described herein, for example comprises a coiled- coil oligomerisation domain.
Such a biotin label can be used to attach the tagged cargo to a further entity via the interaction between biotin and streptavidin. For example in some embodiments the tagged cargo is attached to a streptavidin bead via a biotin tag. In some embodiments the complex of the invention as described herein, i.e. a complex comprising a polypeptide of the invention, for example the cargo-targeting polypeptide or the cargodelivery polypeptide of the invention, as described herein, and a tagged cargo, for example a tagged cargo protein or peptide (for example a peptide tagged antigen), wherein the tagged cargo (for example the peptide tagged antigen) comprises the tag to which the tag binding domain of the polypeptide binds, also comprises a bead via an interaction between a biotin tag located on the tagged cargo and a streptavidin tag located on the bead. Examples of such complexes are given in the Examples.
It will be appreciated that the tagged cargo may comprise the tag at either end of the cargo. For example where the cargo is a protein or peptide, the tag, for example the coiled-coil oligomerisation domain may be located at the N-terminus or the C-terminus. In some embodiments the tag is located internally within the cargo protein or peptide. In some embodiments the cargo protein or peptide comprises a tag at the N-terminus and the C- terminus - in some embodiments the tag at the N-terminus is the same as the tag at the C-terminus, but in other embodiments the tag at the N-terminus is different to the tag at the C-terminus. In some embodiments the cargo protein or peptide comprises only one tag and the tag is located at the N-terminus. In some embodiments the cargo protein or peptide comprises only one tag and the tag is located at the C-terminus.
For example, in some embodiments the tagged cargo protein or peptide comprises an N-terminal K4 coiled-coil domain followed by an OVA peptide as described in SEQ ID NO: 128.
In some embodiments the tagged cargo protein or peptide comprises an OVA peptide followed by a C-terminal K4 coiled-coil domain as described in SEQ ID NO: 129 and 130.
As described elsewhere herein, in some instances it is beneficial if, where the polypeptide, for example a c rgo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises more than one tag binding domain, the tag binding domains, for example the peptide tag binding domains, all have specificity for the same tag (for example specificity for the same peptide tag - for example all of the tag binding domains comprise the same coiled-coil oligomerisation domain which can form a heterodimer with a further coiled-coil oligomerisation domain). In this way for example it is possible to "load" the polypeptide of the invention for the example the cargotargeting polypeptide or cargo-delivery polypeptide of the invention with multiple copies of the tagged cargo (for example to load with multiple copies of a tagged antigen) to be targeted and delivered to the specific target site or cell or tissue. Alternatively, it may be beneficial for several different cargo (for example different neoantigen peptides) to be tagged with the same tag. This strategy would allow for the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention to be "loaded" with multiple different cargo, for example multiple different cargo peptides or cargo proteins comprising multiple different antigens.
In other situations, it may be beneficial for one or more of the tag binding domains (for example the peptide tag binding domains) to have specificity for a different tag (for example a peptide tag), so that the polypeptide of the invention can be "loaded" with a range of tagged cargo, for example a range of tagged proteins for example a range of tagged antigens to be delivered. Alternatively, it may be beneficial if the cargo comprises several different neoantigens (for example different neoantigen peptides) to be present sequentially in one long peptide that is tagged with a single tag for example a single peptide tag. For example, in some embodiments, the polypeptide of the invention comprises at least 1, 2, 3, 4 or more tag binding domains (for example coiled-coil oligomerisation domains), wherein each of the tag binding domains (for example each coiled-coil oligomerisation domains) has specificity for the same tag (for example coiled-coil oligomerisation domain).
In other embodiments, the polypeptide of the invention comprises at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a first tag (for example a first coiled-coil oligomerisation tag domain ), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a second tag (for example a second coiled-coil oligomerisation tag domain). In yet other embodiments, the polypeptide of the invention comprises at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a first tag (for example a first coiled-coil oligomerisation tag domain), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a second tag (for example second coiled-coil oligomerisation tag domain), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a third tag (for example a third coiled-coil oligomerisation tag domain). In yet other embodiments, the polypeptide of the invention comprises at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a first tag (for example a first coiled-coil oligomerisation tag domain), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a second tag (for example a second coiled-coil oligomerisation tag domain), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a third tag (for example a third coiled-coil oligomerisation tag domain), and at least one tag binding domain (for example coiled-coil oligomerisation domain) with specificity for a fourth tag (for example a fourth coiled-coil oligomerisation tag domain).
As will be apparent, the polypeptides and methods described herein allow particular tagged cargo to be targeted or directed towards a specific target. The target may be a specific cell, tissue or other structure which can comprise antigens, for example in some embodiments the cargo are cargo proteins or cargo peptides and are targeted to a particular set of immune cells. The skilled person will be aware that various immune cells express different polypeptides and other ceil surface molecules and it is routine in the field to classify the different populations of immune cells into subpopulations, based on the expression of one or more polypeptides. The polypeptides of interest are often located on the immune cell surface. In one embodiment, the target antigen binding domain is an immune cell binding domain that binds to a molecule, for example a polypeptide, that is located on the surface of the target immune cell.
Some cell surface molecules are receptor molecules, which once ligated by, for example, a polypeptide or antibody directed against said receptor molecules, results in a particular function. The ligation of receptor molecules may be by an agonist or antagonist of said receptor. For example, once the CD40 receptor, which can be found on DC, is bound by an agonist, for example an anti-CD40 antibody, the DC is activated. Accordingly, the choice of immune cell target can not only ensure efficient delivery of the tagged cargo peptide or cargo protein, for example tagged antigen, to the immune cell, but can enable other advantageous functions to be activated, enhanced and/or performed, such as activation of the immune cell, enhancement of cytokine release (or enhancement of other ongoing immune cell functions) and/or internalisation of the tagged antigen.
One particular use for the present invention, and as described herein, is in the targeting of cargo protein or cargo peptides that comprise antigenic peptides to antigen presenting cells (APC). The skilled person will be aware that these immune cells, which include dendritic cells (DC), B cells and macrophages, are able to internalise components, such as polypeptides, and present fragments of the molecule on the cell surface, in complex with MHCI or MHCII molecules. The presentation of antigenic fragments on MHCI and MHCII molecules activates the innate and adaptive immune responses. It is considered that the present invention has particular utility in activating the innate and adaptive immune response through the targeted delivery of antigens to immune cells, and in particular APC such as those described here, including DC.
DC are professional APC that play a central role in the induction and regulation of adaptive immune responses, including the induction of cytotoxic T lymphocyte (CTL) responses. DC can be either plasmacytoid DC (pDC), which mainly reside in the blood and lymphoid organs and are capable of secreting large amounts of cytokines, such as type I interferon, upon activation, and conventional DC (cDC).
By "dendritic cell", we include both conventional dendritic cells (eDCs, also known as classical dendritic cells) and plasmacytoid dendritic cells (pDCs). eDCs include both cDCl and cDC2. By "dendritic cells", we also include both immature dendritic cells and mature, activated, dendritic cells. As discussed above, the selection of DC target determines which DC population that is targeted, how much of the antigen that is taken up and how much is presented on MHC II or MHC I, the latter being critical for cross-presentation to CD8+ T cells.
CD40 is a cell-surface expressed glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) superfamily and plays a central role in the immune system. It is expressed on a variety of immune cells, such as B cells, DC, monocytes and macrophages, but also on other normal tissues including epithelial cells, endothelial cells and fibroblasts, as well as several tumor types, e.g. on B cell lymphomas and carcinomas. Activation of CD40 on DC results in an anti-tumor immune response via tumor-specific T effector cells. CD40 agonists trigger effective anti-tumor responses in pre-clinical models.
Pre-clinical studies have demonstrated proof of concept for agonistic anti-CD40 antibody treatment of several cancer types, including lymphomas, melanoma, hepatoma, osteosarcoma, renal cell carcinoma, breast cancer and bladder cancer. In addition, humanized or human anti-CD40 antibodies have been evaluated in a number of pre-clinical models and consistently demonstrated anti-tumor effects. Notably, SGN- 40, a humanized CD40 monoclonal antibody with partial agonistic effects was evaluated using B cell lymphoma models (Raji and Ramos) in severe combined immunodeficiency (SCID) mice, and demonstrated effects on tumor growth and survival with complete response in approximately 50% of treated mice. CP-870,893, a human agonistic anti-CD40 antibody, showed anti-tumor effects against B cell lymphoma, breast, colon, prostate, and pancreatic cancer in SCID mice. Efficacy was observed in CD40 positive as well as in CD40 negative tumors, thus demonstrating the ability of CP-870,893 to enhance anti-tumor immunity.
TLR3 is part of the Toll-like receptor family and acts as a receptor for danger signals in the form of double-stranded RNA that may be a form of genetic information derived from viruses. Ligation of TLR3 on a DC initiates inflammatory signaling via IRF3 and NF-KB, which activates the DC. Polyinosinic-polycytidylic acid (poly I : C) is a commonly used ligand of TLR3.
Both B cells and DC express high levels of CD40 and may also function as APC. However, it has been demonstrated that DC rather than B cells, macrophages or monocytes are important for generation of antigen-specific T cell responses. These cell populations will, however, act as a sink and may affect the biodistribution. Other additional DC-markers include: XCR-1, CLEC9A, DEC-205, CDlc, Dec-1. Targeting CLEC9A would confer potential advantages since it is a death cell marker and antigen taken up by this receptor ends up in early endosomes and is more likely to result in cross-presentation to CD8+ T cells. CLEC9A is selectively expressed on crosspresenting DC and may be superior to the more widely tested DEC-205 when it comes to inducing CD8+ T cell activation.
Accordingly, in some embodiments the target antigen is present on an immune cell that is an APC. In some embodiments the immune cell is an APC and is selected from the group comprising or consisting of a DC, B cell or a macrophage. In preferred embodiments, the immune cell is a DC; even more preferred is cDCl.
Cargo-targeting or cargo-delivery polypeptides of the present invention may, in some embodiments, be directed to an immune cell target that is capable of mediating any one or more of: (a) activation of the immune cell (including enhancement of an immune cell activity or function); (b) internalisation of the polypeptide; and/or (c) recruitment of DCs, in particular recruitment of conventional type I dendritic cells (cDCl). For example, an immune cell target that is capable of DC activation includes, but is not limited to, CD40 and TLR3.
In some embodiments, the immune cell target may facilitate internalization of the ligand. By internalizing the polypeptides of the present invention, the antigen of interest is capable of being processed by the immune cell in order to be presented on MHC. Examples of immune cell targets that facilitate internalization include, but are not limited to, CLEC9A and DEC-205. CLEC9A is a C-type lectin receptor involved in sensing necrotic cells, and DEC-205 is a type I cell surface protein expressed primarily by DC.
In some embodiments, the immune cell target may facilitate recruitment of DC, preferably recruitment of cDCl. For example, the immune cell target may be XCR1. eDC can be found in tissues throughout the body, and in lymphoid organs. eDC in tissues capture antigen, transport it through the lymphatic system into draining lymph nodes, and present it to T cells. eDC that reside in lymphoid organs can also capture antigen, which may have diffused to the organ through the lymphatics, and present this to T cells. eDC can be further divided into two subsets, sometimes termed cDCl and cDC2. Transcriptional profiling has shown these subsets to be conserved between mice and humans. cDCl express the chemokine receptor XCR1, which allows them to localize close to XCLl-producing CD8+ T cells in lymphoid tissues, and the dead cell receptor CLEC9A. They are specialized at cross-presenting antigen to CD8+ T cells on MHC I and are required for the priming of CTL responses against TAA in mice. Human cDCl display superior cross-presenting abilities in some, but not all, in vitro settings compared to other human DC subsets. Uptake of exogenous antigen, such as TAA, in DC is primarily accomplished by receptor-mediated endocytosis. Cross-presentation of exogenous antigen is favored by a relatively high endosomal pH, and routing of antigen to early rather than late endosomes. While high endosomal pH is a characteristic of cDCl, which endosomal compartment the antigen is targeted to depend on the endocytic receptor engaged.
While pDC are generally not very efficient at presenting antigen to T cells, targeting antigen to uptake receptors on pDC can lead to effective cross-presentation to CD8+ T cells.
In some embodiments, the combination of the polypeptide, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention comprising at least a first target antigen binding domain that is an immune cell binding domain and at least one tag binding domain, and the tagged cargo, for example the tagged cargo protein or tagged cargo peptide, for example that comprises an antigen (for example a peptide comprising both the tag (preferably a coiled-coil oligomerisation domain) and the antigen or neoantigen), when administered to a mammal, for example to a mouse or a human, results in a superior expansion of antigen/neoantigen-specific immune cells, for example CD8+ T cells, compared to treatment with both 1) a polypeptide that comprises the immune cell binding domain but lacks the at least one tag binding domain and 2) the tagged peptide, for example administration of an anti-CD40 antibody (that lacks the tag binding domain) and an antigen/neoantigen peptide separately (i.e. where the antibody and antigen is unable to form a complex) . For example, this results in at least a 1.5 fold increase in expansion of antigen/neoantigen- specific immune cells such as CD8+ T cells, or at least a 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9 or 10 fold increase, for example when administered to a mammal such as a mouse or a human, for example when the agents (as a complex, or administered separately in the absence of the ability to form a complex, i.e. in the absence of the peptide tag, or the peptide tag binding domain) are administered on two occasions with 7 days between, and for example where viable CD45+ CD3+ CD8+ antigen/neoantigen-MHCI tetramer+ T cells are analysed seven days after the second treatment. In addition or instead of the above method, to determine whether a particular polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is able to target the tagged cargo, for example the tagged cargo protein or cargo peptide, for example where the cargo is an antigen, to a relevant cell population, an in vitro experiment can be performed where the tagged cargo protein or peptide, for example tagged antigen is fluorescently labelled. The labelled cargo protein or peptide (for example antigen) is mixed with the cargo-targeting or cargodelivery polypeptide, followed by incubation with relevant cell population. The amount of fluorescently labelled antigen bound to the cells could then be detected by, for example, flow cytometry or fluorescence microscopy.
Accordingly, in one embodiment, the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention results in the localization of a fluorescently labelled cargo, for example a fluorescently labelled cargo protein or peptides, for example labelled antigen, tagged with the corresponding tag (i.e. the tag to which the tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention binds - preferably a coiled- coil oligomerization domain) to the surface of the appropriate cell population ( e.g. the corresponding immune cells to which the target antigen binding domain that is an immune cell binding domain binds), for example when the polypeptide of the invention is first incubated with the fluorescently labelled tagged cargo (e.g. fluorescently labelled tagged antigen) followed by incubation with the immune cell.
The skilled person will be aware of various peptide-based means of targeting a polypeptide specifically to a molecule, the most commonly used of these being antibody-based means. Standard antibodies typically comprise a single, constant region (Fc), and two variable regions which provide target specificity. In a typical monospecific antibody, each of the two variable regions are identical and directed towards the same epitope on the same target, i.e. they are bivalent for the same target, i.e. monospecific.
Bispecific antibodies have been produced.
The term "bispecific" as used herein means the polypeptide is capable of specifically binding at least two different target entities. Bispecific polypeptides, e.g. antibodies, targeting two targets, have the potential to induce specific activation of the immune system in locations where both targets are over expressed. The number of formats engineered is vast and the formats can be grouped according to their general architecture. Brinkmann and Kontermann have proposed a classification of 19 groups (see Brinkmann & Kontermann, 2017, mAbs. 9 : 182-212). The major differences between the groups are in regard to their symmetry, their target valency, their components and the position of these.
Different properties are generally acknowledged for different bispecific format groups. Fragment-based formats that lack an Fc part have limited half-life and cannot mediate Fc effector functions. IgG-like formats display only monovalent binding to each target.
In the field of cancer, the risk of many targeted therapies is that of the cancer adapting or mutating in a way that prevents that target from being functional. Further, the epitopes presented by one person may differ vastly compared with that of another person. Thus, there is an increasing need for off-the-shelf technologies that are readily adaptable to a particular situation and patient.
Accordingly, in some embodiments, the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise a target antigen binding domain that is an immune cell binding domain that binds to at least one immune ceil receptor, optionally wherein the immune cell receptor is CD40, CLEC9A, DEC-205, XCR1 or TLR3. For example, the target antigen binding domain of the polypeptide of the present invention may bind to CD40.
Sequences of exemplary polypeptides of the invention used in the Examples are presented below, and include, where relevant, the coiled-coil oligomerisation domain sequences.
Antibody 1132E3 [Also referred to as A_1132E3, A_1132E3-IgGl, 1132E3- IgGl, IgGl-1132E3]
Heavy chain [SEQ ID NO: 88]
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIGSYGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYVNFGMDYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K Light chain [SEQ ID NO: B9]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSEIAALEKEIAALEKEIAALEK
Antibody 1132E4 [Also referred to as A_1132E4, A_1132E4-IgGl, 1132E4- IgGl, IgGl-1132E4]
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 90]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSEIAALEKEIAALEKEIAALEKEIAALEK
Antibody 1132E5 [Also referred to as A_1132E5, A_1132E5-IgGl, 1132E5- IgGl, IgGl-1132E5]
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 91]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSEIAALEKEIAALEKEIAALEKEIAALEKEIAALE K
Antibody 1132E6 [Also referred to as A_1132E6, A_1132E6-IgGl, 1132E6-
IgGl, IgGl-1132E6]
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 92]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSEIAALEKEIAALEKEIAALEKEIAALEKEIAALE
KEIAALEK
Antibody 1132leucine-zipper
Heavy chain [SEQ ID NO: 88] Light chain [SEQ ID NO: 93]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSALEKELAEAEKELAELEKELAGGCGGALEKK
LAELEKKAAELEKKLA
Antibody 1132HAP1
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 94]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSASENAALEAKNAALKYKIAALKAEIAALEGAP
Antibody 1132HAP2
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 95]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSASKNAALKAENAALEYEIAALEAKIAALKGAP
Antibody 1132HAP3
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 96]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSASEIAALEAEIAALEYENAALEAENAALEGAP
Antibody 1132HAP4
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 97]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSASKIAALKAKIAALKYKNAALKAKNAALKGA P
Antibody 1132HAP5
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 98]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSASKNAALKAEIAALEYKIAALKAENAALEGAP
Antibody 1132HAP6
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 99]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSASENAALEAKIAALKYEIAALEAKNAALKGAP
Antibody 1132HAP7
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 100]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSASKIAALKAKNAALKYENAALEAEIAALEGAP
Antibody 1132HAP8
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 101]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSASEIAALEAENAALEYKNAALKAKIAALKGAP
Antibody 1132K3 Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 102]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSKIAALKEKIAALKEKIAALKE
Antibody 1132K4
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 103]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSKIAALKEKIAALKEKIAALKEKIAALKE
Antibody 1132K5
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 104]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSKIAALKEKIAALKEKIAALKEKIAALKEKIAAL KE
Antibody 1132K6
Heavy chain [SEQ ID NO: 88]
Light chain [SEQ ID NO: 105]
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGECSGGGGSGGGGSKIAALKEKIAALKEKIAALKEKIAALKEKIAAL KEKIAALKE
Antibody 1132E3-IgG2 [Also referred to as A_1132E3-IgG2, 1132E3IgG2, IgG2-1132E3]
Heavy chain [SEQ ID NO: 106] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIGSYGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYVNFGMDYWGQGTLVTVSSASTKGPSV
FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain [SEQ ID NO: 89]
Antibody 1132E4-IgG2 [Also referred to as A_1132E4-IgG2, 1132E4IgG2,
IgG2-1132E4]
Heavy chain [SEQ ID NO: 106]
Light chain [SEQ ID NO: 90]
Bispecific antibodies: the bispecific antibodies used in the Examples comprise an IgG light chain as described in the table below and which comprises the sequence of the relevant coiled-coil oligomerisation domain, and the polypeptide also comprises an IgG heavy chain - Fab light chain of [SEQ ID NO: 117] and a Fab heavy chain of SEQ ID NO: 118.
Figure imgf000053_0001
Figure imgf000054_0001
IgG heavy chain - Fab light chain [SEQ ID NO: 117]
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRRAPGKGLEWVSGIGSYGGG TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYVNFGMDYWGQGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
AAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSELVMTQSPS
SLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQRKPGQPPKLLIYWASTRESGVPDRFTGSG
SGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIKRTVAAPAVFIFPPSDEQLKSGTASV VCLLKNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFN RGEC
Fab heavy chain [SEQ ID NO: 118]
EVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKERPGHGLEWIGDIFPGSGNIHYNE KFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVE VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
The polypeptide of the invention for example the cargo-targeting or cargo-delivery polypeptide of the invention comprises, in some embodiments: a heavy chain of SEQ NO: 88 and light chain selected from the group comprising or consisting of SEQ ID NO: 89-SEQ ID NO: 105; a heavy chain of SEQ ID NO: 106 and a light chain of SEQ ID NO: 89 or of SEQ ID NO: 90; or an IgG heavy chain-Fab light chain comprising or consisting of SEQ ID NO: 117, a Fab heavy chain comprising or consisting of SEQ ID NO: 118, and an IgG light chain that comprises a coiled-coil oligomerisation domain selected from any of [SEQ ID NO: 107-116] or comprises a sequence that has a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences, optionally comprises a sequence with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences and that is able to a) form a multimer, optionally a dimer optionally a heterodimer with a corresponding coiled-coil oligomerisation domain; and b) bind to the target antigen.
In some embodiments, the polypeptide may be an antibody that is selected from ADC-1013; clones 1132/1133, 1140/1135, 1150/1151 and 1107/1108 from WO 2015/091853; CP-870,893, APX005M, ChiLob 7/4, and SEA-CD40.
Accordingly, the polypeptide may be an antibody that comprises or consists of any one or more of the following sequences: AntibodY 132Z1133
Variable heavy chain (VH) amino acid sequence:
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIGSYGGG
TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYVNFGMDYWGQGTLVTV
SS (SEQ ID NO: 1)
Variable light chain (VL) amino acid sequence:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYGRNPPTFGQGTKLEIK (SEQ ID NO: 2)
CDR amino acid sequences:
VH CDRs: CDR1 : GFTFSSYA (SEQ ID NO: 3)
CDR2 : IGSYGGGT (SEQ ID NO: 4)
CDR3 : ARYVNFGMDY (SEQ ID NO: 5)
VL CDRs: CDR1 : QSISSY (SEQ ID NO: 6)
CDR2 : AAS (SEQ ID NO: 7)
CDR3 : QQYGRNPPT (SEQ ID NO: 8)
Variable heavy chain (VH) nucleotide sequence:
GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGC
GCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCC
GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTCTTACGGTGGT
GGTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCC
AAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATAT
TATTGTGCGCGCTACGTTAACTTCGGTATGGACTATTGGGGCCAGGGAACCCTGGTC
ACCGTCTCCTCA (SEQ ID NO: 9)
Variable light chain (VL) nucleotide sequence:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTC
ACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAG
AAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGG
GTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGC
AGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACGGTCGTAACCCGC
CCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 10)
Figure imgf000056_0001
Variable heavy chain (VH) amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGS
TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGPVYSSVFDYWGQGTLV
TVSS (SEQ ID NO: 11)
Variable light chain (VL) amino acid sequence:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK (SEQ ID NO: 12)
CDR amino acid sequences:
VH CDRs: CDR1 : GFTFSSYA (SEQ ID NO: 13)
CDR2 : ISGSGGST (SEQ ID NO: 14)
CDR3 : ARGPVYSSVFDY (SEQ ID NO: 15)
VL CDRs: CDR1 : QSISSY (SEQ ID NO: 16)
CDR2 : AAS (SEQ ID NO: 17)
CDR3 : QQSYSTPYT (SEQ ID NO: 18)
Variable heavy chain (VH) nucleotide sequence:
GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGC
GCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCC
GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGG
TAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTC
CAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTAT
ATTATTGTGCGCGCGGTCCGGTTTACTCTTCTGTTTTTGACTATTGGGGCCAGGGAA
CCCTGGTCACCGTCTCCTCA (SEQ ID NO: 19)
Variable light chain (VL) nucleotide sequence:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTC
ACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAG
AAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGG
GTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGC
AGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGAGTTACAGTACCCCTT
ATACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 20)
Figure imgf000057_0001
Variable heavy chain (VH) amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIGGSSSY
TSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYSYHMDYWGQGTLVTV
SS (SEQ ID NO: 21)
Variable light chain (VL) amino acid sequence:
DIQMTQSPSSLSASVGDHVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYGSAPPTFGQGTKLEIK (SEQ ID NO: 22)
CDR amino acid sequences
VH CDRs: CDR1 : GFTFSSYA (SEQ ID NO: 23)
CDR2 : IGGSSSYT (SEQ ID NO: 24)
CDR3 : ARYYSYHMDY (SEQ ID NO: 25)
VL CDRs: CDR1 : QSISSY (SEQ ID NO: 26)
CDR2 : AAS (SEQ ID NO: 27)
CDR3 : QQYGSAPPT (SEQ ID NO: 28)
Variable heavy chain (VH) nucleotide sequence:
GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGC
GCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCC
GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTGGTTCTTCTTCT
TACACATCTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCC
AAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATA
TTATTGTGCGCGCTACTACTCTTACCATATGGACTATTGGGGCCAGGGAACCCTGGT
CACCGTCTCCTCA (SEQ ID NO: 29)
Variable light chain (VL) nucleotide sequence:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCACGTC
ACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAG
AAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGG
GTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGC
AGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACGGTTCTGCTCCGC
CCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 30)
Figure imgf000058_0001
Variable heavy chain (VH) amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGS
TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRVWGFDYWGQGTLVTVS
S (SEQ ID NO: 31)
Variable light chain (VL) amino acid sequence:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYGVYPFTFGQGTKLEIK (SEQ ID NO: 32)
CDR amino acid sequences:
VH CDRs: CDR1 : GFTFSSYA (SEQ ID NO: 33)
CDR2 : ISGSGGST (SEQ ID NO: 34)
CDR3 : ARRVWGFDY (SEQ ID NO: 35)
VL CDRs: CDR1 : QSISSY (SEQ ID NO: 36)
CDR2 : AAS (SEQ ID NO: 37)
CDR3 : QQYGVYPFT (SEQ ID NO: 38)
Variable heavy chain (VH) nucleotide sequence:
GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGC
GCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCC
GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGG
TAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTC
CAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTAT
ATTATTGTGCGCGCCGTGTTTGGGGTTTTGACTATTGGGGCCAGGGAACCCTGGTCA
CCGTCTCCTCAGG (SEQ ID NO: 39)
Variable light chain (VL) nucleotide sequence:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTC
ACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAG
AAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGG
GTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGC
AGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACGGTGTTTACCCGT
TCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 40)
Figure imgf000059_0001
Variable heavy chain (VH) amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWLSYISGGSSYI
FYADSVRGRFTISRDNSENALYLQMNSLRAEDTAVYYCARILRGGSGMDLWGQGTLVT
VSS (SEQ ID NO: 41)
Variable light chain (VL) amino acid sequence:
QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVYWYQQLPGTAPKLLIYGNINRPSG
VPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDKSISGLVFGGGTKLTVLG (SEQ ID NO: 42)
CDR amino acid sequences:
VH CDRs: CDR1 : GFTFSTYGMH (SEQ ID NO: 43)
CDR2 : GKGLEWLSYISGGSSYIFYADSVRGR (SEQ ID NO: 44)
CDR3 : CARILRGGSGMDL (SEQ ID NO: 45)
VL CDRs: CDR1 : CTGSSSNIGAGYNVY (SEQ ID NO: 46)
CDR2 : GNINRPS (SEQ ID NO: 47)
CDR3 : CAAWDKSISGLV (SEQ ID NO: 48)
Variable heavy chain (VH) nucleotide sequence:
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACTTATGGCATGCACTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTTTCATATATTAGTGGTGGTAGTAGT
TACATTTTCTACGCAGACTCAGTGAGGGGCCGATTCACCATCTCCAGAGACAACTCC
GAGAACGCGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTA TTACTGTGCGAGAATATTAAGAGGCGGGAGCGGTATGGACCTCTGGGGCCAAGGTA CACTGGTCACCGTGAGCTCA (SEQ ID NO: 49)
Variable light chain (VL) nucleotide sequence:
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCTTGCACTGGGAGCAGCTCCAACATCGGGGCGGGTTACAATGTATACTGGTA TCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACATCAATCGGCC CTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGC CATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATA AGAGCATTTCTGGTCTGGTTTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ ID NO: 50)
Accordingly, in some embodiments, the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is selected from the group consisting of: antibodies or antigen binding fragments thereof. By an antibody or an antigen-binding fragment thereof we include substantially intact antibody molecules, as well as chimeric antibodies, humanised antibodies, isolated human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen-binding fragments and derivatives of the same. Suitable antigen-binding fragments and derivatives include Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab)2 fragments), single variable domains (e.g. VH and VL domains) and single domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb], and nanobodies). The potential advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Moreover, antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
In one embodiment, the antigen-binding fragment is selected from the group consisting of: Fv fragments (such as a single chain Fv fragment, or a disulphide-bonded Fv fragment), Fab-like fragments (such as a Fab fragment; a Fab' fragment or a F(ab)2 fragment) and single domain antibodies.
The phrase "an antibody or an antigen-binding fragment thereof" is also intended to encompass antibody mimics (for example, non-antibody scaffold structures that have a high degree of stability yet allow variability to be introduced at certain positions). Those skilled in the art of biochemistry will be familiar with many such molecules, as discussed in Gebauer & Skerra, 2009 (the disclosures of which are incorporated herein by reference). Exemplary antibody mimics include: affibodies (also called Trinectins; Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also called Tetranectins; Innovations Pharmac. Technol. (2006), 27-30); adnectins (also called monobodies; Meth. Mol. Biol., 352 (2007), 95-109); anticalins (Drug Discovery Today (2005), 10, 23-33); DARPins (ankyrins; Nat. Biotechnol. (2004), 22, 575-582); avimers (Nat. Biotechnol. (2005), 23, 1556-1561); microbodies (FEBS J, (2007), 274, 86-95); peptide aptamers (Expert. Opin. Biol. Ther. (2005), 5, 783-797); Kunitz domains (J. Pharmacol. Exp. Ther. (2006) 318, 803-809); affilins (Trends. Biotechnol. (2005), 23, 514-522); affimers (Avacta Life Sciences, Wetherby, UK). Also included within the scope of the invention are chimeric T cell receptors (also known as chimeric T cell receptors, chimeric immunoreceptors, and chimeric antigen receptors or CARs) (see Pule et al., 2003, the disclosures of which are incorporated herein by reference). These are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. Typically, CARs are used to graft the specificity of a monoclonal antibody onto a T cell; with transfer of their coding sequence facilitated by retroviral vectors. The most common form of such molecules is fusions comprising a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to CD3-zeta transmembrane and endodomain. When T cells express this fusion molecule, they recognize and kill target cells that express the transferred monoclonal antibody specificity.
Persons skilled in the art will further appreciate that the invention also encompasses modified versions of antibodies and antigen-binding fragments thereof, whether existing now or in the future, e.g. modified by the covalent attachment of polyethylene glycol or another suitable polymer (see below).
Methods of generating antibodies and antibody fragments are well known in the art. For example, antibodies may be generated via any one of several methods which employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi. et al, 1989; Winter et al., 1991, the disclosures of which are incorporated herein by reference) or generation of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B cell hybridoma technique, and the Epstein-Barr virus (EBV)- hybridoma technique (Kohler et al., 1975,Kozbor et al., 1985; Cote et al., 1983; Cole et al., 1984., the disclosures of which are incorporated herein by reference).
Suitable methods for the production of monoclonal antibodies are also disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988, the disclosures of which are incorporated herein by reference) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982, the disclosures of which are incorporated herein by reference).
Likewise, antibody fragments can be obtained using methods well known in the art (see, for example, Harlow & Lane, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, New York, the disclosures of which are incorporated herein by reference). For example, antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Alternatively, antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
It will be appreciated by persons skilled in the art that for human therapy or diagnostics, human or humanised antibodies are preferably used. Humanised forms of non-human (e.g. murine) antibodies are genetically engineered chimeric antibodies or antibody fragments having preferably minimal-portions derived from non-human antibodies. Humanised antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementary determining region of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired functionality. In some instances, Fv framework residues of the human antibody are replaced by corresponding non-human residues. Humanised antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences. In general, the humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially ail of the complementarity determining regions correspond to those of a non-human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence. Humanised antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986, Riechmann et al., 1988, Presta, 1992, the disclosures of which are incorporated herein by reference).
Methods for humanising non-human antibodies are well known in the art. Generally, the humanised antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. Humanisation can be essentially performed as described (see, for example, Jones et al., 1986, Reichmann et al., 1988, Verhoeyen et al., 1988, US 4,816,567, the disclosures of which are incorporated herein by reference) by substituting human complementarity determining regions with corresponding rodent complementarity determining regions. Accordingly, such humanised antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanised antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies. Human antibodies can also be identified using various techniques known in the art, including phage display libraries (see, for example, Hoogenboom & Winter, 1991, Marks et al., 1991, Cole et al., 1985, Boerner et al., 1991, the disclosures of which are incorporated herein by reference).
It will be appreciated by persons skilled in the art that the bispecific polypeptides, e.g. antibodies, of the present invention may be of any suitable structural format.
Accordingly, in some embodiments, the polypeptide is an antigen-binding fragment selected from the group consisting of: an Fv fragment (such as a single chain Fv fragment, or a disulphide-bonded Fv fragment); a Fab-like fragment (such as a Fab fragment; a Fab' fragment; or a F(ab)2 fragment); and domain antibodies.
In some embodiments, the tag binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, is an Fc region of an antibody or antibody fragment having the ability to specifically bind to the tag, for example to the peptide tag. For example, the tag, for example the peptide tag may be a molecule with affinity to the Fc portion of an antibody, such as an antibody or fragment thereof that is specific to Fc (an anti-Fc antibody or fragment thereof); and Protein A, which is a protein derived from Staphylococcus aureus that binds strongly to IgGs.
In one embodiment, the peptide tag is a 33 amino acid sequence as follows:
Z33: FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD [SEQ ID NO: 51]
Accordingly, in some embodiments the tag binding domain of the polypeptide of the invention is an Fc region of an antibody or antigen binding fragment thereof that is capable of binding to the Z33 peptide sequence of SEQ ID NO: 51.
In one embodiment, the peptide tag is linked to a larger peptide or polypeptide, for example that comprises an antigen or neoantigen, through a linker, for example a GSSSS linker [SEQ ID NO: 55] or a cleavable valine-citrulline linker.
Optionally, the binding domain localized to the Fc region can be engineered by introduction of mutations to enhance the affinity to the Fc-binding peptide tag. Such mutations would improve the stability of the DC-targeting antibody-neoantigen complex and prevent swapping of the Fc in an in vivo setting. However, as stated elsewhere, it is preferred if the tag binding domain is a coiled-coil oligomerization domain and the tag that is used to tag the cargo, for example a cargo protein or peptide is also a coiled-coil oligomerization domain, such that the two coiled- coil oligomerization domains interact when contacted to form a muitimer, for example a dimer, for example a heterodimer.
As described elsewhere herein, a single polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may have multiple tag binding domains of different formats, so for example may have a tag binding domain that is a coiled-coil oligomerization domain, and may also have a tag binding domain that is an Fc region.
In some embodiments the Fc region is not considered to be a tag binding domain.
Also as described elsewhere herein, the tag that is used to tag the cargo for example the cargo protein or cargo peptide may be linked to the cargo via a peptide linker, for example a GS linker or a cleavable valine-citrulline linker or a meta I loprotease cleavable linker as described elsewhere herein.
Also contemplated are embodiments wherein the polypeptide, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention comprises at least 2 tag binding domains (for example at least 2 peptide tag binding domains), for example 3, 4, 5, 6 or more peptide tag binding domains (for example at least 3, 4, 5, 6 peptide tag binding domains). Where at least 2 tag binding domains are present, in some embodiments at least one is selected from the group consisting of: a coiled-coil oligomerisation domain, and at least a second tag binding domain is selected from antibodies or antigen binding fragments thereof; or an Fc region of an antibody having the ability to specifically bind to the peptide tag.
In one embodiment, the tag binding domain (for example the peptide tag binding domain) is a domain other than an Fc region. For instance, the polypeptide may comprise an Fc region, but the Fc region is not the domain that binds to the tagged antigen/neoantigen. In some embodiments the polypeptide may comprise a number of tag binding domains (for example a number of peptide tag binding domains) as described herein, and none of those are the Fc region, i.e. the Fc region is not used to bind to the tagged antigen/neoantigen. In other embodiments, the Fc region is used to bind to the tagged cargo or the Fc region is one of a number of binding domains used to bind to the tagged cargo.
Thus, a number of variations can be envisaged for the present invention, such as where the polypeptide comprises: a) at least a first target antigen binding domain that is an immune cell binding domain that comprises or consists of an IgG antibody; b) at least a first target antigen binding domain that is an immune cell binding domain that comprises or consists of an Fv fragment; c) at least a first target antigen binding domain that comprises or consists of an IgG antibody and at least one tag binding domain that comprises or consists of a Fab- like fragment; d) at least a first target antigen binding domain that comprises or consists of an IgG antibody and at least one tag binding domain that comprises or consists of a domain antibody; e) at least a first target antigen binding domain that comprises or consists of an IgG antibody and at least one tag binding domain that comprises or consists of a coiled-coil oligomerisation domain; f) at least a first target antigen binding domain that comprises or consists of an Fv fragment and at least one tag binding domain that comprises or consists of an IgG antibody; g) at least a first target antigen binding domain that comprises or consists of a Fab-like fragment and at least one tag binding domain that comprises or consists of an IgG antibody; h) at least a first target antigen binding domain that comprises or consists of a domain antibody and at least one tag binding domain that comprises or consists of an IgG antibody; i) at least a first target antigen binding domain that comprises or consists of an IgG antibody and at least one tag binding domain that comprises or consists of an Fc region of an antibody having the ability to specifically bind to the tag; j) at least a first target antigen binding domain that comprises or consists of an Fv fragment and at least one tag binding domain that comprises or consists of an Fc region of an antibody having the ability to specifically bind to the tag; k) at least a first target antigen binding domain that comprises or consists of a Fab-like fragment and at least one tag binding domain that comprises or consists of an Fc region of an antibody having the ability to specifically bind to the tag; l) at least a first target antigen binding domain that comprises or consists of a domain antibody and at least one tag binding domain that comprises or consists of an Fc region of an antibody having the ability to specifically bind to the tag; m) at least a first and second target antigen binding domain that comprises or consists of an IgG antibody wherein the at least a first and second target antigen binding domains bind to the same dendritic cell target, and at least one tag binding domain that comprises or consists of an Fc region of an antibody having the ability to specifically bind to the tag; n) at least a first target antigen binding domain that is an immune cell binding domain and at least a second target antigen binding domain that is an immune cell binding domain that comprise or consist of an IgG antibody, wherein the first and second target binding domain bind to the same immune cell; or o) at least a first target antigen binding domain that is an immune cell binding domain and at least a second target antigen binding domain that is an immune cell binding domain that comprise or consist of an IgG antibody, wherein the first target antigen binding domain and the second target antigen binding domain bind to different targets, but wherein the different targets are present on the same immune cell, optionally wherein the immune cell is a dendritic cell.
In any of the embodiments where the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises antibody like components, the antibody may be an IgG antibody, for example an IgGl, IgG2, IgG3 or IgG4 antibody.
In some embodiments, the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is a bispecific polypeptide, and optionally comprises or consists of a format selected from the group consisting of (wherein the bispecific is comprised of a first target antigen binding domain 1 (Bl) and a second target antigen binding domain 2 (B2) : a) IgG-scFv bispecific antibodies, optionally wherein Bl is an intact IgG and B2 is an scFv attached to Bl at the N-terminus of a light chain; at the C-terminus of a light chain; at the N-terminus of a heavy chain; and/or at the C-terminus of a heavy chain of the IgG. For example, the scFv could be attached to the N-terminus and C- terminus of the light chain; attached to the N-terminus of both the light and heavy chains, etc. b) monovalent bispecific antibodies, such as a DuoBody® (Genmab AS, Copenhagen, Denmark) or 'knob-in-hole' bispecific antibody (for example, an scFv- KIH, scFv-KIHr, a BiTE-KIH or a BiTE-KIHr (see Xu et al., 2015, mAbs 7(l) :231-242); c) scFv2-Fc bispecific antibodies, (such as ADAPTIR™ bispecific antibodies from Emergent Biosolutions Inc); d) BiTE/scFv2 bispecific antibodies; e) DVD-Ig bispecific antibodies; f) DART-based bispecific antibodies (for example, DART2-Fc or DART); g) DNL-Fab3 bispecific antibodies; and h) scFv-HSA-scFv bispecific antibodies i) RUBY™ format antibodies, wherein the antibody comprises:
(i) two copies of a first heavy chain polypeptide and two copies of a first light chain polypeptide, and
(ii) two Fab fragments, the Fab fragments comprising a second heavy chain polypeptide and a second light chain polypeptide and wherein the first Fab fragment is fused to the C-terminus of the first copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment; and the second Fab fragment is fused to the C-terminus of the second copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment, and wherein a) the two copies of a first heavy chain polypeptide and two copies of a first light chain polypeptide form two immune cell binding domains and the two Fab fragments form a first and second tag binding domain; or b) the two copies of a first heavy chain polypeptide and two copies of a first light chain polypeptide form a first and a second target binding domain, and the two Fab fragments form two immune cell binding domains.
For example, the bispecific polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention antibody may be an IgG-scFv antibody. The IgG-scFv antibody may be in either VH-VL or VL-VH orientation. In one embodiment, the scFv may be stabilised by a S-S bridge between VH and VL. In one embodiment, binding domain Bl and binding domain B2 are fused directly to each other.
In some embodiments, Bl is the immune cell binding domain and B2 is the tag binding domain. In other embodiments Bl is the tag binding domain and B2 is the immune cell binding domain.
In an alternative embodiment, binding domain Bl and binding domain B2 are joined via a polypeptide linker. For example, a polypeptide linker may be a short linker peptide between about 10 to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N- terminus of the VH with the C-terminus of the VL, or vice versa.
Thus, the linker may be selected from the group consisting of the amino acid sequence SGGGGSGGGGS [SEQ ID NO: 56], SGGGGSGGGGSAP [SEQ ID NO: 57], NFSQP [SEQ ID NO: 58], KRTVA [SEQ ID NO: 59], GGGSGGGG [SEQ ID NO: 60], GGGGSGGGGS [SEQ ID NO: 61], GGGGSGGGGSGGGGS [SEQ ID NO: 62], GSTSGSGKPGSGEGSTKG [SEQ ID NO: 63] (Whitlow et al. 1993), THTCPPCPEPKSSDK [SEQ ID NO: 64], GGGS [SEQ ID NO: 65], EAAKEAAKGGGGS [SEQ ID NO: 66], EAAKEAAK [SEQ ID NO: 67], or (SG)m, where m = 1 to 7, for example m can be 1, 2, 3, 4, 5, 6 or 7.
In some embodiments, the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises an Fc region or a variant of said region, optionally wherein the region is an IgGl, IgG2, IgG3 or IgG4 region, preferably IgGl or IgG2.
In a further embodiment, the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is a monospecific antibody directed towards an immune cell target. In preferred embodiments the polypeptide comprises a tag binding domain that is a coiled coil oligomerisation domain that can bind to a first tag that is also a coiled-coil oligomerisation domain, and may also comprise an Fc region that has been modified to be capable of binding to a second tag that is not a coiled coil oligomerisation domain; or a bispecific antibody wherein one paratope comprises the immune cell binding domain and the second paratope comprises the tag binding domain (for example the tag binding domain). In preferred embodiments the tag binding domain is a coiled coil oligomerisation domain that can bind to a first tag that is also a coiled-coil oligomerisation domain, and may also comprise an Fc region that has been modified to be capable of binding to a second tag that is not a coiled coil oligomerisation domain.
It will be apparent that in addition to providing a polypeptide, for example a cargotargeting polypeptide or a cargo-delivery polypeptide of the invention capable of targeting a cargo for example a cargo protein or cargo peptide, such as an antigenic peptide or a neoantigen to a particular target site of cell type, for example to an immune cell or population of immune cells, the invention also provides a complex comprising a polypeptide of the invention, for example the cargo-targeting polypeptide or the cargo-delivery polypeptide of the invention, as described herein, and a tagged cargo, for example a tagged cargo protein or peptide (for example a peptide tagged antigen), wherein the tagged cargo (for example the peptide tagged antigen) comprises the tag to which the tag binding domain of the polypeptide binds.
Preferences for features of the complex of the invention, for example in relation to the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, and the tagged cargo for example tagged cargo protein or peptides (for example tagged peptides that comprise the one or more antigens) are as described herein in relation to other aspects of the invention.
Accordingly, the complex of the invention comprises the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and the tagged cargo for example tagged protein or peptide (for example a tagged peptide antigen).
Preferences for the tag, along with the interaction between the tag and the polypeptide of the invention are as described herein in relation to other aspects of the invention.
For example, it is described elsewhere herein that the preferred tag, and tag binding domain, are complementary coiled-coil oligomerisation domains, that can form multimers for example dimers for example heterodimers.
Accordingly, in a preferred embodiment the invention provides a complex comprising a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention and a tagged cargo, for example a tagged cargo protein or cargo peptide, wherein the tagged cargo comprises a tag that is a cargo coiled-coil oligomerisation domain that is an interaction partner for the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention.
Preferences for the coiled-coil oligomerisation domains are as described elsewhere herein. For example preferences for various consensus amino acid sequences are as described elsewhere herein.
In some embodiments the complex does not comprise: a polypeptide according the invention, for example a cargo-targeting or cargodelivery polypeptide of the invention that comprises a tag binding domain that is a coiled-coil oligomerisation domain that is E3 [SEQ ID NO: 71] and a cargo that is attached to a tag that is a coiled-coil oligomerisation domain that is K3 [SEQ ID NO:
75]; a polypeptide according the invention, for example a cargo-targeting or cargodelivery polypeptide of the invention that comprises a tag binding domain that is a coiled-coil oligomerisation domain that is E3 [SEQ ID NO: 71] and a cargo that is attached to a tag that is a coiled-coil oligomerisation domain that is K4 [SEQ ID NO:
76]; a polypeptide according the invention, for example a cargo-targeting or cargodelivery polypeptide of the invention that comprises a tag binding domain that is a coiled-coil oligomerisation domain that is K3 [SEQ ID NO: 75] and a cargo that is attached to a tag that is a coiled-coil oligomerisation domain that is E3 [SEQ ID NO: 71]; a polypeptide according the invention, for example a cargo-targeting or cargodelivery polypeptide of the invention that comprises a tag binding domain that is a coiled-coil oligomerisation domain that is K4 [SEQ ID NO: 76] and a cargo that is attached to a tag that is a coiled-coil oligomerisation domain that is E3 [SEQ ID NO: 71],
In some embodiments the coiled-coil oligomerization domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and the tagged cargo protein or peptide is selected from any of the coiled coil oligomerisation domains described by SEQ ID NO: 71-87 or 119-127, or select from any of the coiled coil oligomerisation domains described by SEQ ID NO: 71-87, or a coiled-coil oligomerisation domain with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences, or wherein any of the coiled-coil oligomerisation domains has a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences and that is able to form a heterodimer with a corresponding coiled- coil oligomerisation domain.
Certain coiled-coil oligomerization domains interact with certain other coiled-coil oligomerization domains. The skilled person will readily be able to determine which coiled-coil oligomerization domains are suitable to be used as a corresponding pair - one on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, and one used as the tag on the tagged cargo protein or peptide.
For example in some embodiments: where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77] or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77] or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is E5 [SEQ ID NO: 73] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77] or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is E6 [SEQ ID NO: 74] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77] or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is K3 [SEQ ID NO: 75] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73] or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is K4 [SEQ ID NO: 76] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73] or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is K5 [SEQ ID NO: 77] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73] or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is K6 [SEQ ID NO: 78] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73] or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is HAP1 [SEQ ID NO: 80] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is HAP2 [SEQ ID NO: 81] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP1 [SEQ ID NO: 80], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is HAP3 [SEQ ID NO: 82] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP1 [SEQ ID NO: 80], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is HAP4 [SEQ ID NO: 83] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP1 [SEQ ID NO: 80], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is HAP5 [SEQ ID NO: 84] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP1 [SEQ ID NO: 80], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is HAP6 [SEQ ID NO: 85] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP1 [SEQ ID NO: 80], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is HAP7 [SEQ ID NO: 86] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP1 [SEQ ID NO: 80] or HAP8 [SEQ ID NO: 87]; or where the coiled-coil oligomerisation domain present on the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention according to any of claims 1-38 is HAP8 [SEQ ID NO: 87] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP1 [SEQ ID NO: 81]; or wherein any of the coiled-coil oligomerisation domains has a sequence with at Ieast85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences, or wherein any of the coiled-coil oligomerisation domains has a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences and that is able to form a heterodimer with a corresponding coiled- coil oligomerisation domain.
In preferred embodiments, the corresponding pairs of coiled-coil oligomerisation domains are as follows: where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K4 [SEQ ID NO: 76] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K5 [SEQ ID NO: 77] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], and where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K6 [SEQ ID NO: 78] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72],
In more preferred embodiments: where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76] or K6 [SEQ ID NO: 78]; and where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76] or K6 [SEQ ID NO: 78].
In more preferred embodiments: where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76]; and where the coiled-coil oligomerisation domain present on the cargo-targeting polypeptide according to any of claims 1-38 is K4 [SEQ ID NO: 76] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E4 [SEQ ID NO: 72].
In some embodiments the complex may comprise a single polypeptide of the invention, for example a single cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and two cargo molecules. For example the invention provides a complex of the invention wherein:
The polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises: at least a first tag binding domain that is a coiled-coil oligomerisation domain and a second tag binding domain that is a coiled-coil oligomerisation domain; and at least a first cargo comprising a first cargo coiled-coil oligomerisation domain and a second cargo comprising a second cargo coiled-coil oligomerisation domain; wherein the first tag binding domain interacts with the first cargo coiled-coil oligomerisation domain; and wherein the second tag binding domain interacts with the second cargo coiled-coil oligomerisation domain.
In some embodiments the first cargo is different to the second cargo.
It will be apparent to the skilled person that the cargo can be any compound, agent, moiety or molecule to which it is possible to attach or connect or otherwise incorporate a tag as described herein.
Accordingly in some embodiments the cargo is selected from any one or more of: a) a protein or a peptide; b) a nucleic acid; c) a toxin; d) a small molecule, for example a small molecule drug, imaging agent, toxin, radionucleotide labelled molecule; e) a virus particle; f) a viral vector; g) crispr editing components; h) lipid vesicles, optionally liposomes and/or exosomes; i) a PAMP or a DAMP; j) bacterial cellular fragments and structures; and/or k) a nanoparticle.
Any of these entities may be conjugated or otherwise connected to a tag as described herein, for example connected to a coiled-coil oligomerisation domain.
In some embodiments, where the cargo is a protein or a peptide, the protein or peptide can be an antigenic protein or peptide. In some embodiments the antigenic protein or peptide has been identified as a neoantigen that has arisen in a tumour or a cell. In some embodiments then the cargo is a neoantigen. In some embodiments the antigenic protein or peptide is or comprises or consists of: a cancer antigen, for example a HPV-associated cancer antigen; an antigenic peptide or protein sequence that is derived from a pathogen, optionally wherein the pathogen is a bacteria, fungus or virus, optionally wherein the virus is Human Papillomavirus (HPV).
In some embodiments then the cargo is: a) A peptide or protein that is, or comprises, an antigen, optionally wherein: i) the antigen has been identified as a neoantigen that has arisen in a tumour or cell; ii) the antigen is a cancer antigen, optionally wherein the cancer antigen is a Human Papillomavirus (HPV)-associated cancer antigen; iii) the antigen is derived from a pathogen, optionally wherein the pathogen is a bacteria, fungus or virus, optionally wherein the virus is Human Papillomavirus (HPV); or b) a nanoparticle that comprises an antigen, optionally comprises an antigen: i) that has been identified as a neoantigen that has arisen in a tumour or cell; ii) that is a cancer antigen, optionally wherein the cancer antigen is a Human Papillomavirus (HPV)-associated cancer antigen; iii) that is derived from a pathogen, optionally wherein the pathogen is a bacteria, fungus or virus, optionally wherein the virus is Human Papillomavirus (HPV).
In some embodiments the nanoparticle is a protein nanoparticle. In some embodiments the cargo is a toxin, optionally wherein the cargo is a toxic protein, toxic peptide or toxic nanoparticle, for example wherein the toxic protein, toxic peptide or toxic nanoparticle is a drug-conjugated protein, peptide or nanoparticle.
In some embodiments the cargo is a viral particle that has been engineered to express the relevant tag, for example the relevant coiled-coil oligomerisation domain.
It will be clear that where the cargo is a protein or peptide, and where the tag is also a protein or peptide, the cargo and the tag may be expressed as one transcript and translated into a single amino acid chain. Generating such constructs is routine for the skilled person. As described elsewhere herein, between the cargo and the tag it is possible to incorporate a linker. Various linkers known to the skilled person are considered to be useful for example GS linkers and metalloprotease cleavage linkers. Cleavable linkers are considered to be advantageous in some uses since it is possible to produce a construct that is cleaved when in an appropriate environment, for example a tumour microenvironment where the appropriate meta I loproteases are present.
In some embodiments, the tag may be attached to the cargo by chemical conjugation. Such an approach may be appropriate where the cargo and the tag are both proteins or peptides, but is particularly suited to situations where the cargo is not proteinaceous and so cannot be generated from a single mRNA transcript that also encodes the tag.
In some instances, the the tag (for example the peptide tag for example the coiled- coil oligomerization domain) may also be adapted to be multifunctional. For example, the tag may facilitate the binding of an antigen or epitope of interest to a polypeptide of the present invention, in addition to acting as an adjuvant. The term "adjuvant" means anything that can elicit or enhance an immune response, for example an immune response directed towards an antigen. Tags that also act as adjuvants may be tags that are epitopes for other immune cells, such as a T cell epitope or B cell epitope. A tag that comprises a sequence that is also a T cell epitope (which may be specific for CD4 or CD8 T cells) means that there may exist T cells in the subject that are specific to the sequence of the tag, and their interaction with said tag may boost or provoke their activity.
A B cell epitope means that the tag may have antibodies directed against it in situ. For example, a tag derived from the tetanus toxin (TTx), such as MTTE, is a B cell epitope that is widely spread within certain populations, i.e. a so-called universal B cell epitope. The term "universal" B cell epitope means that the majority of individuals in a given population have antibodies specific to that particular epitope. This may occur due to herd vaccination (i.e. the vaccination or the majority of a population against a particular disease, such as tetanus, measles, mumps, rubella etc).
Tags comprising particular universal B cell epitopes may be a detriment to the purpose of the present invention. For example, use of the MTTE peptide as a tag may be unworkable for large portions of the population due to patients having anti-MTTE antibodies. A polypeptide of the present invention that is specific to the MTTE peptide (being used as a tag attached to an antigen or epitope of interest) may have patient antibodies directed against it that prevent their intended function. Thus, polypeptides of the present invention are not intended to comprise a second binding domain specific for TTx or the MTTE peptide derived therefrom. As stated above, in all embodiments the peptide tag is not a tetanus derived tag.
The suitability of a tag (for example a peptide tag) may be dependent on a number of factors. For example, a human peptide tag that is a natural human peptide is less likely to provoke an immune response against it (due to it not being considered "foreign" by the immune system). Alternatively, the tag (for example a peptide tag) could be derived from a non-human source, such as bacteria (i.e. a bacterial protein). The structure of the peptide tag is another consideration, such as the secondary and/or tertiary structures. Regarding secondary structures, it may be preferable that the peptide tag comprises largely of a-helices and few to no p-sheets. Thus, in a particular embodiment, a peptide tag may comprise or consist of an a-helical structure. Peptide secondary structure can be predicted using software that is publicly available, such as Jpred4 (http://www.compbio.dundee.ac.uk/jpred/, Drozdetskiy et al., Nucl. Acids Res. 43(W1) : W389-W394, 2015) and PASTA 2.0 (http://protein.bio.unipd.it/pasta2/, Walsh et al., Nucl. Acids Res. 42(W) : W301-W307, 2014), or can be determined experimentally using routine techniques such as circular dichroism spectroscopy (see e.g. Greenfield, N., Nat 5 Protoc. 1(6): 2876-2890, 2006).
In some embodiments the tag is a non-human tag, for example where the tag is a peptide tag, the peptide tag is a non-human peptide.
In one embodiment the complex of the invention comprises a polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention as described herein wherein the polypeptide of the invention comprises an Fc region, and a tagged cargo, for example a tagged cargo protein or peptide (for example a tagged antigen), wherein the tag portion of the tagged cargo (for example a peptide tagged antigen) binds to the Fc region of the polypeptide of the invention. In one embodiment the tag has the sequence of [SEQ ID NO: 51] or a sequence with at least 80%, 85%, 90%, 95%, 98% or 100% sequence identity to SEQ ID NO: 51 and wherein the tag retains the ability to bind to the Fc region.
As stated above, the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise more than one tag binding domain (for example more than one peptide tag binding domain), and may be capable of binding to more than one copy of the same tag, or to multiple different tags (for example multiple different peptide tags). Accordingly, in one embodiment the complex comprises a polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, wherein the polypeptide comprises at least two tag binding domains (for example two tag binding domains that are coiled coil oligomerisation domains) capable of binding to at least two different tags (for example at least two different coiled coil oligomerisation domains), and wherein the complex comprises at least two tagged cargos, for example at least two tagged proteins or peptides (for example at least two peptide tagged antigens). The polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention may comprise at least 2, 3 or 4 tag binding domains (for example at least 2, 3 or 4 peptide tag binding domains), and the complex may comprise at least 2, 3 or 4 tagged antigens (for example peptide tagged antigens), for example tagged neoantigens.
Accordingly, a single complex may comprise multiple copies of a first tagged cargo for example a first tagged cargo protein or peptide, (for example a first peptide tagged antigen), for example a first tagged neoantigen; or may comprise at least 2, 3, or 4 different tagged cargo for example at least 2, 3 or 4 different tagged cargo proteins or peptides (for example may comprise at least 2, 3, or 4 different peptide tagged antigens), for example at least 2, 3 or 4 different tagged neoantigens or peptide tagged neoantigens.
The skilled person will understand what is meant by the term neoantigen, and we include the meaning of newly formed antigens that have not been previously recognized by the immune system in an individual. The genetic instability of tumor cells often leads to the occurrence of a large number of mutations, and expression of non-synonymous mutations can produce neoantigens, i.e. antigens that are specific to that tumor and to that individual. Neoantigens are highly immunogenic as they are not expressed in normal tissues. They can activate CD4+ and CD8+ T cells to generate an immune response and have the potential to become new targets of tumor immunotherapy. Neoantigens can arise from altered tumor proteins formed as a result of tumor mutations or from viral proteins.
We also include the meaning of antigens to which the individual has already been exposed, but which are more highly expressed by a tumour cell. In this case stimulation of the immune system by the polypeptide of the invention would be expected to raise an immune response that would be directed primarily to areas in which the antigen is most highly expressed - i.e. the tumour.
In preferred embodiments the antigen is a peptide, protein or fragment thereof that comprises an antigenic peptide or protein sequence. Accordingly, it is considered to be advantageous if the complex of the invention comprises a neoantigen that has arisen in a particular tumour, since administration of the complex will result in the neoantigen being targeted to the desired immune cell, for example an APC such as a DC, where the immune cell can be activated, the neoantigen can be internalised and subsequently presented to other relevant cells of the immune system, ultimately directing the immune system against the tumour.
In one embodiment therefore the antigen is a neoantigen (for example a peptide neoantigen), and the complex of the invention comprises a polypeptide of the invention, and at least one tagged neoantigen such as at least one tagged peptide neoantigen. As discussed herein, it can be advantageous to administer multiple antigens, for example multiple neoantigens. In one embodiment the complex of the invention comprises a polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and 2, 3, 4 or more different tagged cargo that are or comprise or consist of antigens, for example peptide tagged antigens such as peptide tagged neoantigens. The peptide tagged neoantigens may be tagged with the same peptide tag, or the neoantigens may be tagged with different peptide antigens.
In particular embodiments, the antigen (for example the peptide tagged antigen) is a peptide that comprises an antigenic amino acid sequence that has been identified as a neoantigen that has arisen in a tumour or cell. The skilled person is readily able to sequence the genomic material of a given tumour to identify suitable neoantigens.
In some embodiments the tagged cargo protein or peptide that comprises or consists of a tagged antigen, for example the peptide tagged antigen comprises an antigenic peptide that is a cancer antigen. The skilled person will appreciate which of the known cancer antigens it would be useful to target to immune cells, such as APC, for example DC. For example, in one embodiment the cancer antigen is an HPV-associated cancer antigen. In other embodiments, the neoantigen is a personalized neoantigen and requires the identification of suitable neoantigens in the individual tumour(s).
It will be clear to the skilled person that the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, complex and methods described herein are useful for targeting any cargo, for example a cargo protein or peptide, for example an antigenic peptide to any cell type, for example to immune cells, such as APC, for example DC. For example, the polypeptide, for example a cargo- targeting polypeptide or a cargo-delivery polypeptide of the invention, complex and methods described herein may be ultimately aimed at the treatment or prevention of cancer and may comprise cancer antigens or cancer neoantigens, as described herein. However, it will be clear that the present invention is also useful for the treatment or prevention of pathogenic infections, for example infection with a bacteria, fungus or virus. The skilled person will recognize that suitable antigens such as antigenic peptides from the pathogen should be tagged with the appropriate tag such as a peptide tag, to which the polypeptide of the invention binds. In this way, the necessary antigens are targeted to immune cells, such as APC, for example DC, initiating the appropriate immune response. Accordingly, in some embodiments the tagged cargo comprises or consists of a tagged antigen, and wherein the antigen is a peptide that comprises an antigenic amino acid sequence that is derived from a pathogen, for example wherein the pathogen is a bacteria, fungus or virus, for example wherein the virus is HPV.
The present invention also provides a method of forming the complex of the invention, wherein the complex is formed by contacting a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide according to the invention (i.e. a polypeptide capable of binding to a target antigen for example to an immune cell and capable of binding to a tagged cargo for example to a tagged antigen) with a tagged cargo (for example a tagged antigen) in vitro, wherein the tagged cargo (for example tagged antigen) comprises the corresponding tag (for example a coiled-coil oligomerisation domain) to which the tag binding domain of the polypeptide binds. Preferences for features of the method of forming the complex of the invention, for example in relation to the tagged cargo (for example tagged antigen) and to the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention are as described herein in relation to other aspects of the invention. For example, it is preferred if both the tag binding domain of the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and the tag that is used to tag the cargo are both complementary coiled-coil oligomerisation domains that are capable of interacting with one another and forming a multimer for example a dimer for example a heterodimer.
The method can also be used to prepare a complex that comprises more than one cargo for example more than one cargo antigen (for example more than one antigenic peptide) as will be apparent to the skilled person, for example by contacting the polypeptide of the invention with a number of tagged cargo (for example a number of tagged antigens). As discussed, the tagged cargo (for example tagged antigens, e.g. antigens tagged with a coiled-coil oligomerisation domain tag) may be tagged with the same tag, or with a different tag. The skilled person will understand that it is necessary that the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention comprises the appropriate corresponding tag binding domain(s) so that the polypeptide can bind to the tag(s).
Where the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is to be bound to a number of different tagged cargo, for example tagged antigens (whether the tags are the same or different), the contacting may be performed simultaneously, i.e. the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention can be contacted to a mixture of different tagged cargo, such as a mixture of different tagged peptide antigens; or the contacting can be performed sequentially, wherein the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention is contacted to a first tagged cargo (for example a first tagged antigen), and is subsequently contacted to a second tagged cargo, and a third and fourth as appropriate (.e.g. a second, third and fourth tagged antigen).
It will be clear to the skilled person that since the complex of the invention is considered to have therapeutic utility, the invention provides a pharmaceutical composition comprising a complex of the invention. Preferences for features of the pharmaceutical composition, for example in relation to the tagged antigen (for example peptide tagged antigen) and to the polypeptide of the invention are as described herein in relation to other aspects of the invention.
As will be apparent to the skilled person, the pharmaceutical composition will comprise the necessary excipients and carriers to ensure that the active agent, i.e. the complex, is maintained and supported and delivered in a functional state.
As discussed herein, in some situations it is considered advantageous if more than one cargo, for example more than one antigen (for example more than one peptide tagged antigen, for example peptide tagged neoantigen), is administered to a patient. As described above, this could be achieved by preparing a complex wherein the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention binds to a number of different tagged cargo, for example a number of different peptide antigens or neoantigens - i.e. each individual complex of polypeptide/antigen comprises more than one antigen. However, an alternative means of delivering multiple cargo proteins or peptides, for example delivering multiple antigens or neoantigens is to prepare a composition or pharmaceutical composition that comprises a number of different complexes of the invention, for example the pharmaceutical composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different complexes, wherein each complex is associated with a different cargo (for example different antigen or neoantigen), i.e. each individual complex only comprises one cargo (e.g. one antigen), but the pharmaceutical composition comprises a number of different complexes.
Accordingly, in one embodiment, the invention provides a pharmaceutical composition wherein the composition comprises more than one different complex according to the invention, and wherein the cargo , for example tagged antigen(s) of each complex are different, for example wherein the sequence of the tag (for example the coiled-coil oligomerisation domain) is the same and the sequence of the cargo protein or cargo peptide (for example antigen) is different; or wherein the sequence of the tag (for example the coiled-coil oligomerisation domains) is different and the sequence of the peptide is different.
The invention also provides a nucleic acid encoding the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention.
The invention also provides a nucleic acid encoding a tagged cargo protein or peptide of the invention.
The invention also provides a tagged cargo protein or peptide wherein the cargo protein or peptide comprises a first domain that is a coiled-coil oligomerisation domain and a second domain.
The invention provides a nucleic acid that encodes the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and which also encodes the tagged cargo protein or peptide of the invention.
The invention also provides a vector comprising any one or more the nucleic acids of the invention.
The invention provides a cell comprising any one or more of the nucleic acids of the invention or the vectors of the invention. Preferences for the nucleic acids, vectors and cells of the invention are as described herein in relation to other aspects of the invention.
It will be clear that the complexes of the invention have advantageous therapeutic properties. Accordingly, the invention provides a number of medical uses relating to the administration of the complex or pharmaceutical composition of the invention or nucleic acid of the invention or vectors of the invention or cells of the invention . Preferences for features of the medical uses, for example in relation to the tagged peptide and to the polypeptide of the invention are as described herein in relation to other aspects of the invention.
The invention provides a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in medicine.
The invention also provides a method of treatment or method of preventing a disease, wherein the method comprises the administration of a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or ceil of the invention.
The invention also provides the use of a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in a method of manufacture of a medicament for the treatment or prevention of disease.
Therapeutic agents of the present invention are intended to be pre-formed as complexes prior to administration, with the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention and the tagged cargo forming a complex with one another via the tag binding domain prior to administration. By preforming such complexes, it is considered to be more likely that the target antigen binding portion of the complex will direct the cargo (for example antigen) to the same target (for example immune cells). If the two (or more) components, wherein the first component is a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, i.e. comprising a target antigen binding domain (e.g. an immune cell specific domain) and a tag binding domain (for example a coiled-coil oligomerization domain) and the second component is the tagged cargo (for example the tagged antigen), were to be administered separately, they will traffic independently of each other and would need to essentially rely on chance events inside the body to bring the two in close proximity to allow complex formation For example, a tagged antigen that is not bound to a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention, i.e. with a domain specific to a target antigen (for example specific to an immune cell target), such as a DC target (e.g. CD40), may fail to traffic to the DC and thus not be processed and presented to generate an adaptive immune response. Conversely, such a polypeptide (e.g. anti-CD40 polypeptide) that is not bound to the tagged cargo (for example tagged antigen) provides no specific cargo to be processed and presented to generate an adaptive immune response, but may risk activating DC via CD40 against other epitopes they may present instead.
Thus, the present invention requires that the complexes are preformed prior to administration, to ensure the cargo (e.g. antigen or neoantigen) reaches the intended target for example target cells based on the target antigen binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention.
It will be clear to the skilled person that the complex and pharmaceutical compositions of the invention have a particular use in the treatment or prevention of cancer. In particular, where the cargo is a neoantigen, identified from a patient's tumour, the complex and pharmaceutical composition are particularly useful in the treatment of cancer.
Accordingly, the invention provides a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in a method of treating or preventing cancer, for example wherein the tagged cargo (for example a tagged antigen) comprises an antigenic sequence that has been identified as a neoantigen that has arisen in a tumour or cell; or a cancer antigen.
The invention provides a method of treating or preventing cancer, wherein the method comprises administering a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention. The invention also provides the use of a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention in the manufacture of a medicament for the treatment of cancer.
The polypeptide and complexes, nucleic acid of the invention, or vector of the invention or cell of the invention thereof may also be used as a therapeutic or prophylactic vaccine.
Accordingly the invention also provides a vaccine comprising any one or more of the polypeptide and complexes, nucleic acid of the invention, or vector of the invention or cell of the invention. In some embodiments the vaccination is a cancer vaccine. In some embodiments the vaccine is a vaccine against a contagious disease.
The invention also provides a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in a method of treating or preventing a pathogenic infection, for example for treating or preventing a bacterial, fungal or viral infection, wherein the tagged cargo comprises an antigenic peptide sequence derived from a bacteria, fungus or virus, for example from HPV.
The invention provides a method of treating or preventing a pathogenic infection, for example for treating or preventing a bacterial, fungal or viral infection, wherein the method comprises administering a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention wherein the cargo comprises an antigenic peptide sequence derived from a bacteria, fungus or virus, for example from HPV.
The invention also provides the use of a complex of the invention, or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention in the manufacture of a medicament for the treatment or prevention of a pathogenic infection, for example for treating or preventing a bacterial, fungal or viral infection, wherein the cargo comprises an antigenic peptide sequence derived from a bacteria, fungus or virus, for example from HPV.
The present invention lends itself to be used in methods of personalised therapy, particularly for the treatment of cancer. For example, a tissue sample from a tumour or other sample, for example a blood sample where the cancer is a blood cancer, or a sample of exosomes, can be analysed to identify neoantigens that have arisen specifically in the cancer cell or tissue, and not in other healthy tissues. Such analysis needs to be performed for each individual patient, producing a truly tailored therapy.
Once these neoantigens have been identified, tagged versions of the antigens can easily be produced using, for example, standard laboratory cloning and expression techniques to create a tagged antigen - for example an antigen tagged with a coiled- coil oligomerisation domain. Once the tagged antigens, for example coiled-coil oligomerisation domain tagged antigens have been produced, they can be contacted with the or a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, producing one or more complexes that can be administered to the subject. It will be clear that the actual polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention, for use in the treatment of multiple patients, can be universal. The actual target antigen binding domain (for example the immune cell binding domain) and tag binding domains (for example coiled-coil oligomerisation domains) do not need to vary. The only variable part of the system is the cargo, for example the cargo protein or peptide sequence, for example the neoantigen sequence.
Accordingly, the invention provides a complex of the invention or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in a method of personalised therapy, wherein a tumourspecific antigen such as a neoantigen or viral antigen have been identified as having arisen in a patient (i.e. is specific for the tumour, either by virtue of certain mutations occurring in the tumour DNA, or by virally transmitted antigens), and wherein the tagged cargo protein or peptide comprises the neoantigen.
In some embodiments the personalised therapy is for the treatment or prevention of cancer.
In some embodiments, the information regarding suitable neoantigens for use in the personalised treatment of a patient are already available. In other embodiments, the step of analysing the sample from the patient and obtaining the necessary information regarding suitable neoantigens is part of the method of the invention.
Accordingly, the invention provides a complex of the invention or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for use in a method of personalised therapy, for example for the personalised treatment of cancer, wherein the method of personalised therapy involves the initial step of identifying a neoantigen that has arisen in a patient.
The invention also provides a method of personalised therapy, wherein the method comprises administering a complex of the invention or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention for example for the personalised treatment of cancer. In some embodiments, the method involves the initial step of identifying a neoantigen that has arisen in a patient.
The invention provides the use of a complex of the invention or a pharmaceutical composition of the invention, nucleic acid of the invention, or vector of the invention or cell of the invention in the manufacture of a medicament for personalised therapy, for example for the personalised treatment of cancer. In some embodiments, the therapy involves the initial step of identifying a neoantigen that has arisen in a patient.
The invention also provides an in vitro method of producing a complex comprising a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargodelivery polypeptide of the invention and a tagged cargo for example a tagged cargo protein or peptide (for example a tagged antigen), wherein the tagged cargo (for example tagged antigen) comprises the corresponding tag (for example peptide tag) to which the tag binding domain of the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention binds, wherein the method comprises contacting the polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention with a tagged cargo (for example tagged antigen) in vitro or ex vivo. Preferences for the in vitro or ex vivo method of producing a complex of the invention, for example in relation to the tagged peptide and to the polypeptide of the invention, are as described herein in relation to other aspects of the invention.
The complex of the invention is considered useful for in vivo administration. However, the complex of the invention is also useful in an in vitro or ex vivo method of activating an immune cell, wherein the method comprises contacting the immune cell with a complex of the invention or a pharmaceutical composition of the invention, wherein the immune cell comprises the antigen to which the target antigen binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention binds. Accordingly, the invention provides an in vitro or ex vivo method of activating an immune cell, wherein the method comprises contacting the immune cell with a complex of the invention or a pharmaceutical composition of the invention, wherein the immune cell comprises the antigen to which the target antigen binding domain of the polypeptide, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention . Preferences for this in vitro or ex vivo method, such as the tagged peptide and the polypeptide of the invention are as described herein in relation to other aspects of the invention.
It will be clear to the skilled person that various embodiments of the invention lend themselves to being provided in kit form. For example, the invention provides a kit for the in vitro preparation of a complex according to the invention wherein the kit comprises one or more polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the inventions of the invention and one or more tagged cargo, for example one or more tagged antigens; and/or comprises one or more nucleic acids or vectors or cells according to the invention. Preferences for features of the kits of the invention are as described herein in relation to other aspects of the invention.
In some embodiments, the kit comprises a buffer suitable for the in vitro formation of the complex.
The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For example, the invention provides a complex that comprises a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention in RUBY™ format, that has 1 target antigen binding domain capable of binding to CD40 and one K3 coiled-coil oligomerisation domain peptide tag binding domain that can form a heterodimer with the E4 coiled-coil oligomerisation domain, and wherein the complex comprises a cargo that is a neoantigen that was identified in a patient, wherein the neoantigen is tagged with the E4 coiled-coil oligomerisation domain.
The invention also provides a complex formed by contacting a polypeptide of the invention, for example a cargo-targeting polypeptide or a cargo-delivery polypeptide of the invention that comprises a first and a second tag binding domain, wherein the first and second tag binding domain each binds to a different tag (i.e. a first and second tag), with a mixture of 2 tagged cargo proteins or peptides that are 2 tagged peptide neoantigens, wherein one peptide neoantigen has been tagged with the first tag and the other peptide neoantigen has been tagged with the second tag.
Figure Legends:
Figure 1. Human CD40 transgenic (hCD40tg) mice were given either anti-CD40- Fc(OVA) antibody or anti-CD40 antibody and the OVA peptide separately. Control mice received vehicle only. The treatments were given on two occasions, 7 days between. Seven days after the second treatment, inguinal lymph nodes were collected for flow cytometry analysis of viable CD45+ CD3+ CD8+ OVA-MHCI tetramer+ T cells. The graphs show frequency (±SEM) of OVA (SIINFEKL)-MHCI tetramer+ among CD8+ T cells.
Figure 2. Human CD40 transgenic (hCD40tg) mice were given OVA peptide-conjugated Fc-binding peptide (Z33) or free OVA peptide (SIINFEKL) in combination with anti- CD40 antibody, isotype control antibody, or Poly I:C adjuvant. Control mice received vehicle only. The treatments were given on two occasions, 7 days between. Seven days after the second treatment, inguinal lymph nodes were collected for flow cytometry analysis of viable CD45+ CD3+ CD8+ OVA-MHCI tetramer+ T cells. The graphs show frequency (±SEM) of OVA (SIINFEKL)-MHCI tetramer+ among CD8+ T cells.
Figure 3. Human CD40 transgenic (hCD40tg) mice were inoculated with EG7-OVA cells s.c. on the right flank on day 0, followed by s.c. treatment with either anti-CD40- Fc(OVA) antibody or vehicle control on the left flank on day 1 and 8. The graphs show the mean (±SEM) tumor volume.
Figure 4. Human CD40 transgenic (hCD40tg) mice were inoculated with EG7-OVA cells s.c. on the right flank on day 0, followed by s.c. treatment with anti-CD40-Fc(OVA) antibody, or anti-CD40 antibody and OVA peptide given separately, or vehicle control, on the left flank on day 1 and 8. The graphs show the mean (±SEM) tumor volume.
Figure 5. Binding of the different peptides to antibodies fused with coiled coil peptides, A) binding of K3 and K4 to monoclonal antibodies fused with E3, B) binding of HAP2 to monoclonal antibodies fused with HAP1, HAP2,HAP3, HAP5 and HAP6, C) binding of K4 to IgGl monoclonal antibodies fused with E3, E4, E5 and E6, D) binding of K4 to IgG2 monoclonal antibodies fused with E3, E4, E5 and E6, E) binding of K6 to IgGl monoclonal antibodies fused with E3, E4, E5 or E6, F) binding of K6 to IgG2 monoclonal antibodies fused with E3, E4, E5 or E6.
Figure 6. Representative results of binding kinetics of the different peptides to antibodies fused with coiled coil peptides, A) binding of K3 to a monoclonal antibody fused with E3, B) binding of K4 to monoclonal antibody fused with E3, C) binding of K4 to monoclonal antibody fused with E4, D) binding of K6 to monoclonal antibody fused with E3, E) binding of K6 to monoclonal antibody fused with E4. In A, biotinylated peptides were coupled to streptavidin sensors and assayed against antibody in solution whereas antibodies were coupled to FAB2G sensors and assayed against peptides in solution (B-E).
Figure 7. ELISA results for bispecific antibodies fused with different peptides simultaneously binding two antigens.
Figure 8. CD40 activation by A) monoclonal antibodies and B) bispecific antibodies fused with E3 and complexed with K4, CHO cells expressing Fc gamma receptor I (CHO- CD64), CHO cells expressing EpCAM (CHO-EpCAM) and CHO control cells were used.
Figure 9. Shows ELISA results for monoclonal antibodies fused with different peptides binding to antigen 1.
Figure 10. Binding kinetics of mAbs fused with coiled peptides against human Fey receptors.
Figure 11. Binding kinetics of mAbs fused with coiled peptides against mouse Fey receptors.
Figure 12. Sensorgrams illustrating binding of A_1132E4IgGl, A___1132E4IgGl + K6- Ova and mAb control (A_1132IgGl) without any peptide to human Fey receptors.
Figure 13. Sensorgrams illustrating binding of A_1132E4IgGl, A___1132E4IgGl + K6- Ova and mAb control (A__1132IgGl) without any peptide to mouse Fey receptors.
Figure 14. A (parts A-F) Sensorgrams illustrating binding kinetics of antibodies with coiled coils to human FcRn. A and D) mAbE4, B and E) mAbE4K46 and C and F) mAb control without any peptide. Dissociation was performed at pH 6.0 (A-C) and 7.4 (D- F). B (parts G-L) Sensorgrams illustrating binding kinetics of antibodies with coiled coils to mouse FcRn. G and J) mAbE4, H and K) mAbE4K46 and I and L) mAb control without any peptide. Dissociation was performed at pH 6.0 (G-I) and 7.4 (J-L).
Figure 15. Percentage of OVA-specific CD8+ T-ceiis in i LN of male hCD40tg. Figure 16. Temperature stability evaluation for all the constructs shown as differences in High Molecular Weight species (HMWs).
Figure 17. Precipitation curves for mAbs with coiled coil peptides and controls at different PEG concentrations.
Figure 18. ELISA results for monoclonal antibodies fused with different peptides binding after 7 days incubation in human serum or BSA.
Figure 19. Exemplary schematics showing the relative arrangements of the light and heavy chains, and coiled-coil oligomerisation domains of some potential formats. It will be clear that other arrangements are also possible.
Figure 20. FACS analysis of antibody-peptide complexes binding to cells. K4-Ova Biotin peptides were used in these experiments. Cells only A), Cells + 0.5pg K4 Ova-biotin B), Cells + 5pg 1132E4 only C), Cells+5ugll32E4+0.5 pg K4-Ova-biotin D), Cells+ lugll32E4+0.1 pg K4-Ova-biotin E) and Cells+0.2ugll32E4+0.02 pg K4-Ova- biotin F).
Figure 21. FACS analysis of antibody-peptide complexes binding to cells. K6-Ova Biotin peptides were used in these experiments. Cells only A), Cells + 0.5pg K6 Ova-biotin
B), Cells + 5pgll32E4 only C), Cells+5ugll32E4+0.5 pg K6-Ova-biotin D), Cells+ lugll32E4+0.1 pg K6-Ova-biotin E) and Cells+0.2ugll32E4+0.02 pg K6-Ova- biotin F).
Figure 22. FACS analysis of Antibody-peptide-bead complexes binding to cells. Antibody-peptide-bead complexes were prepared first before addition to cells. Unstained A), Cells+ beads-anti SA PE stained B), Cells+ beads-anti Fc APC stained
C), Cells-- beads+mAbpep-anti SA PE stained D), Cells+ beads+mAbpep-anti Fc APC stained E) and Cells+ beads+mAbpep-anti SA and anti Fc APC stained F),
Figure 23. Percentage of OVA-specific CD8+ T-cells in i LN of male hCD40tg
Figure 24. A - Tumor growth over time. B - Survival over time.
Figure 25. Results of binding kinetics of the different peptides to antibodies fused with coiled coil peptide E4, A) binding of K4-Ova to an IgGl monoclonal antibody fused with E4, B) binding of K4-Ova to an IgG2 monoclonal antibody fused with E4, C) binding of K4-Ova to an IgGl monoclonal antibody control without E4, D) binding of Ova-K4-1 to an IgGl monoclonal antibody fused with E4, E) binding of Ova-K4-1 to an IgG2 monoclonal antibody fused with E4, F) binding of Ova-K4-1 to an IgGl monoclonal antibody control without E4, G) binding of Ova-K4-2 to an IgGl monoclonal antibody fused with E4, H) binding of Ova-K4-2 to an IgG2 monoclonal antibody fused with E4, I) binding of Ova-K4-2 to an IgGl monoclonal antibody control without E4.
The invention is also defined by reference to the following numbered embodiment paragraphs:
1. A polypeptide comprising at least one immune cell binding domain and at least one tag binding domain, wherein the at least one immune cell binding domain is capable of specifically binding to an immune cell target; and wherein the at least one tag binding domain is capable of specifically binding to a peptide tag, wherein the peptide tag is not derived from tetanus toxin.
2. The polypeptide according to embodiment 1 wherein the polypeptide comprises more than one immune cell binding domain, optionally comprises 2, 3, or 4 immune cell binding domains.
3. The polypeptide according to any one of embodiments 1 or 2 wherein the polypeptide comprises more than one peptide tag binding domain, optionally comprises 2, 3, or 4 peptide tag binding domains.
4. The polypeptide according to embodiment 3 wherein each of the peptide tag binding domains binds to the same peptide tag.
5. The polypeptide according to embodiment 3 wherein one or more of the more than one peptide tag binding domains binds to a different peptide tag.
6. The polypeptide according to any one of embodiments 1-5 wherein the immune cell is an antigen presenting cell, such as a dendritic cell (DC), B cell and/or macrophage (preferably DC).
7. The polypeptide according to any one of embodiments 1-6 wherein the immune cell binding domain is an agonist of the immune cell target.
8. The polypeptide according to any one of embodiments 1-7 wherein the immune cell target is capable of mediating: activation of the immune cell; and/or internalisation of the polypeptide; and/or recruitment of conventional type I dendritic cells (cDCl).
9. The polypeptide according to any one of embodiments 1-8 wherein the immune cell binding domain binds to an immune cell receptor, optionally wherein the immune cell receptor is CD40, CLEC9A, DEC-205, XCR1 or TLR3.
10. The polypeptide according to embodiment 9 wherein the at least one immune cell binding domain binds to CD40. 11. The polypeptide according to any one of embodiments 1-10 wherein the immune cell binding domain is an antibody selected from: ADC-1013; clones 1132/1133, 1140/1135, 1150/1151 and 1107/1108 from WO 2015/091853; CP- 870,893, APX005M, ChiLob 7/4, SEA-CD40; wherein:
ADC-1013 comprises one or more sequences selected from SEQ ID NO: 41-48; 1132/1133 comprises one or more sequences selected from SEQ ID NO: 1-8; 1140/1135 comprises one or more sequences selected from SEQ ID NO: 11-18;
1150/1151 comprises one or more sequences selected from SEQ ID NO: 21-28;
1107/1108 comprises one or more sequences selected from SEQ ID NO: 31-38.
12. The polypeptide according to any one of embodiments 1-11 wherein the peptide tag binding domain is capable of binding to a FLAG tag (DYKDDDDK) or a peptide probe sequence, optionally wherein the peptide probe sequence is a coiled-coil peptide tag E3 comprising the amino acid sequence (EIAALEK)x3.
13. The polypeptide according to any one of embodiments 1-12 wherein the peptide tag is a non-human peptide.
14. The polypeptide according to any one of embodiments 1-13 wherein the immune cell binding domain is selected from the group consisting of: antibodies or antigen binding fragments thereof.
15. The polypeptide according to any one of embodiments 1-14 wherein the peptide tag binding domain is: a) selected from group consisting of: antibodies or antigen binding fragments thereof; and/or b) is not an Fc region.
16. The polypeptide according to any one of embodiments 14 or 15 wherein the antigen-binding fragment is selected from the group consisting of: an Fv fragment (such as a single chain Fv fragment, or a disulphide-bonded Fv fragment); a Fab-like fragment (such as a Fab fragment; a Fab' fragment; or a F(ab)2 fragment); and domain antibodies.
17. The polypeptide according to any one of embodiments 1-14 and 16 wherein the peptide tag binding domain is an Fc region of an antibody having the ability to specifically bind to the peptide tag.
18. The polypeptide according to any one of embodiments 1-17 wherein the polypeptide comprises at least 2 peptide tag binding domains, wherein at least one peptide tag binding domain is selected from the group consisting of: antibodies or antigen binding fragments thereof; and wherein at least one peptide tag binding domain is an Fc region of an antibody having the ability to specifically bind to the peptide tag. 19. The polypeptide according to any one of embodiments 1-18 wherein the polypeptide comprises: a) at least one immune cell binding domain that comprises or consists of an IgG antibody and at least one peptide tag binding domain that comprises or consists of an IgG antibody; b) at least one immune cell binding domain that comprises or consists of an IgG antibody and at least one peptide tag binding domain that comprises or consists of an Fv fragment; c) at least one immune cell binding domain that comprises or consists of an IgG antibody and at least one peptide tag binding domain that comprises or consists of a Fab-like fragment; d) at least one immune cell binding domain that comprises or consists of an IgG antibody and at least one peptide tag binding domain that comprises or consists of a domain antibody; e) at least one immune cell binding domain that comprises or consists of an IgG antibody and at least one peptide tag binding domain that comprises or consists of a coiled-coil peptide tag (such as E3 ((EIAALEK)x3)) that binds to a probe (such as K3 ((KIAALKE)x3) or K4 ((KIAALKE)x4), wherein the probe is connected to the neoantigen; f) at least one immune cell binding domain that comprises or consists of an Fv fragment and at least one peptide tag binding domain that comprises or consists of an IgG antibody; g) at least one immune cell binding domain that comprises or consists of a Fab- like fragment and at least one peptide tag binding domain that comprises or consists of an IgG antibody; h) at least one immune cell binding domain that comprises or consists of a domain antibody and at least one peptide tag binding domain that comprises or consists of an IgG antibody; i) at least one immune cell binding domain that comprises or consists of an IgG antibody and at least one peptide tag binding domain that comprises or consists of an Fc region of an antibody having the ability to specifically bind to the peptide tag; j) at least one immune cell binding domain that comprises or consists of an Fv fragment and at least one peptide tag binding domain that comprises or consists of an Fc region of an antibody having the ability to specifically bind to the peptide tag; k) at least one immune cell binding domain that comprises or consists of a Fab- like fragment and at least one peptide tag binding domain that comprises or consists of an Fc region of an antibody having the ability to specifically bind to the peptide tag; l) at least one immune cell binding domain that comprises or consists of a domain antibody and at least one peptide tag binding domain that comprises or consists of an Fc region of an antibody having the ability to specifically bind to the peptide tag; m) at least two immune cell binding domains that comprise or consist of an IgG antibody wherein the at least two dendritic cell binding domains bind to the same dendritic cell target, and at least one peptide tag binding domain that comprises or consists of an Fc region of an antibody having the ability to specifically bind to the peptide tag; n) at least two immune cell binding domains that comprise or consist of an IgG antibody wherein the at least two dendritic cell binding domains bind to the same dendritic cell target, and at least one peptide tag binding domain that comprises or consists of a coiled-coil peptide tag (such as E3 ((EIAALEK)x3)) that binds to a probe (such as K3 ((KIAALKE)x3) or K4 ((KIAALKE)x4), wherein the probe is connected to the neoantigen, optionally wherein the IgG antibody is an IgGl, IgG2, IgG3 or IgG4 antibody.
20. The polypeptide according any one of embodiments 1-19 wherein the polypeptide is a bispecific polypeptide, and optionally comprises or consists of a format selected from the group consisting of: a) IgG-scFv bispecific antibodies; b) monovalent bispecific antibodies; c) scFv2-Fc bispecific antibodies; d) BiTE/scFv2 bispecific antibodies; e) DVD-Ig bispecific antibodies; f) DART-based bispecific antibodies; g) DNL-Fab3 bispecific antibodies; and h) scFv-HSA-scFv bispecific antibodies i) RUBY™ format antibodies, wherein the antibody comprises:
(i) two copies of a first heavy chain polypeptide and two copies of a first light chain polypeptide, and
(ii) two Fab fragments, the Fab fragments comprising a second heavy chain polypeptide and a second light chain polypeptide and wherein the first Fab fragment is fused to the C-terminus of the first copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment; and the second Fab fragment is fused to the C-terminus of the second copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment, and wherein a) the two copies of a first heavy chain polypeptide and two copies of a first light chain polypeptide form two immune cell binding domains and the two Fab fragments form a first and second target binding domain; or b) the two copies of a first heavy chain polypeptide and two copies of a first light chain polypeptide form a first and a second target binding domain, and the two Fab fragments form two immune cell binding domains.
21. The polypeptide according to any one of embodiments 1-20 wherein the polypeptide comprises an Fc region or a variant of said region, optionally wherein the region is an IgGl, IgG2, IgG3 or IgG4 region, optionally IgGl or IgG2.
22. The polypeptide according to any of embodiments 1-21 wherein the polypeptide is a monospecific antibody directed towards the immune cell target, and wherein the Fc region has been modified to be capable of binding to the peptide tag; a bispecific antibody wherein one paratope comprises the immune cell binding domain and the second paratope comprises the peptide tag binding domain, and optionally wherein the Fc region of the antibody has been modified to be capable of binding to a peptide tag.
23. A complex comprising a polypeptide according to any one of embodiments 1- 22 and a tagged peptide antigen, wherein the tagged peptide antigen comprises the peptide tag to which the tag binding domain of the polypeptide binds.
24. The complex of embodiment 23 wherein the polypeptide comprises at least two peptide tag binding domains capable of binding to at least two different peptide tags, and wherein the complex comprises at least two tagged peptide antigens. 25. The complex according to any one of embodiments 23 or 24 wherein the tagged peptide antigen comprises an antigenic sequence that has been identified as a neoantigen that has arisen in a tumour or cell.
26. The complex according to any one of embodiments 23 and 24 wherein the tagged peptide antigen comprises an antigenic peptide sequence that is a cancer antigen, optionally wherein the cancer antigen is a Human Papillomavirus (HPV)- associated cancer antigen.
27. The complex according to any one of embodiments 23 or 24 wherein the tagged peptide antigen comprises an antigenic sequence that is derived from a pathogen, optionally wherein the pathogen is a bacteria, fungus or virus, optionally wherein the virus is Human Papillomavirus (HPV).
28. A complex formed by contacting a polypeptide according to any of embodiments 1-22 with a tagged peptide antigen in vitro, wherein the tagged peptide antigen comprises the peptide tag to which the tag binding domain of the polypeptide binds.
29. A pharmaceutical composition comprising a complex according to any of embodiments 23 and 28.
30. The pharmaceutical composition according to embodiment 29 wherein the composition comprises more than one different complex according to any of embodiments 23-28, and wherein the sequence of the tagged peptide antigen(s) of each complex are different, optionally wherein the sequence of the tag is the same and the sequence of the peptide antigen is different; or wherein the sequence of the tag is different and the sequence of the peptide is different.
31. A complex according to any one of embodiments 23-28 or a pharmaceutical composition according to any one of embodiments 29 or 30 for use in medicine.
32. A complex according to any one of embodiments 23-28 or a pharmaceutical composition according to any one of embodiments 29 or 30 for use in a method of treating or preventing cancer, optionally wherein the tagged peptide antigen comprises an antigenic sequence that has been identified as a neoantigen that has arisen in a tumour or cell; or a cancer antigen.
33. A complex according to any one of embodiments 23-28 or a pharmaceutical composition according to any one of embodiments 29 or 30 for use in a method of treating or preventing a pathogenic infection, optionally for treating or preventing a bacterial, fungal or viral infection, wherein the tagged peptide antigen comprises an antigenic sequence derived from a bacteria, fungus or virus, optionally from Human Papillomavirus (HPV).
34. A complex according to any one of embodiments 23-28 or a pharmaceutical composition according to any one of embodiments 29 or 30 for use in a method of personalised therapy, wherein a neoantigen has been identified as having arisen in a patient, and wherein the tagged antigenic peptide comprises the neoantigen.
35. The complex for use according to embodiment 34 wherein the personalised therapy is for the treatment or prevention of cancer.
36. The complex for use according to any of embodiments 34 or 35 wherein the method of personalised therapy involves the initial step of identifying a neoantigen that has arisen in a patient.
37. A method for the treatment or prevention of a disease wherein the method comprises administering a complex according to any one of embodiments 23-28 or a pharmaceutical composition according to any one of embodiments 29 or 30.
38. A method for the treatment or prevention of cancer, wherein the method comprises administering a complex according to any one of embodiments 23-28 or a pharmaceutical composition according to any one of embodiments 29 or 30, optionally wherein the tagged peptide antigen comprises an antigenic sequence that has been identified as a neoantigen that has arisen in a cell, optionally a tumour cell; or a cancer antigen.
39. A method for the treatment or prevention of a pathogenic infection, wherein the method comprises administering a complex according to any one of embodiments 23- 28 or a pharmaceutical composition according to any one of embodiments 29 or 30, optionally wherein the method is for the treatment of prevention of a bacterial, fungal or viral infection, wherein the tagged peptide antigen comprises an antigenic sequence derived from a bacteria, fungus or virus, optionally from Human Papillomavirus (HPV).
40. A method for the personalised treatment or prevention of a disease in a patient, wherein the method comprises administering a complex according to any one of embodiments 23-28 or a pharmaceutical composition according to any one of embodiments 29 or 30, optionally wherein the tagged peptide antigen comprises an antigenic sequence that has been identified as a neoantigen that has arisen in a cell, optionally a tumour cell; or a cancer antigen.
41. A method for personalised therapy, comprising administering a complex according to any of embodiments 23-28 or a pharmaceutical composition according to any of embodiments 29 or 30 to a patient, wherein a neoantigen has been identified as having arisen in the patient, and wherein the tagged antigenic peptide comprises the neoantigen.
42. The method according to embodiment 41 wherein the personalised therapy is for the treatment or prevention of cancer. 43. The method according to any one of embodiments 41 or 42 wherein the method of personalised therapy involves the initial step of identifying a neoantigen that has arisen in a patient.
44. A method of producing a complex comprising a polypeptide according to any one of embodiments 1-22 and a tagged peptide antigen, wherein the tagged peptide antigen comprises the peptide tag to which the tag binding domain of the polypeptide binds, wherein the method comprises contacting the polypeptide according to any of embodiments 1-22 with a tagged peptide antigen in vitro.
45. An in vitro or ex vivo method of activating an immune cell, wherein the method comprises contacting the immune cell with a complex according to any of embodiments 23-28 or a pharmaceutical composition according to any of embodiments 29 or 30, wherein the immune cell comprises the immune cell target to which the immune cell binding domain of the polypeptide binds.
46. A nucleic acid encoding the polypeptide according to any of embodiments 1-22.
47. A vector comprising the nucleic acid according to embodiment 46.
48. A cell comprising the nucleic acid according to embodiment 46 or the vector according to embodiment 47.
49. A kit for the in vitro preparation of a complex according to any of embodiments 23-28 wherein the kit comprises a polypeptide according to any of embodiments 1-22 and a tagged peptide antigen.
50. The kit according to embodiment 49 wherein the kit comprises a buffer suitable for the in vitro formation of the complex.
Features of the invention are as illustrated below in the following Examples.
Example 1 - Generation of coiled coil tagged antibodies
Design
Coiled coil peptide sequences were connected via a peptide linker to antibody sequences. The coiled coil peptides used in the studies were: E3, E4, E5, E6, K3, K4, K5, K6, Leucine zipper, HAP1, HAP2, HAP3, HAP4, HAP5, HAP6, HAP7, HAP8. Their sequences can be found in Table 1. The E3, E4, E5, E6, Leucine zipper, HAP1, HAP2, HAP3, HAP4, HAP5, HAP6, HAP7 and HAP8 coiled coil peptides were connected to the C-terminus of the antibody light chain. Both IgGl and IgG2 isotypes were tested. The antibody used in these studies was 1132, a CD40 agonistic antibody that has been shown to activate antigen presenting cells.
Manufacturability A control IgGl antibody (called 1188) and monoclonal antibodies coupled with coiled coil peptides were expressed using transient Expi293 HEK (Life technologies) cultures at different volumes ranging from 600 pL - 30 mL according to manufacturer's instructions. Purification of the antibodies from supernatants was made on protein A using the NGC system (BioRad) or Predictor MabSelectSure 50pl 96 well plates (GE Healthcare). Cells were transfected with two different vectors encoding separately for each of the two polypeptides chains (i.e. the immunoglobulin heavy chain and the light chain to which a coiled coil peptide was fused). Protein aggregation was measured with SE-HPLC in a 1260 Infinity II system (Agilent Technologies) using a TSK gel Super SW mAB HTP 4pm, 4.6x150mm column (TOSOH Bioscience) and 100 mM Sodium Phosphate, pH 6.8, 300mM NaCI as mobile phase at ambient temperature and a flow rate of 0.35 ml/min.
Results
All coiled coil antibody fusions could be expressed and purified. Expression yields from 30 mL HEK productions were, for the majority of the tested coiled coil antibody fusions, high and in the range observed for the monoclonal control (Table 2). The only exception was 1132HAP4 that produced at somewhat lower yield. The aggregation levels (measured as high molecular weight species (HMWS)) after protein A purification were for the majority of the constructs very low (<3%) (Table 2). A similar trend was observed for both IgGl and IgG2 antibodies. However, antibodies fused with longer peptides E5 and E6 or the leucin zipper variant were aggregated (aggregation levels 13-60%).
Figure imgf000103_0001
Figure imgf000104_0001
Table 1. Protein sequences for coiled coil peptide pairs tested
Figure imgf000104_0002
Figure imgf000105_0001
Table 2. Manufacturability and purity data of monoclonal antibodies fused with coiled coil
Example 2 - Coiled coil binding studies-ELISA
Material and methods
The aim of the study was to evaluate the oligomerization capacity between antibodies fused to coiled coil peptides and free coiled coil peptides using ELISA.
Plates were coated with 0.5 pg/mL antigen, rh CD40 Fc Chimera (1493-CDB, R&D Systems) in PBS over night at 4°C. After washing in PBS/0.05% Tween 20 (PBST), the plates were blocked with PBS/2% BSA for at least 30 minutes at room temperature before being washed again. Antibodies fused with peptides (IgGl-1132E3 (IgGlmAbE3), IgG2-1132E3 (IgG2mAbE3), IgGl-1132E3 (IgGlmAbE3), IgG2-1132E3 (IgG2mAbE3), IgGl-1132HAPl (IgGlmAbHAPl), IgGl-1132HAP2 (IgGlmAbHAP2), IgGl-1132HAP3 (IgGlmAbHAP3), IgGl-1132HAP5 (IgGlmAbHAP5), IgGl-1132HAP6 (IgGlmAbHAP6), IgGl-1132E4 (IgGlmAbE4), IgG2-1132E4 (IgG2mAbE4), IgGl- 1132E5 (IgGlmAbE5), IgG2-1132E5 (IgG2mAbE5), IgGl-1132E6 (IgGlmAbE6), IgG2-1132E6 (IgG2mAbE6)) or naked antibodies used as control (IgGl-1132 (IgGlmAb), IgG2-1132 (IgG2mAb)) serially diluted in PBS/0.5% BSA were then added and allowed to bind for at least 1 hour at room temperature. After washing, plates were incubated with 0.5 pg/mL of one of the following biotinylated coiled coil peptides K3, K4, K5, K6 or HAP2 for at least 1 hour at room temperature. Plates were washed and the complexed antibodies with CD40 and biotinylated peptide were detected with HRP-labelled streptavidin, SuperSignal Pico Luminescent was used as substrate and luminescence signals were measured using Fluostar Optima.
Results
K4 showed good binding to antibodies coupled with peptides E3 (Figure 5A) whereas no binding was observed for K3 or HAP2 to antibodies fused to E3 (Figure 5A) or HAP1, 2, 3, 5 or 6 respectively (Figure 5B). K4 displayed binding to antibodies fused with peptides E4, E5 and E6 both for IgGl and IgG2 subclasses (Figure 5C and D) although at lower degree compared to antibodies fused to E3. K6 displayed good binding to antibodies fused with peptides E3, E4, E5 and E6 both for IgGl and IgG2 subclasses (Figure 5E and F). In none of the analyses did the control antibody (1332 without coiled coil peptide) bind to the free coiled coils demonstrating that the free coiled coil tested do not bind to naked antibodies or either IgGl or IgG2 isotype.
Example 3 - Coiled coil binding studies, Octet
Material and methods
Kinetic measurements were performed using the Octet RED96 platform (ForteBIo). Streptavidin or FAB2G (Anti-Fab 2nd generation) sensors were used. Streptavidin sensors were coated with 0.25 pg/ml (of K3 or HAP2) or 0.5pg/ml (of K4 or K6) peptides. Peptide coated streptavidin sensors were assayed against antibodies serially diluted 1/z in lx Kinetic buffer. (The tested antibodies were (IgGl-1132E3 (IgGlmAbE3), IgG2-1132E3 (IgG2mAbE3), IgGl-1132E3 (IgGlmAbE3), IgG2-1132E3 (IgG2mAbE3), IgGl-1132E4 (IgGlmAbE4), IgG2-1132E4 (IgG2mAbE4), IgGl- 1132HAP3 (IgGlmAbHAP3), IgGl-1132HAP6 (IgGlmAbHAP6), IgGl-1132HAP7 (IgGlmAbHAP7)). The association was followed for 300 seconds and the dissociation in lx Kinetic buffer for 300 seconds. Sensor tips were regenerated using 10 mM glycine, pH 1.7. Alternatively, antibodies fused with coiled coil peptides (IgGl-1132E4 (IgGlmAbE4) or IgGl-1132E6 (IgGlmAbE6)) were loaded to FAB2G (Anti-Fab 2nd generation) sensors and assayed against K4 or K6 in solution. Antibodies were diluted at 400nM in lx Kinetic buffer and captured on 8 parallel sensors for 300seconds. After setting a new baseline, the captured antibodies were assayed against K4 or K6 for 120 seconds followed by dissociation for 300 seconds in Kinetic buffer. The peptides K4 or K6 were diluted 1/2 in lx Kinetic buffer starting at 0.3 pM. Sensor regeneration using lOmM Glycine pH 1.7 was performed before capture of the next antibody. Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, inter-step correlation by alignment against dissociation was performed and the data were smoothed by a Savitzky-Golay filter in the data analysis software (v.9.0.0.14). The processed data were fitted using a 1: 1 Langmuir binding model with X2 as a measurement of fitting accuracy.
Results
Of the four peptides studied, K3, K4 and K6 showed good binding to antibodies coupled with peptides E3 or E4. No binding was observed for HAP2 at all. A faster dissociation observed for K3 (Figure 6A) was improved by increasing the length of the peptide to K4 and K6 (
Figure imgf000107_0001
Table 3, Figure 6B, C, D and E) as well as increasing the length of the peptide fused to the antibody to E4 (Figure 6C and E). Overall, KD values at low nanomolar or below were measured for the interaction between K4 or K6 against antibodies fused with E3 or E4 (Table 4). A similar affinity trend was observed for both IgGl and IgG2 (
Figure imgf000108_0001
Table 3).
Figure imgf000108_0002
Table 3 Binding kinetics of K4 and K6 in coated on SA sensors
Figure imgf000109_0001
Table 4. Binding kinetics of K4 and K6 in solution (antibodies coupled to FAB2G sensors)
Example 4-Bispecific antibodies
Material and methods
Design
Coiled coils were also fused to bispecific antibodies to evaluate compatibility with other antibody modalities than IgG. E3, Leucin zipper, HAP1, HAP2, HAP3, HAP4, HAP5, HAP6, HAP7 and HAP8 coiled coil peptides were fused connected via a peptide linker to the C-terminal IgG light chain of a bispecific antibody called 1132-3174 consisting of a CD40 antibody 1132 and an EpCAM antibody 3174 in the RUBY format, RUBY™ bsAb are of the Appended IgG class of bsAb formats with FAb domains linked through their light chains to the C -terminal end of IgG molecules.
Manufacturability
The bispecific antibody 1132-3174 and the 1132-3174 variants connected with coiled coil peptides were expressed using transient Expi293 HEK (Life technologies) cells in 30 mL according to manufacturer's instructions. Purification of the antibodies from supernatants was made on protein A using the NGC system (BioRad). Cells were transfected with three different vectors encoding separately for each of the three polypeptides chains (LI, Hl and H2). Aggregation was measured as presence of HMWS with SEC-HPLC in a 1260 Infinity II system (Agilent Technologies) using a TSK gel Super SW mAB HTP 4pm, 4,6x150mm column (TOSOH Bioscience) and 100 mM Sodium Phosphate, pH 6.8, 300mM NaCI as mobile phase at ambient temperature and a flow rate of 0.35 ml/min.
Dual ELISA Plates were coated with 0.5 pg/mL CD40 (recombinant human CD40 Fc Chimera (1493- CDB, R&D Systems)) in PBS over night at 4°C. After washing in PBS/0.05% Tween 20 (PBST), the plates were blocked with PBS/2% BSA for at least 30 minutes at room temperature before being washed again. Samples serially diluted in PBS/0.5% BSA were then added and allowed to bind for at least 1 hour at room temperature. After washing, plates were incubated with 0.5 pg/mL recombinant human EpCAM Fc-Biotin (10694-H02H-B, Sino Biological) for at least 1 hour at room temperature. Dual complexed bsAb with CD40 and EpCAM were detected with HRP-labelled streptavidin. SuperSignal Pico Luminescent was used as substrate and luminescence signals were measured using Fl uostar Optima. H
Octet measurements
Kinetic measurements were performed using the Octet RED96 platform (ForteBio). Biotinylated antigens were coupled to streptavidin sensors (ForteBio) at a concentration of 0.25 pg/mL for mouse Ig human CD40-Biotin (502-030, Ancell) or 0.5 pg/mL for recombinant human EpCAM Fc-Biotin (10694-H02H-B Sino Biological). Antibodies were serially diluted 1/z in lx Kinetic buffer (ForteBio) with start concentrations of lOnM or 25 nM and analysed for binding to antigen-coupled sensors. Association was followed for 300 seconds followed by dissociation in lx Kinetic buffer for 300 seconds. Sensor tips were regenerated using 10 mM glycine, pH 1.7. Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, inter-step correlation by alignment against dissociation was performed and the data were smoothed by a Savitzky-Golay filter in the data analysis software (v.9.0.0.14), The processed data were fitted using a 1: 1 Langmuir binding model with X2 as a measurement of fitting accuracy.
Results
Expression and protein quality after transient expression in 30mL HEK cultures for bispecific antibodies fused to different peptides were similar to the expression control, 1188, a monoclonal antibody, except for bsAbHAP4 for which a monomeric fraction below 90% (Table 5), was indicated as measured main peak area by SE-HPLC. Similar to what was displayed by monoclonal antibodies fused with various coiled coil peptides, fusing these peptides to bispecific antibodies seems not to interfere with their manufacturability and stability.
Figure imgf000110_0001
Figure imgf000111_0001
Table 5. Manufacturability data of bispecific antibodies fused with coiled coil peptides
All bispecific antibodies displayed good simultaneous binding to their two target antigens (CD40 and EpCAM) (7A-C). Compared to the bispecific control that is not connected to any coiled coils peptide no difference in binding is observed for the majority of coiled coil (E3, leucin zipper, HAP1, HAP2, HAP7 and HAP8) fused bsAbs, as shown from the ELISA results, indicating that the presence of these coiled coils do not interfere with the bispecific antibodies' target binding capacity. Similar to ELISA, octet studies showed that the target binding for bsAbs fused with coiled coils peptides differed by around or less than 10-fold compared to the target binding of the bispecific antibody control against the same two antigens, as shown in Table 6 and Table 7.
Figure imgf000111_0002
Table 6. Binding kinetics of bsAb fused with coiled peptides against CD40
Figure imgf000112_0002
Figure imgf000112_0001
Table 7. Binding kinetics of bsAb fused with coiled peptides against EpCAM
Example 5 - In silico immunogenicity predictions
Material and methods
In silico immunogenicity predictions were performed by submitting the amino acid sequences to the AbEpiAnalyser tool (EIR Sciences). For each peptide, a sequence coding of a linker S(G4S)x2 before the coiled coil peptide coding sequence were used for in silico analysis. Each of the peptides was used as a self reference to analyze the other peptides (e.g E3 was used as self peptide to analyze the rest of the peptides). Briefly, for each 15-mer peptide in the submitted sequence, a percentile rank is generated by comparing the predicted affinity against the affinity of a large set of 15- mers randomly selected. The rank threshold is preferred over affinity as different alleles show biases towards high or low binding affinity. Peptides with a percentile rank < 10 were included in the binding predictions, as recommended by IEDB. AbEpiAnalyser analyzes the potential immunogenicity of antibodies and sequence-modified proteins by first predicting the binding of each 15-mer peptide using the NetMHCII3. 1 algorithm to a large panel of human MHC class II alleles and subsequently calculates a positionspecific risk score. The score reflects the frequency of the HLA-DR, HLA-DP and HLA- DQ binding sub-peptides overlapping a given position in the defined population. The position-specific risk scores are summed over each sequence. A cutoff value of 0.1 is set for the for position-specific risk scores (i.e. all position-specific risk scores < 0.1 are set to 0). By subtracting the HLA binding properties of the germline or self protein, potential T-cell neo-epitopes are identified. In this analysis, the world average population of HLA alleles has been used, where the North American population has a weight of 52.7%, European 25.3%, North East Asian 9.2%, South East Asian 5.9%, South Central American 1.7%, Oceanian 1.3%, Western Asian 1.3%, Sub-Saharan African 1.0%, South Asian 0.7%, North African 0.6%, and Australian 0.1%. Results are presented as position-specific risk scores.
Results
Risk score between zero and almost 14 were obtained for the different coiled coil antibody fusions (Table 8). HAP1 and HAP4 displayed the highest immunogenicity risk scores. The risk scores for the E peptides were zero indicating that these coiled coils are particularly beneficial for fusion to antibodies from an immunogenicity perspective.
In all, the studies demonstrate that many of the evaluated coiled coils do not carry peptides predicted to have high risk of inducing immunogenic responses and should therefore be suitable candidates for fusion to antibodies.
Table 8. Summary of risk scores obtained for the different peptides
Figure imgf000113_0001
Example 6 - Retained CD40 functionality and binding
Material and methods ill CD40 functionality assay
Retained cell binding was evaluated using a CD40 bioassay (Promega). Briefly, CD40 Effector Cells were incubated overnight at 37°C. Serially diluted mono or bispecific antibody coiled coil fusion samples (1132E3 (mAbE3) and (1132-3174E3 (bsAbE3) respectively) complexed to soluble coiled coil counterparts (K4) were added followed by addition of CHO cells expressing Fc gamma receptor 1 (Fcyl (CD64)) or human EpCAM (for bsAb antibodies) or CHO-control cells. Plates were incubated at 37°C for 6 hours. Bio-Gio™ Reagent was added and plates incubated at room temperature for 5- 15 minutes. This was followed by measuring luminescence signals using Fluostar Optima. The samples tested consisted of 1132E3 IgGl (mAbE3) or 1132-3174E3 (bsAbE3) complexed with soluble K4 peptide, mAbE3 and bsAbE3 in the absence of soluble K4 peptide and monoclonal and bispecific antibody controls consisting of 1132 mAb or 1132-3174 bsAb that were not fused to any coiled coil peptides. To allow formation of mAbE3/K4 complexes, mAbE3 diluted to 12pg/ml (80nM) was mixed with K4 diluted to 0.64pg/ml (160nM) to a final concentration of mAbE3/K4 40nM to 80nM. The complexes were incubated for 1 hour before serial dilution and addition to cells. mAbE3 and mAb control were diluted to a start concentration of 6pg/ml (40nM). Similarly, to allow formation of bsAbE3/K4 complexes, bsAbE3 diluted to 24pg/ml, (96nM) was mixed with K4 diluted to 0.77pg/ml (192nM) to a final concentration of bsAb48nM/K4 96nM. The complexes were incubated for 1 hr before serial dilution and addition to cells. bsAbE3 and bsAb control were diluted to a start concentration of 12pg/ml (48nM).
CD40 mono ELISA
Plates were coated with 0.5 pg/mL of CD40 in PBS over night at 4°C. After washing in PBS/0.05% Tween 20 (PBST), the plates were blocked with PBS/2% BSA for at least 30 minutes at room temperature before being washed again. Antibodies fused to coiled coil peptides (1132E3 (mAbE3), 1132Leucin zipper (mAbLeucine Zipper), 1132HAP1 (mAbHAPl), 1132HAP3 (IgGlmAbHAP3), 1132HAP4 (ImAbHAP4), 1132HAP5 (mAbHAP5), 1132HAP6 (mAbHAP6), 1132HAP7 (mAbHAP7), 1132HAP8 (mAbHAP8)) or naked antibodies used as control (1132 (mAb)) serially diluted in PBS/0.5% BSA were then added and allowed to bind for at least 1 hour at room temperature. After washing, plates were incubated with a secondary anti kappa antibody for at least 1 hour at room temperature. After washing, SuperSignal Pico Luminescent was used as substrate and luminescence signals were measured using Fluostar Optima.
Results In the presence of CHO cells expressing Fcyl (CD64) or EpCAM, the anti-CD40 monoclonal or bispecific antibodies (connected or not to the coiled coil peptide E3 and regardless of the addition of soluble coiled coil peptide K4) induce CD40 pathway- activated luminescence, which can be detected in a dose-dependent manner by addition of Bio-Gio™ Reagent and quantitation with a luminometer. Both monoclonal (8A) and bispecific antibodies (8B) coupled with coiled coil peptides activated CD40 effector cells. This indicates that neither fusion of coiled coil peptides (E3 in this case) nor complex formation with the soluble coiled coil peptide counterpart (K4) affect the functionality of the antibodies.
ELISA binding studies showed good binding for both mAbs (9A-C) and bsAbs (Figure 9D-F) coupled with coiled coils peptides to their antibody target, CD40. This indicates the fusion of coiled coil peptides to the antibodies does not interfere with the antibody target binding capacity.
Example 8-Binding to Fc gamma receptors
Material and methods
Kinetic measurements were performed using the Octet RED96 platform (ForteBio). Antibody samples of antibody fused with coiled coil peptide E4 (A_1132E4IgGl) and complexed with coiled coil peptide K6 connected to a peptide antigen of ovalbumin (A___1132E4IgGl + K6-Ova) or control antibody not connected or bound to any coiled coil peptide (A_1132IgGl) were diluted to 200nM in lx Kinetic buffer and captured on 8 parallel FAB2G (Anti-Fab 2nd generation) sensors for 300sec. To generate the A_1132E4IgGl + K6-Ova complex, the coiled coil fused antibody (A__1132E4IgGl) was diluted to 400nM, mixed with K6-Ova diluted at 800nM to obtain a complex at a final concentration of 200nM antibody and K6-Ova 400nM. After setting a new baseline, the captured antibodies were assayed against human or mouse Fc gamma receptors for 60 seconds followed by dissociation for 60 seconds. Fc gamma receptors were serially diluted 1/z in seven dilutions in lx Kinetic buffer (ForteBio) starting at 0.1 pM. Sensor regeneration using lOmM Glycine pH 1.7 was performed before capture of the next antibody. Data generated was referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, inter-step correlation by alignment against dissociation was performed and the data was smoothed by a Savitzky-Golay filter in the data analysis software (v.9.0.0.14). The processed data was fitted using a 1: 1 Langmuir binding model. For the receptors with a high off rate, only the initial 5-10 seconds were used for curve fitting and data processing.
Results Interaction between human and mouse Fc gamma receptors and mAbs with coiled peptides was assessed by Octet. From the kinetic measurements obtained (Figure 10, Figure 11, Figure 12 and Figure 13) a similar Fey receptor interaction for mAbs with coiled peptides and mAb control without coiled coil peptide was observed. As expected, high affinity binding to human FcyRI was observed for the molecules. In addition, low binding to hFcGRIIa(H167), hFcGRIIa(R167), hFcGRIIbc, hFcGRIIIAF176 and hFcGRIIIAV176 was observed. No binding to hFcGRIIbc was detected. For the mouse Fc gamma receptors, good binding to mFcgRI and mFcGRIV and no binding to mFcGRIIB and mFcGRIII was observed. Overall, these results are an indication that fusing coiled coil peptides to the antibodies has minimal interference with FcgR interactions.
Example 9-Binding to FcRn
Material and methods
Kinetic measurements were performed using the Octet RED96 platform (ForteBio). Antibody fused with coiled coil peptide E4 (A__1132E4IgGl) and complexed with coiled coil peptide K6- connected to a peptide antigen of ovalbumin (A_1132E4IgGl + K6- Ova) or control antibody not connected or bound to any coiled coil peptide (A___1132IgGl) were used. Antibody samples were diluted to 2 ug/ml (13.5 nM) in 1 x kinetic buffer and captured on 8 parallel FAB2G (Anti-Fab 2nd generation, PALL ForteBio) sensors for 300 seconds. After setting a new baseline using sample buffer (100 mM Sodium phophatase, 150 mM NaCI, 0.05 % Tween 20 pH 6), the captured antibodies were assayed against serially diluted (1/z in seven dilutions starting at 1.6pm) human FcRn (ITF02-200, Immunitrack) or mouse FcRn (ITF08-200, Immunitrack) for 60 seconds. This was followed by dissociation in sample buffer at pH 6.0 or 7.4 for 60 seconds. Sensor regeneration using lOmM Glycine pH 1.7 was performed before capture of the next antibody. To create a 50/50 1132E4+K6-Ova mixture of 27 nM, A__1132E4 was mixed with 54nM K6-Ova to obtain a complex at final concentration of 13,5 nM A_1132E4, 27nM K6-Ova. Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, interstep correlation by alignment against dissociation was performed and the data were smoothed by a Savitzky-Golay filter in the data analysis software (v.9.0.0.14). The processed data were fitted using a 1 : 1 Langmuir binding model. For pH 7.4 where a high off rate was obtained, only the initial 5-10 seconds were used for curve fitting and data processing.
Results The binding interaction of antibodies connected to coiled coil peptides against FcRn was analyzed. Since it is important for antibodies to bind FcRn at a slightly acidic pH (about pH 6.0) and be recycled to the cell surface where they are released at the neutral pH (about 7,4) of blood, the analyses were performed with dissociation buffers at either pH 6.0 or 7.4. Binding kinetic measurements for A_1132E4-IgGl (i.e. the antibody connected to the coiled coil peptide E4 but with no added coiled coil tag), A___1132E4- IgGl + K6-Ova (i.e. the antibody connected to the coiled coil peptide E4 and after addition of the coiled coil tag K6) and the naked antibody control A___1132-IgGl against both human and mouse FcRn are shown in Table 9 and corresponding sensograms are shown in Figure 14. All antibodies regardless of the presence of coiled coil peptides displayed binding kinetic values as expected against both human and mouse FcRn, No difference in FcRn binding properties were observed for the antibodies that are connected to coiled coil peptides E4 and the antibody that is not, indicating that connecting coiled coil E4 does not impact the FcRn binding capacity to antibodies. Furthermore, addition of the neoantigen model antigen ovalbumin peptide (Ova) tagged to coiled coil peptide K6 did not appear to interfere with FcRn binding either, as demonstrated by similar KD values and binding curves for samples with and without addition of K6-Ova. hFcRn dissociation pH 6.0
Figure imgf000117_0001
hFcRn dissociation pH 7.4
Figure imgf000117_0002
mFcRn dissociation pH 6.0
Figure imgf000117_0003
mFcRn dissociation pH 7.4
Figure imgf000117_0004
Figure imgf000118_0001
Table 9. Binding kinetics of mAbs fused with coiled peptides and controls against human and mouse FcRn at different pH
Example 10-In vivo data and stability
Material and methods
Material and methods
The ability of the CD40 agonistic antibody 1132E4 in combination with four different peptides, carrying the peptide antigen sequence for ovalbumin SIINFEKL as is or connected to coiled coil peptides K4 or K6 or the peptide Z33 (which binds to the Fc region of human IgGl antibodies), to induce OVA-specific CD8+ T cell expansion in comparison to the same OVA-peptides used as monotherapies was studied in vivo. On day 0 and day 7, human CD40 transgenic mice were subcutaneously treated with either vehicle (PBS) or molar equivalent amounts of the peptides (14.1, 19.3, 18.9 and 3.3 pg of K4-Ova, K6-Ova, Z33-Ova and ovalbumin peptide control (SIINFEKL), respectively), alone or in combination with 33 pg of 1132E4. Seven days after the second treatment (D14), all mice were sacrificed and their inguinal lymph nodes closest to the treatment site (iLN ) were removed. The frequency of OVA-specific CD8+ T cells out of total CD45+ immune cells in the iLN was assessed using flow cytometry.
Results
Treatment of huCD40tg mice with 1132E4 plus K4-Ova peptide resulted in an expansion of OVA-specific CD8+ T cells that was significantly increased compared to treatment with 1132E4 plus Z33-Ova peptide, 1132E4 plus SIINFEKL peptide, or treatment with K4-Ova peptide alone (Figure 15). The superior ability of K4-Ova to induce OVA-specific T cell expansion was dependent on combination with 1132E4, since the K4-Ova peptide alone did not induce increased expansion of OVA-specific T cells compared to other OVA peptides. Similar results were seen with K6-Ova peptide in combination with 1132E4, which induced a statistically significant increase in OVA- specific T cell expansion compared to K6-Ova peptide alone and 1132E4 plus SIINFEKL peptide, but not 1132E4 plus Z33-Ova peptide.
Stability
Materials and methods
Storage stability Storage stability evaluation was performed by freeze-thawing or incubating the samples, of monoclonal antibodies connected to coiled coil peptides (A__1132IgGl, A_1132IgG2, A_1132E4IgGl, A___1132E4IgG2) or control antibody not connected to a coiled coil peptide (A_1132), at room temperature (RT) or 40°C for up to 2 weeks. Freeze-thawing was performed by incubating proteins (the same proteins as listed above) at -80°C and thawing them in a water bath set at 37°C after 24, 48 and 72 hours. SEC-HPLC analysis was performed only on samples thawed after 24 and 72 hours. For proteins incubated at 40°C, SEC-HPLC analysis was performed for samples at weeks 1 and 2. In addition, all samples were visually inspected for any particles at each of the three-time points.
Colloidal stability
Samples, of monoclonal antibodies connected to coiled coil peptides (A_1132IgGl, A__1132IgG2, A_1132E4IgGl, A_1132E4IgG2) or control antibody not connected to a coiled coil peptide (A__1132) as well as an assay control, were mixed with polyethylene glycol (PEG) solutions with different PEG concentrations ranging from 8%-36%. Samples were added to 96-well filter plates in duplicates and incubated over night at room temperature. Filtrates obtained by centrifugation at 12000 g for 15 minutes were collected and spun down before the absorbance at 280 nm was measured (to determine loss of protein) and compared to measurements taking before incubation with PEG and to the controls.
Serum stability
To further assess their binding stability, serum stability was performed. Protein samples, of monoclonal antibodies connected to coiled coil peptides (A_1132IgGl, A__1132IgG2, A__1132E4IgGl, A___1132E4IgG2) or control antibody not connected to a coiled coil peptide (A__1132), were incubated in BSA (PBS + 0.1% BSA) or 50% human serum at 37°C for 7 days. Mono ELISA was performed after seven days to assess binding.
Results
Very minimal degradation was displayed by ail the antibodies incubated at room temperature and 40°C (Figure 16A). Similarly, proteins displayed negligible degradation after freeze-thawing (Figure 16B).
Protein solubility can be determined by exposing protein samples to different concentrations of PEG. Addition of PEG to the protein solution leads to precipitation as a result of exclusion volume effects. PEG being a long-chain polymer occupies more space in the solution than a protein of a similar molecular mass and this lowers protein solubility resulting into protein precipitation. The antibodies with coiled coil peptides showed good colloidal stability comparable to the antibody control, not connected to a coiled coil peptide, and performed better than the assay control which as expected had about 50% of the protein precipitated at PEG concentration of approximately 8-9 % (Figure 17).
The serum stability assay showed no loss in binding after seven days incubation in human serum, compared to BSA for any the antibodies connected to coiled coil peptides or the control antibody without coiled coil peptide (Figure 18)
In all, the stability studies indicated that the stability of antibodies connected to coiled coil peptides E3 and E4 is high and very similar to the stability of the naked antibody not connected to a coiled coil peptide. It was thus concluded that coiled coil peptide E3 and E4 do not affect the stability of antibodies in a negative way.
Exampie 11 Antibody-peptide complex formation and cell binding
Assessing the formation of complexes between I) CD40 binding antibody 1132E4, which is connected to a coiled coil pair E4, and ii) K4-Ova-biotin or K6-Ova-biotin, which are coiled coil partners to E4 connected to the human ovalbumin immunopeptide (SIINFEKL) and labelled N-terminally with biotin. Complex cell binding was also evaluated.
Material and methods
Molar equivalents of biotinylated K4 Ova or K6-Ovapeptides and 1132E4 were used. K4-Ova-biotin or K6-Ova-biotin were diluted to lpg/ml from which 1/5 serial dilutions were prepared. In addition, 1132E4 was diluted to lOpg/ml from which 1/5 serial dilutions were prepared. K4-Ova-biotin or K6-Ova biotin were mixed with 1132E4 on a 1 : 1 ratio to obtain complex concentrations ranging from 5ug/0.5ug to 0,00032 /0, 000032 1132E4/K4-Ova-Biotin or 1132E4/K6-Ova-Biotin. To facilitate complex formation, incubation was performed for Ihour at room temperature with gentle rotation.
B16-F10 CD40 expressing cells and B16-F10 wt cells were used. Cells were seeded at 300000 cells/well in 25pl. The cells were incubated with 25pl antibody-peptide complexes. Cells without any treatment, cells treated with only the peptide (K4-Ova- biotin or K6-Ova-biotin) and cells treated with only the antibody were used as controls. Incubation was performed for 30 minutes at 4°C. Staining was performed using PE Streptavidin (BD Pharmingen #554061) followed by FACS analysis. Results
Staining was observed for both CD40 expressing and wt cells incubated with K4-Ova- Biotin or K6-Ova-Biotin peptides alone (Figure 20 and 21 B). When the peptides were complexed with the antibody, no binding was observed to the B16 wt cells as indicated in lower rows, Figures 20 and 21 D-F. In the CD40 expressing cells, staining was observed where cells were incubated with peptide complexes (Figure 20 and 21 D-F). The results indicate that the antibody peptide complexes were formed and they bound specifically to CD40 expressing cells.
Example 12 -Neoantigen carrying beads
Evaluation of the use of the coiled coil technology for complex formation of antibodies to neoantigen carrying beads.
Material and methods
Antibody-peptide-beads complexes were prepared by mixing equimolar concentrations of 1132E4IgG2 and K6-Ova-biotin at a ratio of 1 : 1 to obtain a complex at 5ug/0.5ug antibody/K6-peptide complex. This was followed by addition of Streptavidin MicroBeads. To obtain an antibody-peptide-bead mixture, a 12pL beads/500ul antibody-peptide mixture was prepared. An antibody-peptide mixture without beads was prepared as control.
Two 1:4 serial dilutions of the antibody-peptide-beads complexes and control mixtures were prepared. The solutions were incubated at room temperature with gentle rotation to allow complex formation. B16-CD40 or B16-wt cells were prepared at 8x10^6 cells/ml for each cell line. Additional controls were prepared at 12 L beads/8xl0^6 B16-CD40 or B16-wt cells. Finally, 300 000 cells/well were plated followed by addition of the antibody K6-peptide-beads complexes or controls and incubation at 4°C for 30 minutes. Washing with FACS buffer was performed before staining with two secondary antibodies PE anti-Streptavidin (# 410503, Biolegend) and anti-human IgG Fcg-PE (# 109-605-098, Jackson immuno Research) followed by incubation at 4°C for 30 minutes. Washing with FACS buffer was performed and cells fixed in 200 pL CellFix followed by FACS analysis.
Results
Dual staining with anti-streptavidin and anti-Fc detection reagents were observed in mixtures containing streptavidin beads, biotinylated coiled coil peptides (K6-Ova- biotin), CD40 antibody carrying E4 (a coiled coil partner to K6) and CD40 expressing cells (Figure 22F). This indicates that the antibody-K6-Ova biotin-beads complex was formed and able to bind CD40 on the cells. Single stainings with anti-streptavidin and anti-Fc detection reagents (Figure- 22D-E,) show the detection reagents are specific for their targets. No staining was observed with PE anti-Streptavidin and anti-human IgG Fcg-PE in wt cells incubated with beads (lower column, Figure 22 A-F). This indicates that the beads did not unspecifically bind the cells. The results show that it is possible to attach beads to CD40mAb-K6-Ova biotin complex without interfering with cell binding.
Example 13 - Activation of ova-specific CD8+ T cells
These studies aimed at confirming that 1132E4-IgGl and 1132E4-IgG2 in combination with various ova-peptides can induce expansion of ova-specific CD8+ T cells and to compare effects of peptides (or components thereof) used as monotherapies.
Material and methods
Forty-four non-tumor bearing male huCD40tg mice (ICH) of Alligator's own strain were used in the experiment. On day 0 and day 7, non-tumor bearing male huCD40tg mice were subcutaneously treated with either vehicle (PBS), or molar equivalent amounts of the peptides (14.1, 19.3, 18.9 and 3.3 pg of K4-Ova, K6-Ova, Z33-Ova and ovalbumin peptide control (SIINFEKL) alone or in combination with 33 pg of 1132E4 (huCD40) in IgGl or IgG2 format, or 1132 CD40 mAb alone. Seven days after the second treatment (D14), all mice were sacrificed and their inguinal lymph nodes closest to the treatment site (iLN) were removed for FACS analysis of OVA-specific CD8+ T cells.
Results
Treatment of huCD40tg mice with 1132E4-IgGl plus K4-Ova peptide or 1132E4-IgG2 plus K4-Ova peptide resulted in an expansion of OVA-specific CD8+ T cells that was significantly increased compared to treatment with 1132 wt antibody plus Z33-Ova peptide, 1132E4-IgG2 plus SIINFEKL peptide (i.e. no coiled coil part), 1132 wt antibody plus SIINFEKL peptide or treatment with peptides alone (K4-Ova, Z33-Ova or SIINFEKL) (Figure 23). The superior ability of K4-Ova to induce OVA-specific T cell expansion was dependent on combination with 1132E4-IgGl or 1132E4-IgG2, since the K4-0va peptide alone did not induce increased expansion of OVA-specific T cells compared to other OVA peptides.
Example 14 - Anti-tumor efficacy studies
These studies were aimed at evaluating the anti-tumor efficacy of the coiled-coil molecule 1132E4 in IgG2 format in combination with E4-ova-peptide and assess possible superiority of 1132E4+ K4-OVA over peptide components given as monotherapies.
Material and methods
On day 0, 0.2e6 MB49-EpCAM + -ovalbumin+ cells, growing in log phase, were injected subcutaneously in the right outer flank of human CD40 transgenic mice in a volume of 100 pl PBS. On day 10 and day 17, mice were subcutaneously treated in the inside of the left hindleg with either vehicle (PBS), 1.5 pg free OVA-peptide (SIINFEKL), 60 pg of 1132E4-IgG2, 6.5 pg K4-OVA or 60 pg of 1132E4-IgG2 in combination with 6.5 pg K4-OVA. The peptide doses given correspond to equal molar amounts.
The tumor growth was observed and measured with a caliper in width (w), length (I) and height (h) of which the tumor volume was calculated (w/2 x 1/2 x h/2 x n x (4/3)). Tumor measurements were performed until the end of the experiment, when the tumor volume of the last mouse was approaching the ethical limit of 2000 mm3.
Results
The mixture of 1132E4+ K4-OVA induced antitumor effects leading to reduced tumor growth and prolonged survival (Figure 24). No or minor effects were observed for the treatments with either 1132E4 alone or peptide components and no antibody (i.e. K4 or SIINFEKL). These results show that the CD40 agonistic antibodies connected via coiled coil to neoantigens have anti-tumor effect against tumors carrying said neoantigens.
Example 15 - Additional Coiled coil peptide structures, Octet
To evaluate whether the positioning of the Ova peptide (SIINFEKL) affects the binding kinetics of the K4-Ova coiled coil peptides.
Design The K4-0va where the ova fused with the ova peptide on the C terminal was synthesized with higher purity (>98%) compared to ova-K4-l(>70%) or Ova-K4-2 (>70%) peptides. In the ova-K4-l, the Ova peptide is fused on the N-terminal. For the ova-K4-2, the ova peptide is linked to the N-terminal. In addition, the ova peptide is surrounded by hydrophobic amino acids Lysine and Tryptophan.
Figure imgf000124_0001
Material and methods
Kinetic measurements were performed using the Octet RED96 platform (ForteBIo). FAB2G (Anti-Fab 2nd generation) sensors were used. Antibodies were diluted in lOOnM lx Kinetic buffer and captured on 8 parallel sensors for 300sec. After setting a new baseline, the captured antibodies were assayed against K4-Ova, Ova-K4-1 or Ova-K4- 2 for 120 seconds followed by dissociation for 300 seconds in Kinetic buffer. K4-Ova, Ova-K4-1 or Ova-K4-2 was diluted in seven 1 :2 dilutions starting at 0.3 pM. Sensor regeneration using lOmM Glycine pH 1.7 was performed before capture of the next antibody. Data generated were referenced by subtracting a parallel buffer blank, the baseline was aligned with the y-axis, inter-step correlation by alignment against dissociation was performed and the data were smoothed by a Savitzky-Golay filter in the data analysis software (v.9.0.0.14). The processed data were fitted using a 1 : 1 Langmuir binding model with X2 as a measurement of fitting accuracy.
Results
All the three peptides studied (K4-Ova, Ova-K4-1 or Ova-K4-2) showed good binding to an antibody coupled with peptide E4 (Figure 25). No binding was measured for the three peptides to A_1132-IgGl without peptide E4 used as a control. Overall, KD values at low nanomolar were obtained for the interaction between K4-Ova, Ova-K4-1 or Ova- K4-2 against antibodies fused with E4 (Error! Reference source not found.10). Table 10 Binding kinetics of K4-Ova, Ova-K4-1 and Ova-K4-2 coated on FAB2G (Anti-
Fab 2nd generation) sensors
Figure imgf000125_0001
Example 16 - Generation of IgGl and IgG2 coiled coil antibodies
Production, purification and QC of 1132E4 IgGl and 1132E4 IgG2 antibodies, which bind to CD40 and are connected to coiled coil E4.
Expression
A control IgGl antibody (called 1188) and monoclonal antibodies coupled with coiled coil peptides, 1132E4 IgGl and 1132E4 IgG2, were expressed using transient Expi293 HEK (Life technologies) cultures in 30 mL according to manufacturer's instructions. Purification of the antibodies from supernatants was made on protein A using the NGC system (BioRad). Cells were transfected with two different vectors encoding separately for each of the two polypeptides chains (i.e. the immunoglobulin heavy chain and the light chain to which a coiled coil peptide was fused). Protein aggregation was measured with SE-HPLC in a 1260 Infinity II system (Agilent Technologies) using a TSK gel Super SW mAB HTP 4 m, 4.6x150mm column (TOSOH Bioscience) and 100 mM Sodium Phosphate, pH 6.8, 300mM NaCI as mobile phase at ambient temperature and a flow rate of 0.35 ml/min.
Results
All coiled coil antibody fusions could be expressed and purified. Expression yields for the tested coiled coil antibody fusions in 30 mL HEK productions were high and in the range observed for the monoclonal control (Table 11). The antibodies could be purified to a high degree using protein A purification as measured by SE-HPLC showing high % main peak area. This concludes that the connected coiled coil E4 does not affect the expression or purification of the antibodies negatively.
Table 11 Manufacturability and purity data of monoclonal antibodies fused with coiled coil peptides
Figure imgf000126_0001
Example 17: Priming of ovalbumin-specific CD8+ T cells by anti-CD40-
Fc(OVA) antibody
Background and aim
The OVA peptide SIINFEKL [SEQ ID NO: 70] was conjugated to the Fc of an anti-CD40 antibody (bivalent, monospecific) by use of Z33, a 33 amino acid long Fc-binding peptide, which binds between the CH2 and CH3 region of the Fc domain. The SIINFEKL peptide was in turn covalently linked to the Z33 peptide via a GSSSS linker. The purpose with this anti-CD40-Fc(OVA) antibody is to evaluate a concept wherein a peptide is conjugated to an anti-CD40 agonist and how such conjugation impacts T cell responses to the peptide. Ultimately, the OVA peptide can be exchanged to one or more tumor antigens to induce an immune response directed against tumor cells expressing such antigen(s).
Thus, the aim of this experiment was to evaluate the effect of the anti-CD40-Fc(OVA) antibody on the priming of OVA-specific CD8+ T cells, compared to an anti-CD40 antibody and OVA peptide administered separately.
Materjals an^^
Figure imgf000127_0001
Human CD40 transgenic (hCD40tg) mice, 8-9 weeks of age, were given a mixture of 33 pg anti-CD40 antibody and 18.9 pg Z33-OVA complex s.c. on two occasions, 7 days between. Additional cohorts of mice were instead given 33 pg anti-CD40 antibody and 3.3 pg OVA peptide separately or vehicle (PBS) control.
Seven days after the second treatment, mice were sacrificed and inguinal lymph nodes collected. The lymph nodes were mashed through cell strainers to obtain single cell suspensions and the cells were subsequently Fc blocked and stained with an antibody cocktail containing fluorescently-labelled anti-mouse antibodies for CDllb, CD19, MHCII, NK1.1 (dump channel), and CD45, CD3, CD4 and CD8, as well as OVA (SIINFEKL) MHCI tetramer. The cells were also stained with Fixable Viability Stain 780 (BD Biosciences) to assess the cell viability. Samples were analysed by flow cytometry in order to determine the frequency of viable CD45+ CD3+ CD8+ OVA-MHCI tetra mer+ T cells.
Results and concl^
Figure imgf000127_0002
The data (shown in Figure 1) demonstrate that treatment with anti-CD40-Fc(OVA) antibody results in a superior expansion of OVA-specific CD8+ T cells, compared to treatment with anti-CD40 antibody and OVA peptide separately. These data thus support that conjugation of an OVA peptide to an anti-CD40 antibody, results in more potent T cell priming compared to when the antibody and the peptide are administered separately.
Example 18: Effect on priming of OVA-specific T cells in vivo
Background and aim The OVA peptide-linked Fc-binding peptide Z33 is capable of binding to antibodies, thereby forming an antibody-antigenic peptide complex. By using Z33-OVA in combination with a CD40 agonistic antibody, the antigenic peptide is targeted to CD40- expressing antigen-presenting cells such as dendritic cells (DC), which enhances crosspresentation of the antigen and increases priming of antigen-specific T cells. The purpose with this Fc-binding peptide is to generate a concept where T cell priming is improved when the Fc-binding peptide is combined with a DC-targeting and activating antibody. The aim of this experiment was to evaluate the effect on T cell priming when Z33-OVA or free OVA peptide is combined with either a CD40 agonistic antibody, an isotype control antibody, or an adjuvant to which Z33 does not bind.
Materials and methods
Human CD40 transgenic (hCD40tg) mice, 12-14 weeks of age, were given a mixture of 18.9 pg Z33-OVA peptide or a molar equivalent dose of 3.3 pg free OVA peptide (SIINFEKL) and either 33 pg anti-CD40 antibody, 33 pg isotype control antibody, or 50 pg Poly I : C (a TLR3 ligand used as adjuvant) s.c. on two occasions, 7 days between. An additional cohort of mice were instead given vehicle (PBS) control.
Seven days after the second treatment, mice were sacrificed and inguinal lymph nodes collected. The lymph nodes were mashed through cell strainers to obtain single cell suspensions and the cells were subsequently Fc blocked and stained with an antibody cocktail containing fluorescently-labelled anti-mouse antibodies for CDllb, CD19, MHCII, NK1.1 (dump channel), and CD45, CD3, CD4 and CD8, as well as OVA (SIINFEKL) MHCI tetramer. The cells were also stained with Fixable Viability Stain 780 (BD Biosciences) to assess the cell viability. Samples were analysed by flow cytometry in order to determine the frequency of viable CD45+ CD3+ CD8+ OVA-MHCI tetra mer+ T cells.
Results and conclusions
The data (shown in Figure 2) demonstrate that when Z33-OVA or OVA peptide were combined with anti-CD40 antibody, Z33-OVA induced superior expansion of OVA- specific CD8+ T cells. In contrast, Z33-OVA and OVA peptide induced similar levels of OVA-specific CD8+ T cell expansion when combined with isotype control antibody or Poly I:C adjuvant. These data thus support that the DC-targeting and/or agonistic properties of the OVA-conjugated antibody are important for achieving a more potent T cell priming compared to separate administration of peptide and antibody. Further, an immunostimulatory signal alone was not sufficient to induce superior T cell expansion with Z33-OVA compared to OVA peptide, further supporting the notion that conjugating the antigenic peptide to a DC-targeting agonistic antibody is important for achieving an improved T cell priming effect.
Example 19: Anti-tumor efficacy of an OVA-expressing tumor model, comparing anti-CD40-Fc(OVA) to vehicle control
Background and aim
By treating mice subcutaneously with the anti-CD40-Fc(OVA) antibody (i.e. anti-CD40 antibody complexed with the Z33-OVA peptide), an OVA-specific T cell response was induced in draining lymph nodes. Ultimately, the OVA peptide can be exchanged for tumor antigen peptides to induce a tumor-specific immune response against tumors expressing such antigens. This tumor-targeting immune response would be expected to reduce the growth of an established tumor.
Thus, the aim of this experiment was to evaluate the effect of the anti-CD40-Fc(OVA) antibody on the growth of the OVA-expressing tumor EG7-OVA in a therapeutic vaccination setting.
Materials and methods
Human CD40 transgenic (hCD40tg) female mice, 10-12 weeks of age, were inoculated with 1.0 x 106 EG7-OVA cells s.c. on the right flank on day 0. On day 1 and 8, mice were given either a mixture of 100 pg anti-CD40 antibody and 57 pg Z33-OVA peptide (anti-CD40-Fc(OVA) antibody) (which were mixed together prior to administration to allow complexes between the antibody and tagged antigen to form prior to administration to the mice) or vehicle control (Dextrose) s.c. on the left flank. Tumor volume and survival was monitored.
Results a
Figure imgf000129_0001
The data (shown in Figure 3) demonstrate that treatment with anti-CD40-Fc(OVA) antibody results in a significantly reduced tumor volume (p = 0.0022 on day 14, 17 and 21) compared to vehicle control. These data indicate that the potent T cell priming induced by treatment with anti-CD40-conjugated OVA peptide is associated with an anti-tumor effect resulting in delayed growth of an OVA-expressing tumor.
Example 20: Comparison of the anti-tumor efficacy of CD40-Fc(OVA) (premixed combination of anti-CD40 and Z33-OVA) and separate administration of anti-CD40 and OVA in an OVA-expressing tumor model
Figure imgf000130_0001
Treatment with anti-CD40-Fc(OVA) antibody was shown to result in a significantly reduced tumor volume (p = 0.0022 on day 14, 17 and 21) compared to vehicle control (Example 3).
The aim of this experiment was to evaluate the effect of the anti-CD40-Fc(OVA) antibody (i.e. the pre-mixed complex of the anti-CD40 antibody and the Z33-OVA peptide) on the growth of the OVA-expressing tumor EG7-OVA in a therapeutic vaccination setting, compared to the separate administration of the anti-CD40 and OVA peptide (SIINFEKL).
Figure imgf000130_0002
Human CD40 transgenic (hCD40tg) female mice, 9-11 weeks of age, were inoculated with 1.0 x 106 EG7-OVA cells s.c. on the right flank on day 0. On day 1 and 8, mice were given either a mixture of 100 pg anti-CD40 antibody and 19 pg Z33-OVA peptide (anti-CD40-Fc(OVA) antibody) (which were mixed together prior to administration to allow complexes between the antibody and tagged antigen to form prior to administration to the mice) or anti-CD40 and 3.3 pg OVA peptide (a molar equivalent dose to Z33-OVA peptide) s.c. on the left flank. Tumor volume and survival was monitored.
Results and conclusions
The data (shown in Figure 4) demonstrates that treatment with anti-CD40-Fc(OVA) antibody results in a significantly reduced tumor volume (p = 0.0132 on day 11; p = 0.0061 on day 14; p = 0.0349 on day 16; p = 0.0278 on day 18; p = 0.0056 on day 21) compared to separate administration of the anti-CD40 antibody and OVA peptide (SIINFEKL). These data indicate that targeting the OVA peptide to the dendritic cells through the formation of a complex with the anti-CD40 antibody has a significantly enhanced effect on the reduction of tumor growth compared to administration of the OVA peptide in a non-targeted manner. This data validates the hypothesis that targeting antigens to particular immune cells is beneficial over administration of the antigen in the absence of targeting. It will be clear that the OVA antigen used here, the Z33 tag used here, and the CD40 binding domains used here can be replaced with any antigen, tag, or immune cell binding domain.
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2015. 348(6230) : p. 56-61.
2. Picco, G., et al., Targeting DNGR-1 (CLEC9A) with antibody/MUCl peptide conjugates as a vaccine for carcinomas. Eur J Immunol, 2014. 44(7) : p. 1947-55.
3. Zom, G.G., et al., Two in one: improving synthetic iong peptide vaccines by combining antigen and adjuvant in one molecule. Oncoimmunology, 2014. 3(7) : p. e947892.
4. Sanchez-Paulete, A.R., et al., Cancer Immunotherapy with Immunomodulatory Anti-CD137 and Anti-PD-1 Monoclonal Antibodies Requires BATF3-Dependent Dendritic Cells. Cancer Discov, 2016. 6(1): p. 71-9.
5. Reuter, A., et al., Criteria for dendritic cell receptor selection for efficient antibody-targeted vaccination. J Immunol, 2015. 194(6) : p. 2696-705.
6. Chatterjee, B., et al., Internalization and endosomal degradation of receptorbound antigens regulate the efficiency of cross presentation by human dendritic cells. Blood, 2012. 120(10) : p. 2011-20.
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2016. 5: p. 46-58.
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Claims

Claims
1. A polypeptide comprising at least a first target antigen binding domain and at least a first tag binding domain, wherein the first target antigen binding domain is capable of specifically binding to a first target antigen; and wherein the first tag binding domain comprises a first coiled-coil oligomerisation domain.
2. A polypeptide comprising at least a first target antigen binding domain and at least a first tag binding domain, wherein the first target antigen binding domain is capable of specifically binding to a first target antigen; and optionally wherein the first tag binding domain comprises a first coiled-coil oligomerisation domain.
3. The polypeptide according to claim 1 or 2 wherein the first target antigen binding domain comprises a first immunoglobulin light chain and a first immunoglobulin heavy chain, and wherein the first coiled-coil oligomerisation domain is connected to any one or more of: a) the C-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence; b) the C-terminus of the first immunoglobulin heavy chain, optionally connected via a linker sequence; c) the N-terminus of the first immunoglobulin light chain, optionally connected via a linker sequence; and/or d) the N-terminus of the first immunoglobulin heavy chain, optionally connected via a linker sequence.
4. The polypeptide according to claim 3 wherein: a) the first immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR region, regions CDR1, CDR2 and CDR3; and optionally ii) one constant region; and b) the first immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally ii) one constant region.
5. The polypeptide according to claim 3 wherein: a) the first immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and b) the first immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three constant regions.
6. The polypeptide according to any of claims 1-5 wherein the polypeptide comprises a second target antigen binding domain, wherein the second target antigen binding domain comprises a second immunoglobulin light chain and a second immunoglobulin heavy chain.
7. The polypeptide according to claim 6 wherein: a) the second immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and b) the second immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region.
8. The polypeptide according to claim 6 wherein: a) the second immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and b) the second immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three constant regions.
9. The polypeptide according to any of claims 6-8 wherein a) the C-terminus of the second immunoglobulin light chain, is connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence; b) the C-terminus of the second immunoglobulin heavy chain, is connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence; c) the N-terminus of the second immunoglobulin light chain, is connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence; and/or d) the N-terminus of the second immunoglobulin heavy chain, is connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence.
10. The polypeptide according to any of claims 6-9 wherein a) the C-terminus of the second immunoglobulin light chain, is not connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence; b) the C-terminus of the second immunoglobulin heavy chain, is not connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence; c) the N-terminus of the second immunoglobulin light chain, is not connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence; and/or d) the N-terminus of the second immunoglobulin heavy chain, is not connected to a tag binding domain, optionally to a second coiled-coil oligomerisation domain, optionally connected via a linker sequence.
11. The polypeptide according to any of claims 1-10 wherein the polypeptide comprises a third target antigen binding domain, and optionally a fourth target antigen binding domain, wherein the third target antigen binding domain comprises a third immunoglobulin light chain and a third immunoglobulin heavy chain, and the optional fourth target antigen binding domain comprises a fourth immunoglobulin light chain and a fourth immunoglobulin heavy chain.
12. The polypeptide according to claim 11 wherein:
A) the third immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and the third immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region; optionally wherein the polypeptide comprises only one Fc region; or
B) the third immunoglobulin light chain comprises or consists of:
I) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and the third immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three constant regions; optionally wherein the polypeptide comprises only one Fc region; and optionally when present:
C) the fourth immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3CDR regions, regions CDR1, CDR2 and CDR3; and optionally comprises ii) one constant region; and the fourth immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; and optionally comprises ii) one constant region; optionally wherein the polypeptide comprises only one Fc region or
D) the fourth immunoglobulin light chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and the fourth immunoglobulin heavy chain comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and two or three constant regions, optionally wherein the polypeptide comprises only one Fc region.
13. The polypeptide according to any of claims 11 or 12 wherein the C-terminus of the third and/or fourth immunoglobulin light chain and/or the fourth immunoglobulin heavy chain is connected to a third and/or a fourth tag binding domain, optionally to a third and/or fourth coiled coil oligomerisation domain, optionally connected via a linker.
14. The polypeptide according to any of claims 11-13 wherein the C-terminus of the third and/or fourth immunoglobulin light chain and/or the fourth immunoglobulin heavy chain is not connected to a third and/or fourth tag binding domain, optionally not connected to a third and/or fourth coiled coil oligomerisation domain.
15. The polypeptide according to any of claims 11-14 wherein the N-terminus of the third and/or fourth immunoglobulin light chain and/or fourth immunoglobulin heavy chain is connected to a third and/or a fourth tag binding domain, optionally to a third and/or fourth coiled coil oligomerisation domain, optionally connected via a linker.
16. The polypeptide according to any of claims 11-15 wherein the N-terminus of the third and/or fourth immunoglobulin light chain and/or fourth immunoglobulin heavy chain is not connected to a third and/or fourth tag binding domain, optionally not connected to a third and/or fourth coiled coil oligomerisation domain.
17. The polypeptide according to any of claims 1-16 wherein the first and second target antigens are the same, optionally wherein the polypeptide does not comprise a third and fourth target antigen binding domain.
18. The polypeptide according to any of claims 1-16 wherein the first and second target antigens are different, optionally wherein the polypeptide does not comprise a third and fourth target antigen binding domain.
19. The polypeptide according to any of claims 1-18 wherein the first and second target antigens are the same and the third target antigen is a different antigen to the first and second target antigen, optionally wherein the polypeptide does not comprise a fourth target antigen binding domain; or the first and third target antigens are the same and the second antigen is different to the first and third target antigen, optionally wherein the polypeptide does not comprise a fourth target antigen binding domain.
20. The polypeptide according to any of claims 1-19 wherein the first target antigen and the second target antigen and the third target antigen are all different to one another, optionally wherein the polypeptide does not comprise a fourth target antigen binding domain.
21. The polypeptide according to any of claims 1-20 wherein the first and second target antigens are the same as each other and wherein the third and fourth antigens are the same as each other, but wherein the first and second target antigens are different to the third and fourth target antigens; or the first and third target antigens are the same as each other and wherein the second and fourth target antigens are the same as each other, but wherein the first and third target antigens are different to the second and fourth target antigen.
22. The polypeptide according to any of claims 1-21 wherein: a) the first target antigen and the second target antigen and the third target antigen and the fourth target antigen are all the same antigen; or b) the first target antigen and the second target antigen and the third target antigen and the fourth target antigen are all different to one another.
23. The polypeptide according to any of claims 1-22 wherein the polypeptide comprises at least a first target binding domain and a second target binding domain, and wherein the first target binding domain comprises a first immunoglobulin heavy chain that comprises three constant regions and wherein the second target binding domain comprises a second immunoglobulin heavy chain that comprises three constant regions, and wherein the first immunoglobin heavy chain sequence and the second immunoglobin heavy chain sequence are different to one another, and wherein the different amino acid sequences promote the formation of a heterodimer between said first and second immunoglobulin heavy chain polypeptides rather than the formation of a homodimer of the first immunoglobulin heavy chain polypeptide and a homodimer of the second immunoglobulin heavy chain polypeptide.
24. The polypeptide according to claim 23 wherein the polypeptide comprises a) a third target antigen binding domain, wherein the third target antigen binding domain comprises a third immunoglobulin light chain that comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and a third immunoglobulin heavy chain that comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and one constant region; wherein the N-terminus of the third immunoglobulin light chain polypeptide is attached to the C-terminus of the first or second immunoglobulin heavy chain polypeptide; and optionally wherein the polypeptide further comprises: b) a fourth target antigen binding domain, wherein the fourth target antigen binding domain comprises a fourth immunoglobulin light chain that comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR1, CDR2 and CDR3; ii) and one constant region; and a fourth immunoglobulin heavy chain that comprises or consists of: i) a variable region that comprises 3 CDR regions, regions CDR4, CDR5 and CDR6; ii) and one constant region; wherein the N-terminus of the fourth immunoglobulin light chain polypeptide is attached to the C-terminus of the first or second immunoglobulin heavy chain polypeptide.
25. The polypeptide according to any of claims 1-24 wherein: a) the C-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, but, where present, the second, third and fourth immunoglobulin light chain is not connected to a coiled-coil oligomerisation domain; b) the C-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, and where present the C-terminus of the second immunoglobulin light chain is connected to a second coiled-coil oligomerisation domain, but, where present, the third and fourth immunoglobulin light chain is not connected to a coiled-coil oligomerisation domain; c) the C-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, and where present the C-terminus of the second immunoglobulin light chain is connected to a second coiled-coil oligomerisation domain, and the C-terminus of the third immunoglobulin light chain is connected to a third coiled-coil oligomerisation domain but, where present, the fourth immunoglobulin light chain is not connected to a coiled-coil oligomerisation domain; d) the C-terminus of the first immunoglobin light chain is connected to a first coiled-coil oligomerisation domain, and where present the C-terminus of the second immunoglobulin light chain is connected to a second coiled-coil oligomerisation domain, and the C-terminus of the third immunoglobulin light chain is connected to a third coiled-coil oligomerisation domain, and the C-terminus of the fourth immunoglobulin light chain is connected to a fourth coiled-coil oligomerisation domain.
26. The polypeptide according to any of the preceding claims wherein the first coiled-coil oligomerisation domain is able to form a multimer, optionally a dimer optionally a heterodimer with a corresponding first cargo coiled-coil oligomerisation domain, and optionally: when present the second coiled-coil oligomerisation domain is able to form a multimer, optionally a dimer optionally a heterodimer with a corresponding second cargo coiled-coil oligomerisation domain; when present the third coiled-coil oligomerisation domain is able to form a multimer, optionally a dimer optionally a heterodimer with a corresponding third cargo coiled-coil oligomerisation domain; and when present the fourth coiled-coil oligomerisation domain is able to form a multimer, optionally a dimer optionally a heterodimer with a corresponding fourth cargo coiled-coil oligomerisation domain, when present the fifth coiled-coil oligomerisation domain is able to form a multimer, optionally a dimer optionally a heterodimer with a corresponding fifth cargo coiled-coil oligomerisation domain; when present the sixth coiled-coil oligomerisation domain is able to form a multimer, optionally a dimer optionally a heterodimer with a corresponding sixth cargo coiled-coil oligomerisation domain; and when present the seventh coiled-coil oligomerisation domain is able to form a multimer, optionally a dimer optionally a heterodimer with a corresponding seventh cargo coiled-coil oligomerisation domain when present the eighth coiled-coil oligomerisation domain is able to form a multimer, optionally a dimer optionally a heterodimer with a corresponding eighth cargo coiled-coil oligomerisation domain; when present the ninth coiled-coil oligomerisation domain is able to form a multimer, optionally a dimer optionally a heterodimer with a corresponding ninth cargo coiled-coil oligomerisation domain; and when present the tenth coiled-coil oligomerisation domain is able to form a multi mer, optionally a dimer optionally a heterodimer with a corresponding tenth cargo coiled-coil oligomerisation domain; when present the eleventh coiled-coil oligomerisation domain is able to form a multimer, optionally a dimer optionally a heterodimer with a corresponding eleventh cargo coiled-coil oligomerisation domain; and when present the twelfth coiled-coil oligomerisation domain is able to form a multimer, optionally a dimer optionally a heterodimer with a corresponding twelfth cargo coiled-coil oligomerisation domain.
27. The polypeptide according to any of the preceding claims wherein the coiled- coil oligomerisation domain comprises a peptide that has repeated units of an amino acid sequence that comprises a hydrophobic residue at every third or fourth amino acid wherein the hydrophobic residues generate surface(s) on the helix that are hydrophobic and tend to form either parallel or anti-parallel, multimeric, heterodimeric or dimeric helix-helix interactions.
28. The polypeptide according to any of the preceding claims wherein the coiled- coil oligomerisation domain comprises:
A) repeating units of a heptad sequence which follows the consensus of any one or more of: i) abcdefg wherein a and d are hydrophobic amino acids, optionally wherein e and g are polar or charged amino acids; ii) Hpphppp where h is a hydrophobic residue and p is a polar and/or charged residue; iii) Hpphpph where h is a hydrophobic residue and p is a polar and/or charged residue; iv) Hpphhpp where h is a hydrophobic residue and p is a polar and/or charged residue; v) Hpphhph where h is a hydrophobic residue and p is a polar and/or charged residue; vi) Hphhphp where h is a hydrophobic residue and p is a polar and/or charged residue; and/or vii) Hhphphp where h is a hydrophobic residue and p is a polar and/or charged residue;
B) a leucine zipper; and/or
C) repeating units of a non-heptad sequence, optionally with a periodicity of 11/3, 15/4, 18/5 or 25/7, optionally wherein the repeat sequence comprises any one or more of the following consensus sequences: viii) abbcdefg ix) abcabcdefg x) abcdabcdefg xi) abcpppefg xii) abcdefghijk xiii) abcdefghijklmo. where a and d are hydrophobic amino acids, optionally wherein e and g are polar or charged amino acids.
29. The polypeptide according to any of claims 27 or 28 wherein the sequence of each repeat sequence within a coiled-coil oligomerisation domain is identical.
30. The polypeptide according to any of claims 27 or28 wherein at least two repeat sequences within the coiled-coil oligomerisation domain are different.
31. The polypeptide according to any of the preceding claims wherein:
140 the first coiled-coil oligomerisation domain comprises or consists of a sequence that is selected from any one of: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72] ; the E5 coiled-coil oligomerisation domain [SEQ ID NO: 73]; the E6 coiled-coil oligomerisation domain [SEQ ID NO: 74]; the K3 coiled-coil oligomerisation domain [SEQ ID NO: 75]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; the leucine zipper domain of [SEQ ID NO: 79] the HAP1 coiled-coil oligomerisation domain [SEQ ID NO: 80]; the HAP2 coiled-coil oligomerisation domain [SEQ ID NO: 81]; the HAP3 coiled-coil oligomerisation domain [SEQ ID NO: 82]; the HAP4 coiled-coil oligomerisation domain [SEQ ID NO: 83]; the HAP5 coiled-coil oligomerisation domain [SEQ ID NO: 84]; the HAP6 coiled-coil oligomerisation domain [SEQ ID NO: 85]; the HAP7 coiled-coil oligomerisation domain [SEQ ID NO: 86]; the HAP8 coiled-coil oligomerisation domain [SEQ ID NO: 87]; the Leucine zipper 1 coiled-coil oligomerisation domain [SEQ ID NO: 119]; the Leucine zipper 2 coiled coil oligomerisation domain [SEQ ID NO: 120]; the rGCN4-pI coiled coil oligomerisation domain [SEQ ID NO: 121]; the rGCN4-pI' coiled coil oligomerisation domain [SEQ ID NO: 122]; the GCN4-pI coiled coil oligomerisation domain [SEQ ID NO: 123]; the GNC4-PH coiled-coil oligomerisation domain [SEQ ID NO: 124]; the JR2KC coiled-coil oligomerisation domain [SEQ ID NO: 125]; the JR2K coiled-coil oligomerisation domain [SEQ ID NO: 126]; or the JR2E coiled-coil oligomerisation domain [SEQ ID NO: 127]; optionally selected from any one of: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72];
141 the E5 coiled-coil oligomerisation domain [SEQ ID NO: 73] ; the E6 coiled-coil oligomerisation domain [SEQ ID NO: 74]; the K3 coiled-coil oligomerisation domain [SEQ ID NO: 75]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; the leucine zipper domain of [SEQ ID NO: 79] the HAP1 coiled-coil oligomerisation domain [SEQ ID NO: 80]; the HAP2 coiled-coil oligomerisation domain [SEQ ID NO: 81]; the HAP3 coiled-coil oligomerisation domain [SEQ ID NO: 82]; the HAP4 coiled-coil oligomerisation domain [SEQ ID NO: 83]; the HAP5 coiled-coil oligomerisation domain [SEQ ID NO: 84]; the HAP6 coiled-coil oligomerisation domain [SEQ ID NO: 85]; the HAP7 coiled-coil oligomerisation domain [SEQ ID NO: 86]; or the HAP8 coiled-coil oligomerisation domain [SEQ ID NO: 87], optionally selected from any one of: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; optionally selected from any one of: the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; or optionally selected from any one of: the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71];
142 or a coiled-coil oligomerisation domain with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences, optionally a coiled-coil oligomerisation domain with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences and that is able to form a heterodimer with a corresponding coiled-coil oligomerisation domain.
32. The polypeptide according to any of the preceding claims wherein: where present the second, the third, and the fourth coiled-coil oligomerisation domain comprises or consists of a sequence that is selected from any one of: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the E5 coiled-coil oligomerisation domain [SEQ ID NO: 73]; the E6 coiled-coil oligomerisation domain [SEQ ID NO: 74]; the K3 coiled-coil oligomerisation domain [SEQ ID NO: 75]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; the leucine zipper domain of [SEQ ID NO: 79] the HAP1 coiled-coil oligomerisation domain [SEQ ID NO: 80]; the HAP2 coiled-coil oligomerisation domain [SEQ ID NO: 81]; the HAP3 coiled-coil oligomerisation domain [SEQ ID NO: 82]; the HAP4 coiled-coil oligomerisation domain [SEQ ID NO: 83]; the HAP5 coiled-coil oligomerisation domain [SEQ ID NO: 84]; the HAP6 coiled-coil oligomerisation domain [SEQ ID NO: 85]; the HAP7 coiled-coil oligomerisation domain [SEQ ID NO: 86]; the HAP8 coiled-coil oligomerisation domain [SEQ ID NO: 87]; the Leucine zipper 1 coiled-coil oligomerisation domain [SEQ ID NO: 119]; the Leucine zipper 2 coiled coil oligomerisation domain [SEQ ID NO: 120]; the rGCN4-pI coiled coil oligomerisation domain [SEQ ID NO: 121]; the rGCN4-pI' coiled coil oligomerisation domain [SEQ ID NO: 122]; the GCN4-pI coiled coil oligomerisation domain [SEQ ID NO: 123]; the GNC4-PH coiled-coil oligomerisation domain [SEQ ID NO: 124]; the JR2KC coiled-coil oligomerisation domain [SEQ ID NO: 125]; the JR2K coiled-coil oligomerisation domain [SEQ ID NO: 126]; or the JR2E coiled-coil oligomerisation domain [SEQ ID NO: 127]; optionally selected from any one of: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the E5 coiled-coil oligomerisation domain [SEQ ID NO: 73]; the E6 coiled-coil oligomerisation domain [SEQ ID NO: 74]; the K3 coiled-coil oligomerisation domain [SEQ ID NO: 75]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; the leucine zipper domain of [SEQ ID NO: 79] the HAP1 coiled-coil oligomerisation domain [SEQ ID NO: 80]; the HAP2 coiled-coil oligomerisation domain [SEQ ID NO: 81]; the HAP3 coiled-coil oligomerisation domain [SEQ ID NO: 82]; the HAP4 coiled-coil oligomerisation domain [SEQ ID NO: 83]; the HAP5 coiled-coil oligomerisation domain [SEQ ID NO: 84]; the HAP6 coiled-coil oligomerisation domain [SEQ ID NO: 85]; the HAP7 coiled-coil oligomerisation domain [SEQ ID NO: 86]; or the HAP8 coiled-coil oligomerisation domain [SEQ ID NO: 87], optionally selected from any one of: the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; optionally selected from any one of: the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the K4 coiled-coil oligomerisation domain [SEQ ID NO: 76]; the K5 coiled-coil oligomerisation domain [SEQ ID NO: 77]; the K6 coiled-coil oligomerisation domain [SEQ ID NO: 78]; or optionally selected from any one of: the E4 coiled-coil oligomerisation domain [SEQ ID NO: 72]; the E3 coiled-coil oligomerisation domain [SEQ ID NO: 71]; or a coiled-coil oligomerisation domain with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences, optionally a coiled-coil oligomerisation domain with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences and that is able to form a heterodimer with a corresponding coiled-coil oligomerisation domain.
33. The polypeptide according to any of the previous claims wherein the first and second coiled-coil oligomerisation domain are the same, optionally wherein when present the first and second and third; or the first and second and third and fourth coiled-coil oligomerisation domains are the same and form a multimer, optionally a dimer, optionally a heterodimer, with the same partner coiled-coil oligomerisation domain.
34. The polypeptide according to any of the previous claims wherein the polypeptide comprises at least two different coiled-coil oligomerisations domains that each forms a multimer, optionally a dimer, optionally a heterodimer with a different partner coiled-coil oligomerisation domain.
35. The polypeptide according to any of the previous claims wherein the polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 different coiled-coil oligomerisations domains that each forms a multimer, optionally a dimer, optionally a heterodimer with a different partner coiled-coil oligomerisation domain, optionally wherein each coiled-coil oligomerisation domain present is different to one another.
36. The polypeptide according to any of the preceding claims wherein the first target antigen is an antigen present on an immune cell.
145
37. The polypeptide according to claim 36 wherein the immune cell is an antigen presenting cell, optionally a dendritic cell (DC), B cell and/or macrophage (preferably DC).
38. The polypeptide according to any one of claims 36 or 37 wherein the target antigen binding domain is an agonist of the antigen present on the immune cell.
39. The polypeptide according to any one of claims 36-38 wherein the binding of the polypeptide to the target antigen is capable of mediating : activation of the immune cell; and/or internalisation of the polypeptide; and/or recruitment of conventional type I dendritic cells (cDCl).
40. The polypeptide according to any one of claims 36-39 wherein the target antigen binding domain binds to an immune cell receptor, optionally wherein the immune cell receptor is CD40, CLEC9A, DEC-205, XCR1 or TLR.3.
41. The polypeptide according to claim 40 wherein the at least one immune cell binding domain binds to CD40.
42. The polypeptide according to any of the preceding claims wherein the polypeptide comprises: a heavy chain of SEQ NO: 88 and light chain selected from the group comprising or consisting of SEQ ID NO: 89-SEQ ID NO: 105; a heavy chain of SEQ ID NO: 106 and a light chain of SEQ ID NO: 89 or of SEQ ID NO: 90; or an IgG heavy chain-Fab light chain comprising or consisting of SEQ ID NO: 117, a Fab heavy chain comprising or consisting of SEQ ID NO: 118, and an IgG light chain that comprises a coiled-coil oligomerisation domain selected from any of [SEQ ID NO: 107-116] or comprises a sequence that has a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences,
146 optionally comprises a sequence with a sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences and that is able to a) form a multimer, optionally a dimer optionally a heterodimer with a corresponding coiled-coil oligomerisation domain; and b) bind to the target antigen.
43. The polypeptide according to any one of claims 1-41 wherein the target antigen binding domain is an antibody selected from: ADC-1013; clones 1132/1133, 1140/1135, 1150/1151 and 1107/1108 from WO 2015/091853; CP-870,893, APX005M, ChiLob 7/4, SEA-CD40; wherein:
ADC-1013 comprises one or more sequences selected from SEQ ID NO: 41-48; 1132/1133 comprises one or more sequences selected from SEQ ID NO: 1-8; 1140/1135 comprises one or more sequences selected from SEQ ID NO: 11- 18;
1150/1151 comprises one or more sequences selected from SEQ ID NO: 21- 28;
1107/1108 comprises one or more sequences selected from SEQ ID NO: 31-38.
44. The polypeptide according to any of the preceding claims wherein the target antigen binding domain is selected from the group consisting of: an Fv fragment (such as a single chain Fv fragment, or a disulphide-bonded Fv fragment); a Fab-like fragment (such as a Fab fragment; a Fab' fragment; or a F(ab)2 fragment); and domain antibodies.
45. The polypeptide according to any one of the preceding claims wherein the polypeptide comprises: a) at least a first target antigen binding domain that is an immune cell binding domain that comprises or consists of an IgG antibody; b) at least a first target antigen binding domain that is an immune cell binding domain that comprises or consists of an Fv fragment; c) at least a first target antigen binding domain that is an immune cell binding domain that comprises or consists of a Fab-like fragment; d) at least a first target antigen binding domain that is an immune cell binding domain that comprises or consists of a domain antibody;
147 e) at least a first target antigen binding domain that is an immune cell binding domain and at least a second target antigen binding domain that is an immune cell binding domain that comprise or consist of an IgG antibody, wherein the first and second target binding domain bind to the same immune cell target, optionally wherein the immune cell target is a dendritic cell; f) at least a first target antigen binding domain that is an immune cell binding domain and at least a second target antigen binding domain that is an immune cell binding domain that comprise or consist of an IgG antibody, wherein the first and second target binding domain bind to the same immune cell; or g) at least a first target antigen binding domain that is an immune cell binding domain and at least a second target antigen binding domain that is an immune cell binding domain that comprise or consist of an IgG antibody, wherein the first target antigen binding domain and the second target antigen binding domain bind to different targets, but wherein the different targets are present on the same immune cell, optionally wherein the immune cell is a dendritic cell.
46. The polypeptide according any one of claims 1-45 wherein the polypeptide is a bispecific polypeptide, and optionally comprises or consists of a format selected from the group consisting of: a) IgG-scFv bispecific antibodies; b) monovalent bispecific antibodies; c) scFv2-Fc bispecific antibodies; d) BiTE/scFv2 bispecific antibodies; e) DVD-Ig bispecific antibodies; f) DART-based bispecific antibodies; g) DNL-Fab3 bispecific antibodies; and h) scFv-HSA-scFv bispecific antibodies i) RUBY™ format antibodies, wherein the antibody comprises:
(i) two copies of a first heavy chain polypeptide and two copies of a first light chain polypeptide, and
(ii) two Fab fragments, the Fab fragments comprising a second heavy chain polypeptide and a second light chain polypeptide and wherein the first Fab fragment is fused to the C-terminus of the first copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment;
148 and the second Fab fragment is fused to the C-terminus of the second copy of the first heavy chain polypeptide via the light chain polypeptide of the Fab fragment, and wherein a) the two copies of a first heavy chain polypeptide and two copies of a first light chain polypeptide form two immune cell binding domains and the two Fab fragments form a first and second tag binding domain; or b) the two copies of a first heavy chain polypeptide and two copies of a first light chain polypeptide form a first and a second tag binding domain, and the two Fab fragments form two immune cell binding domains.
47. The polypeptide according to any one of claims 1-46 wherein the polypeptide comprises an Fc region or a variant of said region, optionally wherein the region is an IgGl, IgG2, IgG3 or IgG4 region, optionally IgGl or IgG2, optionally where the polypeptide comprises a single Fc region.
48. A complex comprising a polypeptide according to any one of claims 1-47 and at least a cargo wherein the cargo is attached to a cargo coiled-coil oligomerisation domain that is an interaction partner for the coiled-coil oligomerisation domain present on the polypeptide according to any one of claims 1-47.
49. The complex according to claim 48 wherein the polypeptide coiled-coil oligomerisation domain and corresponding cargo coiled-coil oligomerisation domain are able to form a multi mer, optionally a dimer, optionally a heterodimer with one another, optionally wherein the coiled-coil oligomerisation domains comprise:
A) a peptide that has repeated units of an amino acid sequence that comprises a hydrophobic residue at every third or fourth amino acid wherein the hydrophobic residues generate surface(s) on the helix that are hydrophobic and tend to form either parallel or anti-parallel, multimeric, heterodimeric or dimeric helix-helix interactions;
B) repeating units of a heptad sequence which follows the consensus of any one or more of: i) abcdefg
149 wherein a and d are hydrophobic amino acids, optionally wherein e and g are polar or charged amino acids; ii) Hpphppp where h is a hydrophobic residue and p is a polar and/or charged residue; iii) Hpphpph where h is a hydrophobic residue and p is a polar and/or charged residue; iv) Hpphhpp where h is a hydrophobic residue and p is a polar and/or charged residue; v) Hpphhph where h is a hydrophobic residue and p is a polar and/or charged residue; vi) Hphhphp where h is a hydrophobic residue and p is a polar and/or charged residue; and/or vii) Hhphphp where h is a hydrophobic residue and p is a polar and/or charged residue;
C) a leucine zipper; and/or
D) repeating units of a non-heptad sequence, optionally with a periodicity of 11/3, 15/4, 18/5 or 25/7, optionally wherein the repeat sequence comprises any one or more of the following consensus sequences: viii) abbcdefg ix) abcabcdefg x) abcdabcdefg xi) abcf efg xii) abcdefghijk xiii) abcdef hijklmo wherein a and d are hydrophobic amino acids, optionally wherein e and g are polar or charged amino acids.
50. The complex according to any of claims 48 or 49 wherein the cargo is: a) a protein or a peptide; b) a nucleic acid; c) a toxin;
150 d) a small molecule, for example a small molecule drug, imaging agent, toxin, radionucleotide labelled molecule; e) a virus particle; f) a viral vector; g) crispr editing components; h) lipid vesicles, optionally liposomes and/or exosomes; i) a PAMP or a DAMP; j) bacterial cellular fragments and structures; and/or k) a nanoparticle; optionally wherein: the peptide or protein comprises or consists of an antigen; the nanoparticle is a protein nanoparticle; and/or the toxin is a toxic protein, toxic peptide or toxic nanoparticle; the toxic protein, toxic peptide or toxic nanoparticle is a drug-conjugated protein, peptide or nanoparticle.
51. The complex according to claim 50 wherein: a) the antigen i) has been identified as a neoantigen that has arisen in a tumour or cell; ii) is a cancer antigen, optionally wherein the cancer antigen is a Human Papillomavirus (HPV)-associated cancer antigen; and/or iii) is derived from a pathogen, optionally wherein the pathogen is a bacteria, fungus or virus, optionally wherein the virus is Human Papillomavirus (HPV); or b) the nanoparticle that comprises an antigen, optionally comprises an antigen: i) that has been identified as a neoantigen that has arisen in a tumour or cell; ii) that is a cancer antigen, optionally wherein the cancer antigen is a Human Papillomavirus (HPV)-associated cancer antigen; and/or iii) that is derived from a pathogen, optionally wherein the pathogen is a bacteria, fungus or virus, optionally wherein the virus is Human Papillomavirus (HPV).
151
52. The complex according to any of claims 48-51 wherein the cargo is attached to the coiled-coil oligomerisation domain that is an interaction partner for the coiled-coil oligomerisation domain present on the polypeptide via a linker, optionally wherein the linker is a peptide or protein linker, optionally a GS linker, optionally wherein where the cargo is a peptide or protein or a protein nanoparticle and the linker is a peptide or protein linker, the cargo the linker and optionally the coiled-coil oligomerisation domain are transcribed into a single mRNA.
53. The complex according to any of claims 48-52 wherein the cargo is attached to the coiled-coil oligomerisation domain that is an interaction partner for the coiled-coil oligomerisation domain present on the polypeptide via chemical conjugation.
54. The complex according to any of claims 48-53 wherein: where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6 [SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6 [SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E5 [SEQ ID NO: 73] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6 [SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E6 [SEQ ID NO: 74] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K3 [SEQ ID NO: 75], K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6 [SEQ ID NO: 78];
152 where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is K3 [SEQ ID NO: 75] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73]or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is K4 [SEQ ID NO: 76] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73]or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is K5 [SEQ ID NO: 77] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73]or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is K6 [SEQ ID NO: 78] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], E5 [SEQ ID NO: 73]or E6 [SEQ ID NO: 74]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is HAP1 [SEQ ID NO: 80] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is HAP2 [SEQ ID NO: 81] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP1 [SEQ ID NO: 80], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87];
153 where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is HAP3 [SEQ ID NO: 82] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP1 [SEQ ID NO: 80], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is HAP4 [SEQ ID NO: 83] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP1 [SEQ ID NO: 80], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is HAP5 [SEQ ID NO: 84] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP1 [SEQ ID NO: 80], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is HAP6 [SEQ ID NO: 85] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP1 [SEQ ID NO: 80], HAP7 [SEQ ID NO: 86] or HAP8 [SEQ ID NO: 87]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is HAP7 [SEQ ID NO: 86] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP1 [SEQ ID NO: 80] or HAP8 [SEQ ID NO: 87]; or where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is HAP8 [SEQ ID NO: 87] then the cargo coiled-coil
154 oligomerisation domain present on the cargo protein or cargo peptide is selected from any of HAP2 [SEQ ID NO: 81], HAP3 [SEQ ID NO: 82], HAP4 [SEQ ID NO: 83], HAP5 [SEQ ID NO: 84], HAP6 [SEQ ID NO: 85], HAP7 [SEQ ID NO: 86] or HAP1 [SEQ ID NO: 81]; or wherein any of the coiled-coil oligomerisation domains has a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences, or wherein any of the coiled-coil oligomerisation domains has a sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% to any of the above sequences, or has 100% sequence identity to any of the above sequences and that is able to form a heterodimer with a corresponding coiled- coil oligomerisation domain.
55. The complex according to any of claims 48-54 wherein: where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76], K5 [SEQ ID NO: 77]or K6[SEQ ID NO: 78]; where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is K4 [SEQ ID NO: 76] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is K5 [SEQ ID NO: 77] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72], and
155 where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is K6 [SEQ ID NO: 78] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of E3 [SEQ ID NO: 71], E4 [SEQ ID NO: 72],
56. The complex according to any of claims 48-55 wherein: where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E4 [SEQ ID NO: 72] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76] or K6 [SEQ ID NO: 78]; and where the coiled-coil oligomerisation domain present on the polypeptide according to any of claims 1-38 is E3 [SEQ ID NO: 71] then the cargo coiled-coil oligomerisation domain present on the cargo protein or cargo peptide is selected from any of K4[SEQ ID NO: 76] or K6 [SEQ ID NO: 78],
57. The complex of any of claims 48-56 wherein the polypeptide comprises: at least a first coiled-coil oligomerisation domain and a second coiled-coil oligomerisation domain; and at least a first cargo comprising a first cargo coiled-coil oligomerisation domain and a second cargo comprising a second cargo coiled-coil oligomerisation domain; wherein the first polypeptide oligomerisation domain interacts with the first cargo oligomerisation domain; and wherein the second polypeptide oligomerisation domain interacts with the second cargo oligomerisation domain.
58. The complex of claim 57 wherein the first cargo is different to the second cargo.
59. A complex formed by contacting a polypeptide according to any of claims 1-47 with a cargo in vitro, wherein the cargo comprises a coiled-coil oligomerisation domain capable of forming a multimer optionally a dimer optionally heterodimer with one or more coiled-coil oligomerisation domains present on the polypeptide.
60. A nucleic acid encoding the polypeptide according to any of claims 1-47.
156
61. A nucleic acid encoding a cargo protein or cargo peptide or cargo protein nanoparticle connected to a coiled-coil oligomerisation domain as described herein.
62. A vector comprising the nucleic acid according to claim 60 and/or the nucleic acid according to claim 61.
63. A cell comprising the nucleic acid according to claim 60 and/or 61 or the vector according to claim 62.
64. A pharmaceutical composition comprising a complex according to any of claims 48-58, or a polypeptide according to any of claims 1-47, or a nucleic acid according to any of claims 60 or 61, or a vector according to claim 62, or a cell according to claim 63.
65. The pharmaceutical composition according to claim 64 wherein the composition comprises at least two different:
Complexes according to any of claims 48-59; polypeptides according to any of claims 1-47; nucleic acids according to any of claims 60 or 61; vectors according to claim 62; and/or cells according to claim 63.
66. A complex according to any one of claims 48-59 or a pharmaceutical composition according to any one of claims 64 or 65 for use in medicine.
67. A complex according to any one of claims 48-59 or a pharmaceutical composition according to any one of claims 64 or 65 for use in a method of treating or preventing cancer, optionally wherein the cargo comprises: an antigen that comprises an antigenic peptide or protein sequence that has been identified as a neoantigen that has arisen in a tumour or cell; or a cancer peptide or protein antigen; or a toxin.
68. A complex according to any one of claims 48-59 or a pharmaceutical composition according to any one of claims 64 or 65 for use in a method of treating or preventing a pathogenic infection,
157 optionally for treating or preventing a bacterial, fungal or viral infection, wherein the cargo protein or cargo peptide comprises an antigen, and wherein the antigen comprises an antigenic peptide or protein sequence derived from a bacteria, fungus or virus, optionally from Human Papillomavirus (HPV).
69. A complex according to any one of claims 48-59 or a pharmaceutical composition according to any one of claims 64 or 65 for use in a method of personalised therapy, wherein a peptide or protein neoantigen has been identified as having arisen in a patient, and wherein the cargo comprises the peptide or protein neoantigen sequence.
70. The complex for use according to claim 69 wherein the personalised therapy is for the treatment or prevention of cancer.
71. The complex for use according to any of claims 69 or 70 wherein the method of personalised therapy involves the initial step of identifying a neoantigen that has arisen in a patient.
72. A method for the treatment or prevention of a disease wherein the method comprises administering a complex according to any one of claims 48-59 or a pharmaceutical composition according to any one of claims 64 or 65.
73. A method for the treatment or prevention of cancer, wherein the method comprises administering a complex according to any one of claims 48-59 or a pharmaceutical composition according to any one of claims 64 or 65, optionally wherein the antigen comprises an antigenic peptide or protein sequence that has been identified as a neoantigen that has arisen in a cell, optionally a tumour cell; or a cancer protein or peptide antigen.
74. A method for the treatment or prevention of a pathogenic infection, wherein the method comprises administering a complex according to any one of claims 48-59 or a pharmaceutical composition according to any one of claims 64 or 65, optionally wherein the method is for the treatment or prevention of a bacterial, fungal or viral infection, wherein the antigen comprises an antigenic peptide or protein sequence derived from a bacteria, fungus or virus, optionally from Human Papillomavirus (HPV).
158
75. A method for the personalised treatment or prevention of a disease in a patient, wherein the method comprises administering a complex according to any one of claims 48-59 or a pharmaceutical composition according to any one of claims 64 or 65, optionally wherein the cargo comprises an antigenic peptide or protein sequence that has been identified as a neoantigen that has arisen in a cell, optionally a tumour cell; or a cancer protein or peptide antigen.
76. A method for personalised therapy, comprising administering a complex according to any of claims 48-59 or a pharmaceutical composition according to any of claims 64 or 65 to a patient, wherein a peptide or protein neoantigen has been identified as having arisen in the patient, and wherein the cargo comprises the neoantigen peptide or protein sequence.
77. The method according to claim 76 wherein the personalised therapy is for the treatment or prevention of cancer.
78. The method according to any one of claims 76 or 77 wherein the method of personalised therapy involves the initial step of identifying a neoantigen that has arisen in a patient.
79. A method of producing a complex comprising a polypeptide according to any one of claims 1-47 and a cargo, optionally a cargo protein or cargo peptide, wherein the cargo comprises a coiled-coil oligomerisation domain capable of forming a multimer optionally a dimer optionally a heterodimer with the coiled-coil oligomerisation domain present on the polypeptide wherein the method comprises contacting the polypeptide according to any of claims 1-47 with the cargo in vitro or ex vivo, optionally wherein the cargo comprises an antigen.
80. An in vitro or ex vivo method of activating an immune cell, wherein the method comprises contacting the immune cell with a complex according to any of claims 48- 59 or a pharmaceutical composition according to any of claims 64 or 65, wherein the immune cell comprises an immune cell target to which the targeting binding domain of the polypeptide binds.
81. A kit for the in vitro or ex vivo preparation of a complex according to any of claims 48-59 wherein the kit comprises a polypeptide according to any of claims 1-47
159 and a cargo wherein the cargo comprises a coiled-coil oligomerisation domain capable of forming a heterodimer with the coiled-coil oligomerisation domain present on the polypeptide.
82. The kit according to claim 80 wherein the kit comprises a buffer suitable for the in vitro or ex vivo formation of the complex.
83. The polypeptide, complex or method according to any of the preceding claims wherein the coiled-coil oligomerisation domain is located at the N-terminus of the cargo protein or peptide.
84. The polypeptide, complex or method according to any of the preceding claims wherein the coiled-coil oligomerisation domain is located at the C-terminus of the cargo protein or peptide.
85. The polypeptide, complex or method according to any of the preceding claims wherein the tagged cargo comprises one of the following sequences: SEQ ID NO: 128; 129 or 130.
160
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