WO2022258678A1 - Multispecific proteins binding to nkp30, a cytokine receptor, a tumour antigen and cd16a - Google Patents

Multispecific proteins binding to nkp30, a cytokine receptor, a tumour antigen and cd16a Download PDF

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WO2022258678A1
WO2022258678A1 PCT/EP2022/065528 EP2022065528W WO2022258678A1 WO 2022258678 A1 WO2022258678 A1 WO 2022258678A1 EP 2022065528 W EP2022065528 W EP 2022065528W WO 2022258678 A1 WO2022258678 A1 WO 2022258678A1
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domain
polypeptide
protein
abd
binds
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Laurent Gauthier
Yannis Morel
Olivier DEMARIA
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Innate Pharma
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • 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
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • Multispecific proteins that bind and specifically redirect effector cells to lyse a target cell of interest via multiple receptors are provided.
  • the proteins have utility in the treatment of disease, notably cancer or infectious disease.
  • Interleukin 2 is one example of a pluripotent cytokine that acts on a cytokine receptor expressed by NK cells.
  • IL-2 is mainly produced by activated T cells, especially CD4+ T helper cells, and functions in aiding the proliferation and differentiation of B cells, T cells and NK cells.
  • IL-2 is also essential for Treg function and survival.
  • human IL-2 (uniprot: P60568) is synthesized as a precursor peptide of 153 amino acids with a 20 residue signal sequence, that gives rise to a mature secreted IL-2 having the amino acid sequence of SEQ ID NO: 213.
  • Interleukin 2 has four antiparallel, amphiphilic alpha helices.
  • IL-2 works through three different receptors: interleukin 2 receptor alpha (IL- 2Ra; CD25), interleukin 2 receptor beta (IL-2Rb; CD122), and interleukin 2 receptor gamma (IL-2Ry; CD132).
  • IL-2Ra interleukin 2 receptor alpha
  • IL-2Rb interleukin 2 receptor beta
  • IL-2Ry interleukin 2 receptor gamma
  • ⁇ L-2R and IL-2Ry are essential for IL-2 signaling
  • IL-2Ra (CD25) is not necessary for signaling, but can confer high affinity binding of IL-2 to receptors.
  • trimer receptor (I ⁇ -2abg) formed by the combination of IL-2Ra, b, and g is the IL-2 high affinity receptor (KD about 10 pM), and the dimer receptor (IL- 2bg) is an intermediate affinity receptor (KD about 1 nM).
  • Immune cells express dimer or trimer IL-2 receptors. Dimer receptors are expressed on cytotoxic CD8 + T cells and natural killer cells (NK), while trimer receptors are mainly expressed on activated lymphocytes and CD4 + CD25 + FoxP3 + inhibitory regulatory T cells (Treg). Because resting effector T cells and NK cells do not have CD25 on the cell surface, they are relatively insensitive to IL-2. Treg cells consistently express the highest level of CD25 in the body. Due to the low concentrations of IL-2 that typically exists in tissues, IL-2 preferentially activates cells that express the high affinity receptor complex (CD25:CD122:CD132), and therefore under normal circumstances, IL-2 will preferentially stimulate Treg cell proliferation.
  • IL-15, IL-12, IL-7, IL-27, IL-18, IL-21, and IFN-a share many aspects of receptor binding, complex assembly and signaling with IL-2.
  • IL-15, IL-21 and IL-7 like IL- 2 both act on NK cells via the common-g chain receptor (CD132).
  • IL-15 binds to the IL-15 receptor (IL-15R) which is composed of three subunits: IL-15Ra, CD122, and CD132. Two of these subunits, CD122 and CD132, are shared with the receptor for IL-2, but IL-2 receptor has an additional subunit (CD25).
  • IL-15Ra (CD215) specifically binds IL15 with very high affinity, and is capable of binding IL-15 independently of other subunits.
  • IL-21 is another example of a type I cytokine, and its IL-21 receptor (IL-21 R) has been shown to form a heterodimeric receptor complex with the IL-2/IL-15 receptor common gamma chain (CD132).
  • NK cells have the potential to mediate anti-tumor immunity.
  • NK cells have been shown to cause toxicity in mice through their hyper-activation and secretion of multiple inflammatory cytokines when IL-2 was administered together with IFN-a (Rothschilds et al, Oncoimmunology. 2019;8(5):).
  • NK cells were also shown to cause toxicity of the cytokine IL-15 that also signals through IL-2Rby (see WO2020247843 citing Guo et al, J Immunol. 2015;195(5):2353-64).
  • cytokines such as IL-2
  • fuse it to or associate it with a tumor-specific antibody.
  • IL-2 indeed synergized with antitumor antibody in anti-tumor effect in vivo
  • the inclusion of IL- 2 and anti-tumor antigen antibody in the same molecule presented no efficacy or toxicity advantage.
  • the IL-2 moiety entirely governed biodistribution, explaining the observation that immunocytokines recognizing irrelevant antigen performed equivalently to tumor-specific immunocytokines when combined with antibody (Tzeng et al. Proc Natl Acad Sci USA. 2015 Mar 17; 112(11): 3320-332).
  • cytokines have generally focused on single cytokines or simple combinations. More recently, it has been reported that IL-15, IL-18, IL-21, and IFN-a, alone and in combination, and their potential to synergize with IL-2, and that very low concentrations of both innate and adaptive common g chain cytokines synergize with equally low concentrations of IL-18 to drive rapid and potent NK cell CD25 and IFN-g expression (Nielsen et al. Front Immunol. 2016; 7: 101). However, administration of cytokines to humans has involved toxicity, which makes combination treatment with cytokines challenging. Furthermore, little remains known on potential synergies or interaction between cytokine receptor signaling pathways and other activating receptors in NK cells. There is therefore a need for new ways to mobilize NK cells in the treatment of disease, particularly cancer.
  • the present provides multi-specific proteins that bind to NKp30 and a cytokine receptor (e.g. CD122 and/or CD132) on NK cells, and optionally that further bind CD16A on NK cells, and that also bind to an antigen of interest (e.g. a cancer antigen) on a target cell, where in the multi-specific proteins are capable of increasing NK cell cytotoxicity toward a target cell that expresses the antigen of interest (e.g., a cell that contributes to disease, a cancer cell).
  • a cytokine receptor e.g. CD122 and/or CD132
  • an antigen of interest e.g. a cancer antigen
  • the multi-specific protein’s ability to bind, in cis, to NKp30 and to the cytokine receptor (and optionally further through CD16A) at the surface of an NK cell is believed to lead to a particularly advantageous cell surface receptor signalling, in turn resulting a potent anti-tumor response by NK cells.
  • the examples made use of a variant IL-2 cytokine (IL-2v) which was modified to reduce affinity for its receptor(s) on T cells (CD25) but retained substantially full affinity (comparable to wild-type IL-2) for its receptor on NK cells (CD122 and/or CD132).
  • the NKp30 binding domain (exemplified as a VH/VL pair comprised in a Fab or scFv), the CD16A- binding Fc domain and the cytokine were placed adjacent to one another in series within the protein, each separated from the adjacent element (i.e. NKp30 ABD, Fc domain or cytokine) solely by a short peptide linker.
  • NKp30 ABD adjacent element
  • Fc domain or cytokine i.e. NKp30 ABD, Fc domain or cytokine
  • These configurations of multispecific proteins were designed to present the respective antigen binding domains so as to permit co-engagement of NKp30 and cytokine receptor on the same cell surface plane (i.e. in NKp30, cytokine receptor (and further CD16A) are bound in cis).
  • the examples used a Fc domain that binds CD16A, showing that binding to CD16A does not negatively affect the tumor and NK-targeted biodistribution and instead led to triple co-engagement of NKp30, CD16A and cytokine receptor, and in turn permitted the incorporation of a cytokine that retained binding affinity for its receptor on NK cells.
  • cytokines can be used that retain good affinity, optionally substantially full binding affinity, for their receptor on NK cells sufficient to mediate potent signaling in NK cells.
  • cytokines such as the ones described herein generally have an affinity for binding to their receptor on NK cells that is no stronger than that of the affinity of the multispecific protein for NKp30 (affinity can be determined as the KD).
  • the multispecific proteins tested herein all bear an Fc domain that bound FcRn with a long in vivo half-life, with or without CD16A binding.
  • the multispecific proteins mediated considerably higher NK cell cytotoxicity compared to a conventional human lgG1 antibody bearing the same variable regions of model anti-CD20 antibody GA101, despite the latter lgG1 binding to CD20 bivalently.
  • the multispecific proteins mediated considerably higher NK cell cytotoxicity compared to the gold standard antibody obinutuzumab (GA101) in glycoengineered form (Fc enhanced for binding to CD16A).
  • cytokine e.g. a type 1 cytokine such as an IL-2, IL-15, IL-21, IL-7, IL-27 or IL-12 cytokine, an IL-18 cytokine or a type 1 interferon (e.g. IFN-a, IFN-b), to NKp30 promotes cis-presentation to the cytokine’s receptor (e.g. II_2/15bg, IL-21R, IL-7Ra, IL-27Ra, IL-12R, IL-18R, IFNAR), as shown in Figure 1 for the cytokine IL2 and cytokine receptor complex II_2bg).
  • a type 1 cytokine such as an IL-2, IL-15, IL-21, IL-7, IL-27 or IL-12 cytokine, an IL-18 cytokine or a type 1 interferon (e.g. IFN-a, IFN-b)
  • IL2v placed immediately adjacent to (and on the C-terminal side of) the NKp30-binding ABD permitted the triple receptor cis-presentation to occur (the IL2v was connected to the NKp30-binding ABD by a linker peptide of as little as five amino acid residues).
  • use of a dimeric Fc domain as the CD16A-binding ABD provides FcRn, in turn conferring a half-life sufficiently long to induce NK cell tumor infiltration and proliferation of NK cells in vivo.
  • the multispecific proteins directed to NKp30 on NK cells have the advantage that they permit a range of cytokines to be used and/or tested without a requirement for reduced binding affinity for their receptor on NK cells (e.g. CD122).
  • the cytokine therefore may or may not be modified to attenuate or decrease binding affinity for its receptor.
  • the multispecific proteins directed to NKp30 on NK cells can thus make use of ayn one of several cytokines in their wild- type form or where the cytokine retains binding affinity for its receptor on NK cells that is not substantially diminished compared to the wild-type cytokine, particularly where the cytokine does not have substantially reduced activity at its receptor on NK cells, and/or where the cytokine’s affinity for its receptor is no stronger than the affinity of the NKp30 ABD for NKp30.
  • the cytokine ABD e.g. cytokine moiety within the multispecific protein
  • the cytokine moiety induces signalling at its receptor on NK cells (e.g. CD122) that is at least 70% or 80% of that observed with the wild- type form of the cytokine.
  • NK cells e.g. CD122
  • the cytokine ABD e.g. cytokine moiety within the multispecific protein
  • the cytokine moiety has a binding affinity for its receptor on NK cells (e.g.
  • CD122 that is within 3-log, 2-log or 1-log of that of the wild-type form of the cytokine (e.g. the cytokine moiety has a KD for binding to the cytokine receptor that is not more than 3-, 2- or 1- log higher than that observed for the wild-type form of the cytokine).
  • Affinity can be KD for binding to recombinant receptor protein, as determined using SPR. Signaling or receptor binding affinity of cytokines can be specified as being when incorporated into an otherwise equivalent multispecific protein.
  • High efficacy combined with low effect on Treg cells and/or low immune toxicity is a particular advantage of a therapeutic molecule that combines the ability to bind each of NKp30 and cytokine receptor (e.g. CD122), and further CD16A, on an individual NK cell, particularly for a therapeutic agent having a long in vivo half-life.
  • NKp30 and cytokine receptor e.g. CD122
  • CD16A cytokine receptor
  • the multispecific proteins are particularly advantageous due to high potency in enhancing NK cell activity (e.g. NK cell proliferation, activation, cytotoxicity and/or cytokine release, including by tumor-infiltrating NK cells), yet with low immune toxicity (e.g. low systemic increase or release of cytokines IL-6 and TNF-a).
  • NK cell activity e.g. NK cell proliferation, activation, cytotoxicity and/or cytokine release, including by tumor-infiltrating NK cells
  • low immune toxicity e.g. low systemic increase or release of cytokines IL-6 and TNF-a.
  • the present disclosure provides examples using protein formats that permit sufficient distance between NKp30 and cytokine receptor (e.g. CD122) and CD16A binding domains to permit all three receptors to be bound by a single NK cell, thereby providing combined NK cell receptor activation.
  • the combined binding on a single cell may account for the minimal off-target immune toxicity and lack of fratricidal killing of NKp30-expressing and/or CD16-expressing cells (e.g., NK cells) because the multispecific protein is bound by at least one activating receptor in addition to cytokine receptor (e.g. CD122) at the surface of the NKp30 and/or CD16+ effector cell.
  • NKp30-expressing and/or CD16-expressing cells e.g., NK cells
  • CD16-expressing cells e.g., NK cells
  • the multispecific proteins are further advantageous due to their ability to potentiate the activity and/or proliferation of both NKp30 + CD16 + and NKp30 + CD16A _ NK cells.
  • NKp30 + CD16 + and NKp30 + CD16A _ NK cells As shown herein, combined dual binding to NKp30 and CD122, in the absence of binding to CD16A, demonstrates strong potentiation of NK cell activity.
  • the CD16- population represents 5-15% of the total NK cell population, while in some cancer patients the proportion of CD16- NK cells is greatly increased, making up as much as 50% of the total NK cell population.
  • the tumor micro-environment has been shown to affect the phenotype of CD16A + NK cells by either inducing shedding of CD16A from the surface of the cells or promoting conversion from CD16A + to CD16 NK cells.
  • some individuals have mutations in CD16A (e.g. at residue 158 of CD16A) that result in reduced ability to mediate ADCC.
  • Overcoming CD16A deficiencies particular as may occur in the tumor environment, while increasing both the number of activated NKp30+ NK cells in the tumor, is particularly advantageous.
  • multispecific proteins do not require binding or signaling via NKG2D and can be used to potentiate NK cell activity in patients having NK and/or T cells characterized by relatively low levels of surface expression of the activating receptor NKG2D, for example as is known to be a general or common feature in gastric and prostate cancer.
  • a multispecific protein comprising: a NKp30-binding domain that binds to a human NKp30 polypeptide, (b) a binding domain that binds an antigen of interest (e.g. a tumor-associated or cancer antigen; an antigen of interest present expressed by a target cell), (c) an optional CD16A-binding domain (e.g. an Fc dimer) that binds to a human CD16A polypeptide, and (c) an antigen binding domain that binds to a human cytokine receptor polypeptide expressed on NK cells (e.g.
  • a receptor such as CD122 (I L2/15Rb), IL- 21R, IL-7Ra, IL-27Ra, IL-12R, IL-18R, IFNAR (IFNAR1 and/or IFNAR2).
  • a multispecific protein comprising a NKp30-binding domain that binds to a human NKp30 polypeptide, a binding domain that binds an antigen of interest (e.g. a tumor-associated or cancer antigen; an antigen of interest present expressed by a target cell), an Fc domain (e.g.
  • an Fc domain dimer that is bound by human FcRn (and optionally that is further bound by a human CD16A polypeptide), and an antigen binding domain that binds to a human cytokine receptor polypeptide (e.g. CD122 (IL2/15R ), IL-21 R, IL-7Ra, IL-27Ra, IL-12R, IL-18R, IFNAR (IFNAR1 and/or IFNAR2).
  • the antigen binding domain that binds a cytokine receptor can be a variant cytokine having a modification that reduces binding to a receptor counterpart found on non-NK cells (e.g. T cells, Treg cells) compared to its wild-type form.
  • the ABD that binds to a human NKp30 polypeptide and the ABD that binds a human cytokine receptor can be specified as being configured to be capable of adopting a membrane planar binding confirmation.
  • the multispecific protein can be specified as being capable of interacting with, binding to or co-engaging NKp30 and the cytokine receptor, and optionally further CD16A, on the surface of an NK cell.
  • the ABD that binds to a human NKp30 polypeptide and the ABD that binds a human cytokine receptor, and optionally further the Fc domain are specified as being positioned or connected within the multispecific protein in series (e.g. with respect to the N- and C-termini of the multispecific protein).
  • the ABD that binds NKp30 can optionally be specified as being connected to the CD16A- binding domain (e.g. an Fc domain) by an Ig- derived (e.g. a peptide from a hinge domain or heavy or light chain constant domain) or non- Ig-derived domain linker, optionally wherein the domain linker is a flexible polypeptide linker.
  • NKp30 e.g. a Fab, single variable domain or scFv
  • Ig- derived e.g. a peptide from a hinge domain or heavy or light chain constant domain
  • non- Ig-derived domain linker optionally wherein the domain linker is a flexible polypeptide linker.
  • the ABD that binds a cytokine receptor can optionally be specified as comprising a wild-type or variant cytokine connected to the rest of the multispecific protein or to the NKp30 ABD by a domain linker, optionally a flexible polypeptide linker.
  • the cytokine can be specified as being positioned C-terminal to both the NKp30- and CD16A- binding domains on the multispecific protein, and optionally further the cytokine is connected to the rest of the multispecific protein (or e.g., a domain thereof, the NKp30 ABD) via a peptide linker of 15, 10 or 5 residues or less.
  • the NKp30- and CD16A- binding domains can optionally be specified as being placed adjacent to one another on the multispecific protein and optionally connected to one another by a peptide linker (e.g. an immunoglobulin-derived linker such as a hinge-derived linker, a non-immunoglobulin-derived linker, a flexible linker) having a length of 15, 10 or 5 residues or less.
  • a peptide linker e.g. an immunoglobulin-derived linker such as a hinge-derived linker, a non-immunoglobulin-derived linker, a flexible linker having a length of 15, 10 or 5 residues or less.
  • the ABD that binds to a human NKp30 polypeptide is positioned adjacent to the Fc domain within the protein (or on a polypeptide chain thereof), and wherein either the ABD that binds to a human NKp30 polypeptide or the Fc domain are positioned adjacent to the ABD that binds a human cytokine receptor, optionally further wherein the ABD that binds a human cytokine is connected to the ABD that binds to a human NKp30 polypeptide or the Fc domain by a linker peptide having 20 or less than 20 amino acid residues, optionally less than 15 amino acid residues, optionally less than 10 amino acid residues, optionally between 5 and 15 residues, optinally between 5 and 10 residues, optionally between 3 and 5 residues.
  • the ABD that binds a cytokine receptor can be a human cytokine polypeptide, for example CD122 (IL2/15RP), IL-21R, IL-7Ra, IL-27Ra, IL-12R, IL-18R, IFNAR (IFNAR1 and/or IFNAR2).
  • the ABD that binds a cytokine receptor can optionally be a human cytokine polypeptide (e.g. IL-2, IL-15, IL-21) that is modified (e.g.
  • the ABD that binds a human cytokine receptor can be a variant cytokine that displays reduced binding affinity for cytokine receptor present on T cells compared to the non- modified or wild-type cytokine polypeptide.
  • the cytokine polypeptide can be modified to reduce binding to such receptor that is expressed on non-NK cells (e.g. Treg cells, T cells) compared to its wild-type cytokine counterpart.
  • the multispecific protein comprises an NKp30-binding domain or portion thereof fused, optionally via a domain linker, to a cytokine receptor-binding domain, e.g. a cytokine that binds a receptor expressed at the surface of an NK cell.
  • the portion of the NKp30-binding domain comprises a single variable domain (e.g.
  • variable domain fused to a first constant domain that, together with a complementary variable domain (e.g. a second variable domain fused to a second constant region), forms an ABD (e.g., a Fab) that binds NKp30.
  • ABD e.g., a Fab
  • the multispecific protein comprises: (i) a first polypeptide chain comprising, from N- to C-terminal, an NKp30-binding domain or portion thereof comprising a variable domain, a human CH1 or CL constant domain, optionally a domain linker, and a wild- type or variant I L-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or lFN-b polypeptide, and
  • the protein further comprises a dimeric Fc domain and an ABD that binds an antigen of interest.
  • a protein comprising a NKp30 ABD-cytokine unit.
  • the protein is a multispecific protein comprising a V H and a VL that associate to form an ABD that binds a cancer antigen or other antigen of interest, and a NKp30 ABD- cytokine unit (and optionally further a CD16A ABD (e.g. a dimeric Fc domain)).
  • the NKp30 ABD-cytokine unit comprises an NKp30 ABD fused, optionally via a domain linker, to a cytokine that binds a receptor expressed at the surface of an NK cell.
  • a multispecific protein can thus comprise a NKp30 ABD- cytokine unit that is formed from the association of two polypeptide chains and has one of the following structures:
  • V b -2 (CH1 or CL) - L- Cyt (chain 2) or
  • V a-2 (CH 1 or CL) a (chain 1)
  • V a -2 and V b -2 are each a VH domain or a VL domain, wherein one of V a -2 and V b -2 is a VH and the other is a VL and wherein V a -2 and V b -2 form an ABD that binds NKp30;
  • CH1 is a human immunoglobulin CH1 domain and CL is a human light chain constant domain; one of (CH1 or CL) a and (CH1 or CL) b is a CH1 and the other is a CL such that a (CH1/CL) pair is formed;
  • L, L1 and L2 are each an amino acid domain linker, wherein L, L1 and L2 can be different or the same, wherein L1 is a linker connecting the NKp30 ABD-cytokine unit to the rest of the multispecific protein (e.g., a protein comprising a VH and a VL and that associate to form an ABD that binds a cancer antigen); and Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21 , IL-7, IL-27, IL-12, IL-18, IFN-a or lFN-b polypeptide.
  • Cyt is a wild-type or variant human IL-2, IL-15, IL-21 , IL-7, IL-27, IL-12, IL-18, IFN-a or lFN-b polypeptide.
  • the Cyt can be specified as having a free C-terminus (no further domains or amino acid sequences fused to the Cyt at its free terminus).
  • chain 1 can be specified as having a free C- terminus.
  • the NKp30 ABD-cytokine unit comprises: (i) a first polypeptide chain comprising, from N- to C-terminal, an NKp30-binding domain or portion thereof comprising a variable domain comprising an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 123-212 or 250, a human CH1 or CL constant domain, optionally a domain linker, and a wild-type or variant IL- 2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b polypeptide comprising an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 213-248 or to a fragment thereof of at least 40, 50, 60, 80 or 100 contiguous amino acids thereof; and
  • variable domain comprises an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 45-122 or 249, and a human CH1 or CL constant domain.
  • the NKp30 ABD-cytokine unit comprises: (i) a first polypeptide chain comprising, from N- to C-terminal, an NKp30-binding domain or portion thereof comprising a variable domain comprising an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 123-212 or 250, a human CH1 or CL constant domain, optionally a domain linker, and a wild-type or variant IL- 2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b polypeptide comprising an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 213-248 or to a fragment thereof of at least 40, 50, 60, 80 or 100 contiguous amino acids thereof; and
  • variable domain comprises an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 45-122 or 249, and a human CH1 or CL constant domain.
  • the NKp30 ABD-cytokine unit in another embodiment where the NKp30 ABD-cytokine unit is placed on one polypeptide chain of a protein, the NKp30 ABD-cytokine unit can have the structure:
  • V a -2 and V b -2 are each a VH domain or a VL domain, wherein one of V a -2 and V b -2 is a VH and the other is a VL and wherein V a -2 and V b -2 form an ABD that binds NKp30;
  • L1 , L2 and L3 are each an amino acid domain linker, wherein L1 , L2 and L3 can be different or the same, wherein L1 is a linker connecting the NKp30 ABD-cytokine unit to the rest of the multispecific protein (e.g., a protein comprising a VH and a VL and that associate to form an ABD that binds a cancer antigen); and Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
  • L1, L2 and L3 can each be independently specified as having a length of 15, 10 or 5 residues or less.
  • the cytokine or cytokine receptor ABD (either as a free cytokine or as incorporated into a multispecific protein) binds its receptor, as determined by SPR, with a binding affinity (KD) of 1 mM or lower, 200 nM or lower, 100 nM or lower, 50 nM or lower, or 25 nM or lower. In one embodiment, the cytokine or cytokine receptor ABD binds its receptor, as determined by SPR, with a binding affinity (KD) that is 1 nM or higher than 1 nM, optionally that is higher than 10 nM optionally that is higher than 15 nM.
  • KD binding affinity
  • the cytokine or cytokine receptor ABD binds its receptor, as determined by SPR, with a binding affinity (KD) between about 1 nm and about 200 nm, optionally between about 1 nm and about 100 nm, optionally between about 10 nM and about 1 pM, optionally between about 10 nM and about 200 pM, optionally between about 10 nM and about 100 nM, optionally between about 15 nM and about 1 pM, or optionally between about 15 nM and about 200 nM.
  • KD binding affinity
  • the cytokine is a wild-type cytokine or a fragment or variant thereof that has at least 80% of the ability of a wild-type cytokine counterpart to induce signaling in NK cells, optionally wherein signaling is assessed by bringing the isolated cytokine moiety into contact with an NK cell and measuring STAT phosphorylation in the NK cells.
  • the cytokine is a wild-type cytokine or a fragment thereof that retains at least 70%, 80% or 90% of the affinity for its cytokine receptor present on NK cells, compared to the wild-type cytokine counterpart.
  • the cytokine is a variant cytokine, wherein the cytokine retains at least 70%, 80% or 90% of the affinity for its cytokine receptor present on NK cells, compared to the wild-type cytokine counterpart. In one embodiment, the cytokine does not comprise mutations that substantially reduce the affinity of the cytokine for the cytokine receptor present on NK cells. In one embodiment, the multispecific protein (or the cytokine when included in the multispecific protein) exhibits an EC50 for cytokine pathway signaling in NK cells that is lower than that observed with its wild-type cytokine counterpart alone.
  • the multispecific protein (or the cytokine when included in the multispecific protein) exhibits an EC50 for cytokine pathway signaling in NK cells that is lower than that observed with the cytokine alone or in a protein of comparable structure but lacking a NKp30 ABD and/or a CD16 ABD.
  • the EC50 for cytokine pathway signaling in NK cells is at least 10-fold or 100-fold lower, optionally wherein cytokine pathway signaling is assessed by bringing the respective cytokine or multispecific protein into contact with an NK cell and measuring STAT phosphorylation in the NK cells.
  • the multispecific protein is configured such that an Fc domain (or CD16-binding domain), the NKp30-binding domain and the cytokine receptor-binding domain are each capable of binding to their respective NKp30, CD16A or cytokine receptor binding partner when such binding partners are present together at the surface of a cell (e.g. an NK cell).
  • the multispecific protein can be characterized by monovalent binding to NKp30 (e.g. the multispecific protein comprises only one NKp30 ABD), monovalent (or optionally bivalent) binding to antigen of interest, monovalent binding to CD16A (e.g. the multispecific protein comprises only one Fc domain dimer), and monovalent binding to cytokine receptor (e.g., the multispecific protein comprises only one cytokine receptor ABD).
  • the multispecific protein is configured e.g., through placement or configuration of the domain within a multispecific protein, optionally through use of one or more using domain linkers having a maximal potential length of 18 Angstroms (5 amino acid residues), 36 Angstroms (10 residues) or 54 Angstroms (15 residues) when in a stretched configuration such that the NKp30-binding domain and the cytokine receptor-binding domain, and the CD16-binding domain when present and capable of binding CD16, can assume a membrane planar binding conformation such that each of NKp30, CD16A and cytokine receptor are bound at the surface of an NK cell.
  • domain linkers having a maximal potential length of 18 Angstroms (5 amino acid residues), 36 Angstroms (10 residues) or 54 Angstroms (15 residues) when in a stretched configuration such that the NKp30-binding domain and the cytokine receptor-binding domain, and the CD16-binding domain when present and capable of binding CD16, can assume a membrane planar binding conformation such that
  • the multispecific protein can thus be configured such that the cytokine receptor binding domain is placed, topologically within the multimeric protein, terminally (e.g. C- terminal) to both the NKp30-binding domain and the CD16A-binding domain within the multispecific protein.
  • the cytokine receptor ABD can be placed C-terminally on a polypeptide chain of the multispecific protein such that the positioning allows the cytokine receptor ABD to be topologically C-terminal within the multimeric protein).
  • the NKp30 ABD and the CD16A ABD e.g.
  • a dimeric Fc domain can be positioned adjacent to one another topologically in the protein, optionally connected to one another via a short domain linker.
  • the NKp30 ABD, CD16A ABD and cytokine (or parts thereof) can thus be connected or positioned in series in the protein (or on polypeptide chain(s) thereof.
  • the protein comprises a dimeric Fc domain (the Fc domain for example specified as consisting of two Fc monomers placed on separate polypeptide chains).
  • the NKp30 ABD the dimeric Fc domain and the cytokine are advantageously positioned adjacent to one another topologically (within the topology of the multispecific protein).
  • an NKp30-binding domain (or a part thereof, e.g. a VH or VL) and an Fc domain monomer (or CD16A-binding domain) are placed adjacent to one another on a same polypeptide chain(s), e.g. the adjacent NKp30-binding domain and the CD16A-binding domain can be separated by a domain linker but without any intervening protein domain (e.g. without an intervening domain that binds an antigen, and the cytokine moiety can be placed C-terminally thereto.
  • Figures 2A to 20 show exemplary domain configurations.
  • the multispecific protein comprises a Fc domain dimer comprised of a first and second Fc domain monomer positioned on different polypeptide chains (that dimerize via CH3-CH3 association).
  • the first Fc domain monomer can be fused at its C- terminus to an anti-NKp30 ABD (or portion thereof), and the anti-NKp30 ABD (or portion thereof) is in turn fused at its C-terminus to a cytokine.
  • the portion of an anti-NKp30 ABD can be for example a ((VH or VL)-CH1) unit or ((VH or VL)-CL) unit where the ABD is a Fab.
  • the cytokine receptor-binding domain (cytokine receptor ABD), the NKp30-binding domain (NKp30 ABD) and the CD16-binding domain (CD16 ABD) can be specified as being placed within the one or more polypeptide chains that make up the multispecific protein so that the domains are oriented in a configuration in which they are adjacent to one another or in series, from N to C terminal, on the multimeric (e.g. heteromultimeric) protein. Domains can be optionally separated by a domain linker e.g. a linking peptide of 5-20 residues that does not itself bind to a predetermined antigen.
  • a domain linker e.g. a linking peptide of 5-20 residues that does not itself bind to a predetermined antigen.
  • the multispecific protein can be specified as being configured e.g., through placement or configuration of the domain within a multispecific protein, such that the NKp30 ABD and the cytokine receptor ABD (e.g. the cytokine moiety) have the ability to assume a position where they are on the same side or face of the Fc domain dimer within the multispecific protein molecule, so as to enhance the ability to bind NKp30, CD16A and cytokine receptor in a membrane planar binding conformation.
  • the NKp30 ABD and the cytokine receptor ABD e.g. the cytokine moiety
  • This configuration can be readily implemented in any in any of the heterodimeric, heterotri meric or heterotetrameric proteins of the disclosure, for example by positioning the NKp30 ABD (or a part thereof, if the ABD is formed from association of two polypeptide chains) and the cytokine receptor ABD (or a part thereof, if the ABD is formed from association of two polypeptide chains) on the same polypeptide chain together with one of the Fc domain monomers.
  • the multispecific protein can have the formula (Xi)-l_i-(X 2 )-l- 2 -(X 3 ), where one of Xi and X2 is an NKp30 ABD (e.g. a Fab, an scFv, a VHH) or a part thereof (e.g. the part may be an a VH or VL, a VH-CH1, VH-CL, VL-CL, VL-CH1) and the other is a Fc dimer or part thereof (e.g. an Fc monomer), and X 3 is a cytokine, wherein and l_ 2 are each an optional domain linker.
  • NKp30 ABD e.g. a Fab, an scFv, a VHH
  • the part may be an a VH or VL, a VH-CH1, VH-CL, VL-CL, VL-CH1
  • X 3 is a cytokine, wherein and l_
  • the cytokine is positioned at the C-terminus of the polypeptide chain on which it (or a part thereof) is placed. connects Xi and X 2 via a covalent bond (e.g. peptide bond). I_ 2 connects X 2 and X 3 via a covalent bond (e.g. peptide bond). Xi to X 3 can optionally be specified as being arranged from the topological N- to C-terminus of the protein.
  • the multispecific protein can further comprise an ABD that binds an antigen of interest, e.g. connected at the N-terminus oif Xi or positioned N-terminally within the topology of the protein.
  • the different elements Xi, X 2 and X 3 can each be readily distributed onto two or more different polypeptide chains within the protein.
  • the multispecific protein can optionally be characterized as comprising a first polypeptide chain comprising the formula (Xi)-I_i-(X 2 )-I_ 2 -(X 3 ), where one of Xi and X 2 is an NKp30 ABD or part thereof and the other is a Fc monomer, and X 3 is a cytokine or a part thereof, wherein and l_ 2 are each an optional domain linker.
  • the cytokine can thus be positioned at the C-terminus of the polypeptide chain on which it (or a part thereof) is placed.
  • the multispecific protein can further comprise an ABD that binds an antigen of interest, wherein the ABD (or a part thereof) is placed on the first polypeptide chain or on a separate polypeptide chain that associates (e.g. dimerizes) with first polypeptide chain (or with any other chain of the protein).
  • the protein can then comprise one, two or more additional polypeptide chains that provide the complementary domains for the NKp30 ABD (when the NKp30 ABD is a part of an ABD,) the Fc monomer (so as to form an Fc dimer), the cytokine (where the cytokine is a part of a cytokine) and/or the ABD that binds an antigen of interest.
  • additional polypeptide chains can thus associate (e.g. dimerize) with first or other polypeptide chain of the protein via non-covalent interactions and optionally further covalent interactions.
  • the multispecific protein can optionally be characterized as comprising (i) a first polypeptide chain comprising the formula (Xi)-l_i-(X 2a ), where X1 is a first Fc monomer and za is a first part of a NKp30 ABD, and (ii) a second polypeptide chain comprising the formula (X2 b )-l-2-(X3), wherein X2 b is a second part of a NKp30 ABD (e.g.
  • the multispecific protein can further comprise an ABD that binds an antigen of interest, wherein the ABD (or a part thereof) is placed on the first polypeptide chain (e.g. N-terminal to Xi) or on a separate polypeptide chain that associates (e.g. dimerizes) with first polypeptide chain (or with any other chain of the protein).
  • the multispecific protein can further com prise an ABD that binds CD16A, optionally the ABD is a dimeric Fc domain; the ABD that binds CD16A (or a part thereof) ca be placed on the first polypeptide chain (e.g. N-terminal to Xi), and when the ABD is a dimeric Fc domain one of the Fc monomers can be placed on the first polypeptide chain and the second Fc monomer can be placed on a separate polypeptide chain that associates (e.g. dimerizes) with first polypeptide chain.
  • the multispecific protein can be characterized by having only one cytokine receptor binding domain.
  • the multispecific protein can be characterized by having only one NKp30 binding domain.
  • Certain exemplary heteromultimeric proteins can comprise the following general domain organization of structure 1a or 1b, where the CD16 ADC (e.g. Fc domain) and NKp30 ABD are immediately adjacent to other another within the protein, and NKp30 ABD is immediately adjacent to the cytokine receptor ABD (embodied as a cytokine (Cyt)), and wherein the NKp30 ABD is interposed between the CD16 ABD and the Cyt:
  • NKp30 ABD (Fc domain dimer) (NKp30 ABD) (Cyt) (Structure 1b) wherein the NKp30 ABD and the CD16ABD (e.g. Fc domain dimer) are connected by a domain linker and the NKp30 ABD and Cyt are connected by a domain linker.
  • CD16ABD e.g. Fc domain dimer
  • the NKp30 ABD can conveniently be a Fab, a single domain antibody or an scFv.
  • the CD16 ABD can be an Fc domain, a Fc domain dimer, an Fc domain of human lgG1 subtype.
  • the Cyt can be for example an IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b) polypeptide, optionally wherein the polypeptide is a variant cytokine that differs by at least one residue from the wild-type human cytokine counterpart.
  • the protein of structure 1a can comprise an ABD that binds an antigen of interest on a target cell (Antigen ABD) placed terminal to (e.g. N-terminal to) the CD16 ABD (e.g. Fc domain dimer), as in a heteromultimeric protein having the structure 1c or 1d below: (Antigen ABD) n (CD16 ABD) (NKp30 ABD) (Cyt) (Structure 1c) or
  • Structure 1d (Antigen ABD) n (Fc domain dimer) (NKp30 ABD) (Cyt) (Structure 1d) wherein “n” is 1 or 2, and the Antigen ABD and the CD16 ABD (e.g. Fc domain dimer) are connected by a linker, optionally wherein the linker is an immunoglobulin hinge polypeptide, wherein the CD16 ABD and the NKp30 ABD are connected by a linker and the NKp30 ABD and Cyt are connected by a linker.
  • Structure 1d can also be represented as Structure 1 e:
  • the multispecific protein can be characterized as binding monovalently to each of the NKp30 polypeptide and the cytokine receptor, and being capable of directing an NKp30-expressing NK cell to lyse a target cell expressing the antigen of interest.
  • the multi-specific protein can bind (i) to antigen of interest on target cells, (ii) to NKp30 on NK cells, (iii) to CD16A on NK cells and (iv) to the cytokine receptor on NK cells (e.g.
  • NKp30 the protein acts as an NKp30 agonist
  • cytokine receptor the protein acts as a cytokine receptor agonist
  • the multi-specific protein in the presence of NK cells and target cells, can induce the cytotoxicity of, cytokine receptor pathway signaling in (as assessed by STAT signaling) and/or activation of the NK cells, wherein such cytotoxicity, activation and/or signaling is greater (e.g. at least 100-fold or 1000-fold lower ECso value) than that observed when the multi-specific protein is contacted with NK cells in the absence of target cells.
  • the multi-specific protein can bind NKp30 and CD122 on NK cells (e.g. the protein comprises an IL2 or IL15 moiety, optionally an modified or variant IL2 or IL15 with decreased binding to CD25), and, when bound to both NKp30 and CD122, can induce signaling in the NK cells through both NKp30 and CD122.
  • the multi-specific protein can bind NKp30, CD16A and CD122 on NK cells, and, when bound to NKp30, CD16 and CD122, can induce signaling in the NK cells through NKp30, CD16A and CD122.
  • Cytokine receptor signaling can be assessed by measuring STAT5, optionally wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
  • the multi-specific protein can bind NKp30 and IL-21 R on NK cells (e.g. the protein comprises an IL21 moiety), and, when bound to both NKp30 and IL-21R, can induce signaling in the NK cells through both NKp30 and IL-21 R.
  • the multi-specific protein can bind NKp30, CD16A and IL-21 R on NK cells, and, when bound to NKp30, CD16A and IL- 21 R, can induce signaling in the NK cells through NKp30, CD16A and IL-21 R.
  • cytokine signaling is assessed by measuring STAT3, wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
  • the multi-specific protein can bind NKp30 and IL-18R on NK cells (e.g. the protein comprises an IL18 moiety), and, when bound to both NKp30 and IL-18R (IL-18Ra and/or IL-18R ), can induce signaling in the NK cells through both NKp30 and IL-18R.
  • the multi-specific protein can bind NKp30, CD16A and IL-18R on NK cells, and, when bound to NKp30, CD16A and IL-18R, can induce signaling in the NK cells through NKp30, CD16A and IL-18R.
  • cytokine signaling signaling is assessed by measuring STAT3, wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
  • the multi-specific protein can bind NKp30 and IL-7R (e.g. IL-7Ra (CD127) and/or CD132) on NK cells (e.g. the protein comprises an IL-7 moiety), and, when bound to both NKp30 and IL-7R, can induce signaling in the NK cells through both NKp30 and IL-7Ra.
  • the multi-specific protein can bind NKp30, CD16A and IL-7R on NK cells, and, when bound to NKp30, CD16A and IL-7R, can induce signaling in the NK cells through NKp30, CD16A and IL-7R.
  • cytokine signaling signaling is assessed by measuring STAT5, wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
  • the multi-specific protein can bind NKp30 and IL-27R (e.g. IL-27Ra and/or GP130) on NK cells (e.g. the protein comprises an IL-27 moiety), and, when bound to both NKp30 and IL-27R, can induce signaling in the NK cells through both NKp30 and IL-27R.
  • the multi-specific protein can bind NKp30, CD16A and IL-27R on NK cells, and, when bound to NKp30, CD16A and IL-27R, can induce signaling in the NK cells through NKp30, CD16A and IL-27R.
  • NKp30 and/or CD16A can be assessed by a marker of NK cell activation (e.g. a marker used in the Examples, CD69 expression, etc.).
  • cytokine signaling signaling is assessed by measuring STAT 1 , wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
  • the multi-specific protein can bind NKp30 and IL-12R (e.g., II_-12Bb1 and/or II_-12Bb2) on NK cells (e.g. the protein comprises an IL-27 moiety), and, when bound to both NKp30 and IL-12R, can induce signaling in the NK cells through both NKp30 and IL-12R.
  • the multi-specific protein can bind NKp30, CD16A and IL-12R on NK cells, and, when bound to NKp30, CD16A and IL-12R, can induce signaling in the NK cells through NKp30, CD16A and IL-12R.
  • cytokine signaling signaling is assessed by measuring STAT4, wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
  • the multi-specific protein can bind NKp30 and IFNAR on NK cells, and, when bound to both NKp30 and IFNAR (IFNAR1 and/or IFNAR2), can induce signaling in the NK cells through both NKp30 and IFNAR.
  • the multi-specific protein can comprise an IFN-a or IFN-b moiety
  • the multi-specific protein can bind NKp30, CD16A and IFNAR on NK cells, and, when bound to both NKp30, CD16A and IFNAR, can induce signaling in the NK cells through NKp30, CD16A and IFNAR.
  • cytokine signaling signaling is assessed by measuring STAT (e.g., STAT1, STAT2 or lFN regulatory factor (I RF)-9), wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
  • STAT e.g., STAT1, STAT2 or lFN regulatory factor (I RF)-9
  • I RF lFN regulatory factor
  • NKp30 and/or CD16A can be assessed by a marker of NK cell activation (e.g. a marker used in the Examples, CD69 expression, etc.).
  • a marker of NK cell activation e.g. a marker used in the Examples, CD69 expression, etc.
  • the multispecific protein comprises at least a portion of a human Fc domain, e.g. a portion sufficient such that the Fc domain is bound by a human FcRn polypeptide, optionally wherein said FcRn binding affinity as assessed by SPR is within 1-log of that of a conventional human lgG1 antibody.
  • the multispecific proteins advantageously are able to potently mobilize both CD16 + and CD16- NK cells (all NK cells are NKp30 + ).
  • the multispecific protein comprises two or more polypeptide chains, i.e. it comprises a multi-chain protein (also referred as a multimeric protein).
  • a multi-chain protein also referred as a multimeric protein.
  • the multispecific protein or multi-chain protein can be a hetero-dimer, hetero-trimer or hetero- tetramer or may comprise more than four polypeptide chains.
  • any antigen binding domain e.g. the ABD that binds the antigen of interest (e.g. tumor antigen), NKp30, or cytokine receptor
  • the antigen of interest e.g. tumor antigen
  • NKp30 e.g. IL-12
  • cytokine receptor e.g. IL-12 receptor
  • an antigen binding domain can be made of two or more protein domains placed on separate polypeptide chains, such that the antigen binding domain binds its target when two or more complementary protein domains (e.g. as VH/VL pairs) are associated in the multimeric protein.
  • the multispecific protein can bind to the antigen of interest (e.g. cancer antigen) in monovalent or multivalent manner. Where the multispecific protein binds the antigen of interest monovalently, binds NKp30 monovalently and binds the cytokine receptor monovalently, the multispecific protein can be indicated as having a 1 :1:1 configuration. Where the multispecific protein binds the antigen of interest bivalently, binds NKp30 monovalently and binds the cytokine receptor monovalently, the multispecific protein can be indicated as having a 2:1:1 configuration. Representative examples of different 1 :1:1 and 2:1:1 configurations are shown in Figure 2.
  • the multispecific protein can be characterized as having a structure in which the freedom of motion (intrachain domain motion) or flexibility of one or more antigen binding domains (ABDs) is increased, e.g. compared to the ABDs of a conventional human IgG antibody.
  • a multispecific protein comprising a structure that permits the antigen binding site of the first antigen binding domain and the antigen binding site of the second antigen binding domain to be separated by a distance that results in enhanced function, e.g., the ability of the multispecific protein to induce NKp30 signaling and lysis of target cells, e.g., optionally a distance of less than 80 angstrom (A).
  • Multispecific proteins wherein the ABDs possess greater flexibility and/or are separated by an optimized distance may enhance the formation of a lytic NKp30-target synapse, thereby potentiating NKp30-mediated signaling.
  • Such flexibility and/or domain of motion can be readily achieved through the use of linkers (e.g. flexible amino acid based linkers) that separate the NKp30 binding domain from the Fc domain (e.g. the Fc domain dimer, or more generally the rest of the multispecific protein).
  • the multispecific protein can be characterized as having increased freedom of motion of the antigen binding domains (e.g. compared to the ABDs of a conventional human IgG antibody, e.g., a human lgG1 antibody).
  • a multimeric Fc domain-containing protein e.g. a heterodimer or heterotrimer
  • an antigen binding domain e.g., the ABD that binds NKp30
  • the linker can provide flexibility or freedom of motion of one or more ABDs by conferring the ability to bend thereby potentially decreasing the angle between the ABD and the Fc domain (or between the two ABDs) at the linker.
  • both antigen binding domains are linked or fused to the Fc domain via a linker, typically a flexible peptide linker.
  • a linker typically a flexible peptide linker.
  • the protein with increased freedom of motion permits the protein to adopt a conformation in which the distance between the NKp30 binding site and the antigen of interest binding site is less that than observed in proteins in which both binding domains were Fabs, or less than in full length antibodies.
  • An ABD can be connected to an Fc domain monomer (or CH2 or CH3 domain thereof) via a domain linker.
  • the linker can be a polypeptide linker, for example peptide linkers comprising a length of at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, or more.
  • the linkers comprises a length of between 2-4 residues, between 2-5 residues, between 2-6 residues, between 2-8 residues, between 5-10 residues, between 2-15 residues, between 4-15 residues, between 3-15 residues, between 5- 15 residues, between 10-15 residues, between 4-20 residues, between 5-20 residues, between 2-20 residues, between 10-30 residues, or between 10-50 residues.
  • a linker comprises an amino acid sequence derived from an antibody constant region, e.g., an amino acid sequence from a CH1 or CK domain (e..g an an N-terminal sequence from a CH1 or CK domain) or from a hinge.
  • a linker comprises the amino acid sequence RTVA.
  • a linker is a flexible linker predominantly or exclusively comprised of glycine and/or serine residues, e.g., comprising an amino acid sequence (G x S) n where G is 1, 2, 3 or 4 and n is an integer from 1-10, from 1-6 or from 1-4.
  • the linker comprises 1-20 or 1-10 further amino acid residues.
  • heterotrimer having a polypeptide chain 1 , 2 and 3:
  • V a -i, V b -i, V a -2 and V -2 are each a VH domain or a VL domain, wherein one of V a -i and V -i is a V h nand the other is a VL, and wherein V a -i and VM form a first antigen binding domain (ABD) that binds an antigen of interest, wherein one of V a-2 and V -2 is a VH and the other is a VL and wherein V a-2 and V -2 form a second ABD that binds NKp30;
  • ABD antigen binding domain
  • CH1 is a human immunoglobulin CH1 domain and CL is a human light chain constant domain; one of (CH1 or CL) a and (CH1 or CL) c is a CH1 and the other is a CL such that a (CH1/CL) pair is formed; one of (CH1 or CL) b and (CH1 or CL) d is a CH1 and the other is a CL such that a (CH1/CL) pair is formed; Hinge is an immunoglobulin hinge region or portion thereof;
  • L1 and L2 are each an amino acid domain linker, wherein L1 and L2 can be different or the same;
  • CH2 and CH3 are human immunoglobulin CH2 and CH3 domains, respectively.
  • Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21, IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
  • Cyt is a wild-type or variant human IL-2, IL-15, IL-21, IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
  • a heterotrimer having a polypeptide chain 1, 2 and 3:
  • heterodimer having a polypeptide chain 1 and 2:
  • V a -i, V b -i, V a and V are each a VH domain or a VL domain wherein one of V a -i and V -i is a V h nand the other is a VL, and wherein V a -i and VM form a first antigen binding domain (ABD) that binds an antigen of interest), wherein one of V a-2 and V b-2 is a VH and the other is a VL and wherein V a-2 and V b-2 form a second ABD that binds NKp30;
  • ABS antigen binding domain
  • CH1 is a human immunoglobulin CH1 domain and CL is a light chain constant domain; one of (CH1 or CL) a and (CH1 or CL) b is a CH1 and the other is a CL such that a (CH1/CL) pair is formed;
  • Hinge is an immunoglobulin hinge region or portion thereof
  • L1, L2 and L3 are each an amino acid domain linker, wherein L1, L2 and L3 can be different or the same;
  • CH2 and CH3 are human immunoglobulin CH2 and CH3 domains, respectively.
  • Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21, IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
  • a multispecific protein is a heteromultimer, heterodimer, heterotrimer, heterotetramer having a structure or domain arrangement as shown in any of Figures 2A to 20.
  • an ABD e.g., the anti- NKp30 ABD, the ABD that binds the antigen of interest or tumor antigen
  • VH immunoglobulin heavy chain variable domain
  • VL immunoglobulin light chain variable domain
  • a VH can be specified as having the amino acid sequence of a human VH domain.
  • a VL can be specified as having the amino acid sequence of a human VL domain.
  • a VH region comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence encoded by a gene of a human V gene group selected from the group consisting of IGHV1-18, IGHV1-2, IGHV1-24, IGHV1-3, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV1-69-2, IGHV1-69D, IGHV1-8, IGHV2-26, IGHV2-5, IGHV2-70, IGHV2-70D, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21 , IGHV3-23, IGHV3-23D, IGHV3-30, IGHV3-30-3, IGHV3-30-5, IGHV3-33, IGHV3-43, IGHV3-43D, IGHV3-48, IGHV3-
  • a VH region comprises a VH comprising an amino acid sequence (e.g. CDR(s) and/or a human framework region(s), for example according to Kabat numbering) from said gene.
  • a VH region comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence of SEQ ID NOS: 45-122.
  • a VL region comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence encoded by a gene of a human V gene group selected from the group consisting of IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-39, IGKV1-5, IGKV1-6, IGKV1-8, IGKV1-9, IGKV1-NL1 , IGKV1 D-12, IGKV1 D-13, IGKV1 D-16, IGKV1 D-17, IGKV1 D-33, IGKV1 D-43, IGKV1 D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2- 30, IGKV2-40, IGKV2D-26,
  • a VL region comprises a VL comprising an amino acid sequence (e.g. CDR(s) and/or a human framework region(s), for example according to Kabat numbering) from said gene.
  • a VL region comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence of SEQ ID NOS: 123- 212.
  • an ABD comprises an scFv or Fab, wherein the scFv comprises a VH comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, any of 45-122, and 249, a domain linker, and a VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, any of 123-212, and 250; and wherein the Fab comprises one VH comprising an amino acid sequence at least 90% identical to a selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, any of 45-122, and 249, one VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, any of 123-212, and 250, one human CH1 domain comprising an amino acid sequence at least 90%
  • an IL2 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-2 polypeptide of any of SEQ ID NOS: 213-226, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • the IL2 further comprises 2, 3, 4, 5 or more amino acid substitutions that reduce binding to CD25, e.g. substitutions at any of the residues disclosed herein.
  • an IL15 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-15 polypeptide of any of SEQ ID NO: 227, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • an IL12 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-12 polypeptide of any of SEQ ID NOS: 247 and/or 248, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • an IL7 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-7 polypeptide of any of SEQ ID NO: 244, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • an IL27 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-21 polypeptide of any of SEQ ID NOS: 245 and/or 246, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • an IL21 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-27 polypeptide of any of SEQ ID NOS: 229 or 230, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • an IL18 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-18 polypeptide of any of SEQ ID NO: 231 , or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • an IFN-a comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IFN-a polypeptide of any of SEQ ID NOS: 232-242, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • an IFN-b comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IFN-a polypeptide of any of SEQ ID NO: 243, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • an Fc domain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the Fc polypeptide of any of SEQ ID NOS: 30-35, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • a CH1, CH2 and CH3 domain respectively comprise an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the CH1, CH2 or CH3 polypeptide of SEQ ID NO: 26, 27 or 28, respectively, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • a CK or CL domain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the CK polypeptide of any of SEQ ID NO: 29, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • a hinge domain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the CK polypeptide of any of SEQ ID NOS: 36-40.
  • a multispecific protein comprises:
  • an ABD that binds to the antigen of interest wherein the ABD comprises an scFv or Fab, a.
  • the scFv comprises a VH comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and any of 45-122, a domain linker, and a VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25 and any of 123-212; and b.
  • the Fab comprises one VH comprising an amino acid sequence at least 90% identical to a selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and any of 45-122, one VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25 and any of 123-212, one human CH1 domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 26 and one human CL domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 29, wherein the VH is fused to one of the CH1 or CL domains, and the VL is fused to the other of the CH1 or CL domains,
  • an ABD that binds to a human NKp30 polypeptide wherein the ABD comprises an scFv or Fab, a. wherein the scFv comprises a VH comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 45-122 or 249, a domain linker, and a VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 123-212 or 250; and b.
  • the Fab comprises one VH comprising an amino acid sequence at least 90% identical to a selected from SEQ ID NOS: 45-122 or 249 and any of 236-313, one VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 123-212 or 250, one human CH1 domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 26 and one human CL domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 29, wherein the VH is fused to one of the CH1 or CL domains, and the VL is fused to the other of the CH1 or CL domains,
  • Fc domain dimer comprising two Fc domain monomer polypeptides, wherein each Fc domain monomer polypeptide comprises an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 30-35;
  • cytokine polypeptide comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 213-248, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof, fused, via a domain linker, to the C-terminus of one of the polypeptide chains of the multispecific protein.
  • a multispecific protein comprises: a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of a first chain of a heterotrimeric protein described herein, a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of a second chain of a heterotrimeric protein described herein and a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of a third chain of a heterotrimeric protein described herein.
  • a multispecific protein comprises: a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of a first chain of a heterodimeric protein described herein, and a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of a second chain of a heterodimeric protein described herein.
  • a multispecific protein comprises: a polypeptide comprising an amino acid sequence of a first chain of a heterotrimeric protein described herein, a polypeptide comprising an amino acid sequence of a heterotrimeric protein described herein and a polypeptide comprising an amino acid sequence of a third chain of a heterotrimeric protein described herein.
  • a multispecific protein comprises: a polypeptide comprising an amino acid sequence of a first chain of a heterodimeric protein described herein, and a polypeptide comprising an amino acid sequence of a second chain of a heterodimeric protein described herein.
  • a multispecific protein comprises: a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 251 , a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 252 and a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 253.
  • a multispecific protein comprises: a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 257, a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 258; and a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 259.
  • a recombinant nucleic acid encoding a first polypeptide chain, and/or a second polypeptide chain, and/or a third polypeptide chain and/or a fourth polypeptide.
  • the invention provides a recombinant host cell comprising a nucleic acid encoding a first polypeptide chain, and/or a second polypeptide chain and/or a third polypeptide chain, optionally wherein the host cell produces a multimeric or other protein according to the invention with a yield (final productivity or concentration before or after purification) of at least 1 , 2, 3 or 4 mg/L.
  • kits or set of nucleic acids comprising a recombinant nucleic acid encoding a first polypeptide chain of the according to the invention, a recombinant nucleic acid encoding a second polypeptide chain according to the invention, and, optionally, a recombinant nucleic acid encoding a third polypeptide chain according to the invention. Also provided are methods of making dimeric, trimeric and tetrameric proteins according to the invention.
  • any of the methods can further be characterized as comprising any step described in the application, including notably in the “Detailed Description of the Invention”).
  • the invention further relates to methods of identifying, testing and/or making proteins described herein.
  • the invention further relates to a multispecific protein obtainable by any of present methods.
  • the disclosure further relates to pharmaceutical or diagnostic formulations containing at least one of the multispecific proteins disclosed herein.
  • the disclosure further relates to methods of using the subject multispecific proteins in methods of treatment or diagnosis.
  • FIGURES Figure 1 shows the topology of the multispecific NK cell engager (NKCE) protein that binds on one face to a tumor antigen on a tumor cell, and on another face to an NK cell via a triple receptor cis-presentation of II_2bg complex, NKp30 and CD16A.
  • NKCE multispecific NK cell engager
  • IL2v capture on NK cells may improve binding to CD122 and mimic CD25-mediated IL-2 presentation.
  • Figures 2A and 2B show an exemplary multispecific protein in T5 format that binds to NKp30, CD16A and cytokiner receptor (e.g. CD122) on an NK cell, and to tumor antigen (e.g. TA, Tag, CD20) on a tumor cell.
  • Figures 2C-20 show other multimeric protein structures with or without CD16A binding.
  • Figure 3 shows activation of TReg cells by heterotrimer proteins that contained either a wild-type IL-2 or a variant IL2, and that lacked binding to NKp30, CD16A and antigen of interest.
  • the protein containing the variant IL2 showed a strongly decreased ability to activate Treg cells compared to wild-type IL-2 and to the heterotrimer protein containing wild-type IL- 2.
  • Figure 4 shows ability to direct purified NK cells to lyse CD20-positive RAJ I tumor target cells by CD20 x NKp30 binding proteins.
  • GA101-T5-Com_mAb8-IL2v proteins, coengaging CD16A and NKp30 on NK cells and GA101-T6-Com_mAb8-IL2v proteins, engaging only NKp30 were highly potent in mediating NK cell lysis of tumor target cells.
  • Figures 5, 6, 7 and 8 show % of pSTAT5 cells among NK cells, CD4 T cells CD8 T cells and Treg cells, respectively.
  • GA101-T5-Com_mAb8-IL2v, GA101-T6-Com_mAb8-IL2v and GA101-T6-IC-IL2v displayed comparable activation of each cell type.
  • GA101- T5-Com_mAb8-IL2v resulted in a strong increase in potency in the ability to cause an increase in percent of pSTAT5+ cells among the NK cells, compared to that observed in CD4 T cells CD8 T cells and Treg cells.
  • the GA101-T5-Com_mAb8-IL2v protein therefore permitted a selective activation of NK cells over Treg cells, CD4 T cells and CD8 T cells.
  • Figure 9 shows percentage of IFN-g expressing NK cells within PBMCs in presence of RAJI targeted tumor cells, upon treatment with GA101-T5-Com_mAb8-IL2v, GA101-T6- Com_mAb8-IL2v, IC-T6-IC-IL2v that did not bind CD16A or NKp30 on NK cells or human IgG isotype antibody having the GA101 VH/VL domains (HuG1).
  • Figure 10 shows MIR1b MedFI on NK cells within PBMCs in presence of RAJI targeted tumor cells, induced by GA101-T5-Com_mAb8-IL2v, GA101-T6-Com_mAb8-IL2v and IC-T6- IC-IL2v that did not bind CD16A or NKp30 on NK cells.
  • Figures 11A and B show respectively induction of IFN-g production and MIR1b MedFI, by GA101-T5-Com_mAb8-IL2v and GA101-T6-Com_mAb8-IL2v in NK cells, without any effect on IFN-g production and MIR1b MedFI in CD4 T cells, CD8 T cells or in gd T cells within PBMCs in presence of RAJI targeted tumor cells.
  • the term "antigen binding domain” or ”ABD refers to a domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope.
  • said domain can comprise a hypervariable region, optionally a VH and/or VL domain of an antibody chain, optionally at least a VH domain.
  • the binding domain may comprise at least one complementarity determining region (CDR) of an antibody chain.
  • the binding domain may comprise a polypeptide domain from a non-immunoglobulin scaffold.
  • antibody herein is used in the broadest sense and specifically includes full- length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and derivatives, so long as they exhibit the desired biological activity.
  • Various techniques relevant to the production of antibodies are provided in, e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).
  • An “antibody fragment” comprises a portion of a full- length antibody, e.g. antigen-binding or variable regions thereof.
  • antibody fragments include Fab, Fab', F(ab)2, F(ab’)2, F(ab)3, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VH and CH1 domain), and dAb (typically a V H domain) fragments; V H , V L , VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10: 949-57); camel IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments, and one or more isolated CDRs or a functional paratope, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment.
  • scFv single
  • hypervariable region when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • the hypervariable region generally comprises amino acid residues from a "complementarity-determining region” or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. 1991) and/or those residues from a "hypervariable loop" (e.g.
  • the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as “Kabat position”, "variable domain residue numbering as in Kabat” and “according to Kabat” herein refer to this numbering system for heavy chain variable domains or light chain variable domains.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
  • frame or "FR” residues as used herein is meant the region of an antibody variable domain exclusive of those regions defined as CDRs.
  • Each antibody variable domain framework can be further subdivided into the contiguous regions separated by the CDRs (FR1 , FR2, FR3 and FR4).
  • constant region as defined herein is meant an antibody-derived constant region that is encoded by one of the light or heavy chain immunoglobulin constant region genes.
  • constant light chain or “light chain constant region” or “CL” as used herein is meant the region of an antibody encoded by the kappa (CK) or lambda (C l) light chains.
  • the constant light chain typically comprises a single domain, and as defined herein refers to positions 10S- 214 of CK, or CA, wherein numbering is according to the EU index (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda).
  • constant heavy chain or “heavy chain constant region” as used herein is meant the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes to define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively.
  • the constant heavy chain refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 118-447, wherein numbering is according to the EU index.
  • Fab or "Fab region” as used herein is meant a unit that comprises the VH, CH1 , VL, and CL immunoglobulin domains.
  • the term Fab includes a unit that comprises a VH-CH 1 moiety that associates with a VL-CL moiety, as well as crossover Fab structures in which there is crossing over or interchange between light- and heavy-chain domains.
  • a Fab may have a VH-CL unit that associates with a VL-CH1 unit.
  • Fab may refer to this region in isolation, or this region in the context of a protein, multispecific protein or ABD, or any other embodiments as outlined herein.
  • single-chain Fv or “scFv” as used herein are meant antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
  • Fv or “Fv fragment” or “Fv region” as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody.
  • Fc or “Fc region”, as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N- terminal to these domains.
  • Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cy2 (CH2) and Cy3 (CH3) and optionally the hinge between Cy1 and Cy2.
  • Fc polypeptide or “Fc-derived polypeptide” as used herein is meant a polypeptide that comprises all or part of an Fc region.
  • Fc polypeptides herein include but are not limited to antibodies, Fc fusions and Fc fragments.
  • Fc regions according to the invention include variants containing at least one modification that alters (enhances or diminishes) an Fc associated effector function.
  • Fc regions according to the invention include chimeric Fc regions comprising different portions or domains of different Fc regions, e.g., derived from antibodies of different isotype or species.
  • variable region as used herein is meant the region of an antibody that comprises one or more Ig domains substantially encoded by any of the VL (including VK (VK) and VA) and/or VH genes that make up the light chain (including k and l) and heavy chain immunoglobulin genetic loci respectively.
  • VL or VH consists of a "framework" or “FR” region interrupted by three hypervariable regions referred to as “complementarity determining regions” or "CDRs".
  • CDRs complementarity determining regions
  • the extent of the framework region and CDRs have been precisely defined, for example as in Kabat (see “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, (1983)), and as in Chothia.
  • the framework regions of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs, which are primarily responsible for binding to an antigen.
  • the term “specifically binds to” means that an antibody or polypeptide can bind preferably in a competitive binding assay to the binding partner, e.g. NKp30, as assessed using either recombinant forms of the proteins, epitopes therein, or native proteins present on the surface of isolated target cells.
  • a competitive binding assay to the binding partner, e.g. NKp30, as assessed using either recombinant forms of the proteins, epitopes therein, or native proteins present on the surface of isolated target cells.
  • an antibody or polypeptide e.g. a multispecific protein
  • a particular multispecific protein or a particular monoclonal antibody it means that the antibody or polypeptide competes with the particular multispecific protein or monoclonal antibody in a binding assay using either recombinant target (e.g. NKp30) molecules or surface expressed target (e.g. NKp30) molecules.
  • test multispecific protein or antibody reduces the binding of multispecific protein GA101-T5-Com_mAb8-IL2v to a NKp30 polypeptide or NKp30-expressing cell in a binding assay, the test multispecific protein or antibody is said to “compete” respectively with GA101-T5-Com_mAb8-IL2v.
  • affinity means the strength of the binding of an antibody or protein to an epitope.
  • the affinity of an antibody is given by the dissociation constant KD, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody- antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen.
  • KD dissociation constant
  • a “determinant” designates a site of interaction or binding on a polypeptide.
  • epitope refers to an antigenic determinant, and is the area or region on an antigen to which an antibody or protein binds.
  • a protein epitope may comprise amino acid residues directly involved in the binding as well as amino acid residues which are effectively blocked by the specific antigen binding antibody or peptide, /.e., amino acid residues within the "footprint" of the antibody. It is the simplest form or smallest structural area on a complex antigen molecule that can combine with e.g., an antibody or a receptor.
  • Epitopes can be linear or conformational/structural.
  • linear epitope is defined as an epitope composed of amino acid residues that are contiguous on the linear sequence of amino acids (primary structure).
  • formational or structural epitope is defined as an epitope composed of amino acid residues that are not all contiguous and thus represent separated parts of the linear sequence of amino acids that are brought into proximity to one another by folding of the molecule (secondary, tertiary and/or quaternary structures). A conformational epitope is dependent on the 3-dimensional structure.
  • formational is therefore often used interchangeably with ‘structural’.
  • Epitopes may be identified by different methods known in the art including but not limited to alanine scanning, phage display, X-ray crystallography, array- based oligo-peptide scanning or pepscan analysis, site-directed mutagenesis, high throughput mutagenesis mapping, H/D-Ex Mass Spectroscopy, homology modeling, docking, hydrogen- deuterium exchange, among others.
  • Valent or “valency” denotes the presence of a determined number of antigen-binding moieties in the antigen-binding protein.
  • a natural IgG has two antigen-binding moieties and is bivalent.
  • a molecule having one binding moiety for a particular antigen is monovalent for that antigen.
  • amino acid modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence.
  • An example of amino acid modification herein is a substitution.
  • amino acid modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a given position in a protein sequence with another amino acid.
  • substitution Y50W refers to a variant of a parent polypeptide, in which the tyrosine at position 50 is replaced with tryptophan. Amino acid substitutions are indicated by listing the residue present in wild-type protein / position of residue / residue present in mutant protein.
  • a “variant" of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or “parent” polypeptide.
  • the polypeptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence.
  • Constant amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Families of amino acid residues having similar side chains are known in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • identity refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e. , "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md.
  • NCBI National Center for Biotechnology Information
  • An “isolated” molecule is a molecule that is the predominant species in the composition wherein it is found with respect to the class of molecules to which it belongs (i.e., it makes up at least about 50% of the type of molecule in the composition and typically will make up at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the species of molecule, e.g., peptide, in the composition).
  • composition of a polypeptide will exhibit 98%, 98%, or 99% homogeneity for polypeptides in the context of all present peptide species in the composition or at least with respect to substantially active peptide species in the context of proposed use.
  • treatment refers to preventing, alleviating, managing, curing or reducing one or more symptoms or clinically relevant manifestations of a disease or disorder, unless contradicted by context.
  • “treatment” of a patient in whom no symptoms or clinically relevant manifestations of a disease or disorder have been identified is preventive or prophylactic therapy
  • “treatment” of a patient in whom symptoms or clinically relevant manifestations of a disease or disorder have been identified generally does not constitute preventive or prophylactic therapy.
  • NK cells refers to a sub-population of lymphocytes that is involved in non-conventional immunity.
  • NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD56 and/or NKp46 for human NK cells, the absence of the alpha/beta or gamma/delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express "self" MHC/HLA antigens by the activation of specific cytolytic machinery, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response.
  • NK cells any of these characteristics and activities can be used to identify NK cells, using methods well known in the art. Any subpopulation of NK cells will also be encompassed by the term NK cells.
  • active NK cells designate biologically active NK cells, including NK cells having the capacity of lysing target cells or enhancing the immune function of other cells.
  • NK cells can be obtained by various techniques known in the art, such as isolation from blood samples, cytapheresis, tissue or cell collections, etc. Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). Humana Press pp. 219-238 (2000).
  • an agent that has “agonist” activity at NKp30 is an agent that can cause or increase "NKp30 signaling".
  • NKp30 signaling refers to an ability of an NKp30 polypeptide to activate or transduce an intracellular signaling pathway. Changes in NKp30 signaling activity can be measured, for example, by assays designed to measure changes in NKp30 signaling pathways, e.g.
  • reporter genes can be naturally occurring genes (e.g. monitoring cytokine production) or they can be genes artificially introduced into a cell. Other genes can be placed under the control of such regulatory elements and thus serve to report the level of NKp30 signaling.
  • NKp30 refers to a protein or polypeptide encoded by the Ncr3 (natural cytotoxicity triggering receptor 3) gene or by a cDNA prepared from such a gene. Any naturally occurring isoform, allele, ortholog or variant is encompassed by the term NKp30 polypeptide (e.g., an NKp30 polypeptide 90%, 95%, 98% or 99% identical to SEQ ID NO 1 , or a contiguous sequence of at least 20, 30, 50, 100 or 200 amino acid residues thereof).
  • the 201 amino acid residue sequence of human NKp30 is shown below:
  • SEQ ID NO: 1 corresponds to NCBI accession number 014931, the disclosure of which is incorporated herein by reference.
  • the proteins described herein can be conveniently configured and produced using well known immunoglobulin-derived domains, notably heavy and light chain variable domains, hinge regions, CH1 , CL, CH2 and CH3 constant domains, and wild-type or variant cytokine polypeptides. Domains placed on a common polypeptide chain can be fused to one another either directly or connected via linkers, depending on the particular domains concerned.
  • the immunoglobulin-derived domains will preferably be humanized or of human origin, thereby providing decreased risk of immunogenicity when administered to humans.
  • advantageous protein formats are described that use minimal non-immunoglobulin linking amino acid sequences (e.g. not more than 4 or 5 domain linkers, in some cases as few as 1 or 2 domain linkers, and use of domains linkers of short length), thereby further reducing risk of immunogenicity.
  • Immunoglobulin variable domains are commonly derived from antibodies (immunoglobulin chains), for example in the form of associated VL and VH domains found on two polypeptide chains, or a single chain antigen binding domain such as an scFv, a VH domain, a VL domain, a dAb, a V-NAR domain or a VHH domain.
  • an antigen binding domain e.g., ABDi and ABD2
  • the term “antigen-binding protein” can be used to refer to an immunoglobulin derivative with antigen binding properties.
  • the binding protein comprises an immunologically functional immunoglobulin portion capable of binding to a target antigen.
  • the immunologically functional immunoglobulin portion may comprise immunoglobulins, or portions thereof, fusion peptides derived from immunoglobulin portions or conjugates combining immunoglobulin portions that form an antigen binding site.
  • Each antigen binding moiety comprises at least the necessarily one, two or three CDRs of the immunoglobulin heavy and/or light chains from which the antigen binding moiety was derived.
  • an antigen-binding protein can consist of a single polypeptide chain (a monomer).
  • the antigen binding protein comprises at least two polypeptide chains.
  • Such an antigen-binding protein is a multimer, e.g., dimer, trimer or tetramer.
  • antigen binding proteins includes antibody fragments, antibody derivatives or antibody-like binding proteins that retain specificity and affinity for their antigen.
  • antibodies are initially obtained by immunization of a non-human animal, e.g., a mouse, rat, guinea pig or rabbit, with an immunogen comprising a polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment, for which it is desired to obtain antibodies (e.g. a human polypeptide).
  • an immunogen comprising a polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment, for which it is desired to obtain antibodies (e.g. a human polypeptide).
  • the step of immunizing a non-human mammal with an antigen may be carried out in any manner well known in the art for stimulating the production of antibodies in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is herein incorporated by reference).
  • Human antibodies may also be produced by using, for immunization, transgenic animals that have been engineered to express a human antibody repertoire (Jakobovitz et al. Nature 362 (1993) 255), or by selection of antibody repertoires using phage display methods.
  • a XenoMouse (Abgenix, Fremont, CA) can be used for immunization.
  • a XenoMouse is a murine host that has had its immunoglobulin genes replaced by functional human immunoglobulin genes.
  • antibodies produced by this mouse or in hybridomas made from the B cells of this mouse are already humanized.
  • the XenoMouse is described in United States Patent No. 6,162,963, which is herein incorporated in its entirety by reference.
  • Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in (Ward et al. Nature , 341 (1989) p. 544, the entire disclosure of which is herein incorporated by reference).
  • Phage display technology McCafferty et al (1990) Nature 348:552-553
  • V variable domain gene repertoires from unimmunized donors. See, e.g., Griffith et al (1993) EMBO J. 12:725- 734; US 5,565,332; US 5,573,905; US 5,567,610; and US 5,229,275).
  • combinatorial libraries comprise variable (V) domain gene repertoires of human origin, selection from combinatorial libraries will yield human antibodies.
  • an antigen binding domain can be obtained from a humanized antibody in which residues from a complementary-determining region (CDR) of a human antibody are replaced by residues from a CDR of the original antibody (the parent or donor antibody, e.g. a murine or rat antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody.
  • CDRs of the parent antibody some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted in whole or in part into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs.
  • An antigen binding domain can thus have non-human hypervariable regions or CDRs and human frameworks region sequences (optionally with back mutations).
  • Antibodies will typically be directed to a pre-determined antigen.
  • examples of antibodies include antibodies that recognize an antigen expressed by a target cell that is to be eliminated, for example a proliferating cell or a cell contributing to a disease pathology.
  • examples include antibodies that recognize tumor antigens, microbial (e.g. bacterial or parasite) antigens or viral antigens.
  • antigen binding domains used in the proteins described herein can be readily derived from any of a variety of non-immunoglobulin scaffolds, for example affibodies based on the Z-domain of staphylococcal protein A, engineered Kunitz domains, monobodies or adnectins based on the 10th extracellular domain of human fibronectin III, anticalins derived from lipocalins, DARPins® (designed ankyrin repeat domains, multimerized LDLR-A module, avimers or cysteine-rich knottin peptides. See, e.g., Gebauer and Skerra (2009) Current Opinion in Chemical Biology 13:245-255, the disclosure of which is incorporated herein by reference.
  • an antigen binding domain can conveniently comprise a VH and a VL (a VH/VL pair).
  • the VH/VL pair can be integrated in a Fab structure further comprising a CH1 and CL domain (a CH1/CL pair).
  • a VH/VL pair refers to one VH and one VL domain that associate with one another to form an antigen binding domain.
  • a CH1/CL pair refers to one CH1 and one CL domain bound to one another by covalent or non-covalent interactions, preferably non-covalent interactions, thus forming a heterodimer (e.g., within a protein such as a heterotrimer, heterotetramer, heteropentamer that can comprise one or more further polypeptide chains).
  • the constant chain domains forming the pair can be present on the same or on different polypeptide chain, in any suitable combination.
  • Exemplary CDRs or VH and VL domains that bind NKp30 can be derived from the anti- NKp30 antibodies provided herein, or can be selected from known anti-NKp30 antibodies, for example the CDRs, VH and VL domains of PCT publication no. WO2019/226617, the disclosure of which is incorporated herein by reference.
  • Variable regions can be used directly, or can be modified by selecting hypervariable or CDR regions from the NKp30 antibodies and placing them into the desired VL or VH framework, for example human frameworks.
  • Antigen binding domains that bind NKp30 can also be derived de novo using methods for generating antibodies. Antibodies can be tested for binding to NKp30 polypeptides.
  • a polypeptide that binds to NKp30 will be capable of binding NKp30 expressed on the surface of a cell, e.g. native NKp30 expressed by a NK cell.
  • the antibody can be a function-conservative variant of mAb8. “Function- conservative variants” are those in which a given amino acid residue in a protein (e.g. an antibody or antibody fragment) has been changed without altering the overall conformation and function of the protein, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
  • Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm.
  • a “function-conservative variant” also includes a polypeptide which has at least 60% amino acid identity with the antibody capable of specifically binding to a NKp30 polypeptide as defined hereinabove as determined by BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, still preferably at least 90%, and even more preferably at least 95%, and which has the same or substantially similar properties or functions as the antibodies capable of specifically binding to a NKp30 polypeptide as defined hereinabove.
  • anti-NKp30 VH and VL domains can comprise all the amino acid sequence of SEQ ID NO: 249 and SEQ ID NO: 250 respectively.
  • anti-NKp30 VH domain comprises the CDR1, CDR2 and CDR3 of the VH domain of SEQ ID NO: 249, optionally wherein CDR numbering is according to Kabat, Chothia or IMGT.
  • anti-NKp30 VL domain comprises the CDR1, CDR2 and CDR3 of the VL domain of SEQ ID NO: 250, optionally wherein CDR numbering is according to Kabat.
  • any of the CDR1 , 2 or 3 can be specified as having an amino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR or set of CDRs.
  • Antigen binding domains that bind an antigen of interest can be selected based on the desired predetermined antigen of interest (e.g. an antigen other than NKp30), and may include for example cancer antigens such as antigens present on tumor cells and/or on immune cells capable of mediating a pro-tumoral effect, e.g. a monocyte or a macrophage, optionally a suppressor T cell, regulatory T cell, or myeloid-derived suppressor cell (for the treatment of cancer); bacterial or viral antigens (for the treatment of infectious disease); or antigens present on pro-inflammatory immune cells, e.g. T cells, neutrophils, macrophages, etc. (for the treatment of inflammatory and/or autoimmune disorder).
  • cancer antigens such as antigens present on tumor cells and/or on immune cells capable of mediating a pro-tumoral effect, e.g. a monocyte or a macrophage, optionally a suppressor T cell, regulatory T cell, or myeloid-derived suppress
  • bacterial antigen includes, but is not limited to, intact, attenuated or killed bacteria, any structural or functional bacterial protein or carbohydrate, or any peptide portion of a bacterial protein of sufficient length (typically about 8 amino acids or longer) to be antigenic. Examples include gram-positive bacterial antigens and gram-negative bacterial antigens.
  • the bacterial antigen is derived from a bacterium selected from the group consisting of Helicobacter species, in particular Helicobacter pyloris; Borrelia species, in particular Borrelia burgdorferi ; Legionella species, in particular Legionella pneumophilia ; Mycobacteria s species, in particular M. tuberculosis, M.
  • avium M. intracellulare, M. kansasii, M. gordonae ; Staphylococcus species, in particular Staphylococcus aureus; Neisseria species, in particular N. gonorrhoeae, N. meningitidis; Listeria species, in particular Listeria monocytogenes; Streptococcus species, in particular S. pyogenes, S. agalactiae; S. faecalis; S. bovis, S.
  • pneumoniae anaerobic Streptococcus species; pathogenic Campylobacter species; Enterococcus species; Haemophilus species, in particular Haemophilus influenzae; Bacillus species, in particular Bacillus anthracis; Corynebacterium species, in particular Corynebacterium diphtheriae; Erysipelothrix species, in particular Erysipelothrix rhusiopathiae; Clostridium species, in particular C. perfringens, C.
  • Enterobacter species in particular Enterobacter aerogenes, Klebsiella species, in particular Klebsiella 1S pneumoniae, Pasteurella species, in particular Pasteurella multocida, Bacteroides species; Fusobacterium species, in particular Fusobacterium nucleatum; Streptobacillus species, in particular Streptobacillus moniliformis; Treponema species, in particular Treponema per pneumonia; Leptospira; pathogenic Escherichia species; and Actinomyces species, in particular Actinomyces Israeli.
  • viral antigen includes, but is not limited to, intact, attenuated or killed whole virus, any structural or functional viral protein, or any peptide portion of a viral protein of sufficient length (typically about 8 amino acids or longer) to be antigenic.
  • Retroviridae e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV- 11 l/LAV, or HIV-Ill; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies
  • Retroviridae e.g., human immunodeficiency
  • cancer antigen and “tumor antigen” are used interchangeably and refer to antigens (other than the cytokine receptor expressed on NK cells, NKp30, and CD16) that are differentially expressed by cancer cells or are expressed by non- tumoral cells (e.g. immune cells) having a pro-tumoral effect (e.g. an immunosuppressive effect), and can thereby be exploited in order to target cancer cells.
  • Cancer antigens can be antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, or expressed at lower levels or less frequently, by normal cells.
  • cancer antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens.
  • Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations.
  • Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses.
  • Still other cancer antigens can be expressed on immune cells capable of contributing to or mediating a pro-tumoral effect, e.g. cell that contributes to immune evasion, a monocyte or a macrophage, optionally a suppressor T cell, regulatory T cell, or myeloid-derived suppressor cell.
  • the cancer antigens are usually normal cell surface antigens which are either over expressed or expressed at abnormal times, or are expressed by a targeted population of cells.
  • the target antigen is expressed only on proliferative cells (e.g., tumor cells) or pro- tumoral cells (e.g. immune cells having an immunosuppressive effect), however this is rarely observed in practice.
  • target antigens are in many cases selected on the basis of differential expression between proliferative/disease tissue and healthy tissue.
  • Example of cancer antigens include: Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), Crypto, CD4, CD19, CD20, CD30, CD38, CD47, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), a Siglec family member, for example CD22 (Siglec2) or CD33 (Siglec3), CD79, CD123, CD138, CD171 , PSCA, L1-CAM, PSMA (prostate specific membrane antigen), BCMA, CD52, CD56, CD80, CD70, E-selectin, EphB2, Melanotransferrin, Mud 6 and TMEFF2.
  • ROR1 Receptor Tyrosine Kinase-like Orphan Receptor 1
  • Crypto Crypto
  • CD4 CD19
  • CD20 CD30
  • CD38 CD47
  • Glycoprotein NMB CanAg
  • Her2 ErbB2/Neu
  • a Siglec family member for example CD22 (Siglec2) or
  • cancer antigens also include Immunoglobulin superfamily (IgSF) such as cytokine receptors, Killer-lg Like Receptor, CD28 family proteins, for example, Killer-lg Like Receptor 3DL2 (KIR3DL2), B7-H3, B7-H4, B7-H6, PD-L1.
  • IgSF Immunoglobulin superfamily
  • Examples also include MAGE, MART-1/Melan-A, gp100, major histocompatibility complex class l-related chain A and B polypeptides (MICA and MICB), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A/GA733, protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), nectins (e.g.
  • nectin-4 major histocompatibility complex class l-related chain A and B polypeptides
  • MICA and MICB proteins of the UL16-binding protein
  • RAET1 proteins of the retinoic acid early transcript-1
  • CEA carcinoembryonic antigen
  • PSA prostate specific antigen
  • T-cell receptor/C D3-zeta chain MAGE-family of tumor antigens, GAGE-family of tumor antigens, anti-Mullerian hormone Type II receptor, delta-like ligand 4 (DLL4), DR5, ROR1 (also known as Receptor Tyrosine Kinase-Like Orphan Receptor 1 or NTRKR1 (EC 2.7.10.1), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC family, VEGF, VEGF receptors, Angiopoie
  • a multispecific protein can be specified as excluding or not requiring a stromal modifying moiety, e.g., a moiety capable of altering or degrading a component of, the stroma such as an ECM component, e.g., a glycosaminoglycan, e.g., hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparin sulfate, heparin, entactin, tenascin, aggrecan and keratin sulfate; or an extracellular protein, e.g., collagen, laminin, elastin, fibrinogen, fibronectin, and vitronectin.
  • ECM component e.g., a glycosaminoglycan, e.g., hyaluronan (also known as hyaluronic acid or
  • the stromal modifying moiety can be a hyaluronan degrading enzyme, an agent that inhibits hyaluronan synthesis, or an antibody molecule against hyaluronic acid.
  • a multispecific protein can be specified as excluding a mesothelin targeting moiety or mesothelin-binding ABD.
  • a multispecific protein can be specified as excluding a PD-L1 targeting moiety, a HER3 targeting moiety, an IGFIR targeting moiety or a hyaluronidase 1 targeting moiety, or a combination a stroma targeting moiety or ABD and a cancer-antigen targeting moiety.
  • a cancer antigen or antigen of interest can be specified as being other than a PD- L1, a HER3, an IGFIR or hyaluronidase 1.
  • exemplary VH and VL pairs can be selected from antibodies trastuzumab, pertuzumab or margetuximab:
  • exemplary VH and VL pairs can be selected from the VH and VL pair from blinatumomab.
  • exemplary VH and VL pairs can be selected from VH and VL pair from rituximab and obinutuzumab :
  • exemplary VH and VL pairs can be selected from the EGFR-binding VH and VL pair from cetuximab, panitumumab, nimotuzumab, depatuxizumab and necitumumab: Cetuximab VH:
  • Cetuximab VL DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSG SGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 13).
  • Panitumumab VH DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSG SGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 13).
  • DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGWLQQKPGKSFKGLIYHGTNLDDGVPSR FSGSGSGTDYTLTISSLQPEDFATYYCVQYAQFPWTFGGGTKLEIK (SEQ ID NO: 21).
  • exemplary VH and VL pairs can be selected from the BCMA-binding VH and VL pair from belantamab, teclistamab, elranatamab or pavurutamab:
  • exemplary VH and VL pairs can be selected from the PD-L1 -binding VH and VL pair from antibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1 B12, 7H1, 11 E6, 12B7, and 13G4 shown in US Patent no. 7,943,743, the disclosure of which is incorporated herein by reference, or of any of the antibodies MPDL3280A (atezolizumab, TecentriqTM, see, e.g., US patent no.
  • exemplary VH and VL pairs can be selected from the B7-H3 -binding VH and VL pairs of enoblituzumab, of TRL4542 shown in PCT publication no. W02018/129090, of 8H9 shown in PCT publication no. WO2018/209346, or of any of the antibodies of PCT publication nos. WO2016/106004, W02017/180813, W02019/024911, WO2019/225787,
  • single domain B7H3 ABDs that can be used include AffibodyTM formats described in PCT publication W02020/041626 and single domain antibodies (sdAb) of PCT publication nos. W02020/076970 and WO2021/247794.
  • sdAb single domain antibodies
  • exemplary VH and VL pairs can be selected from the B7-H6-binding VH and VL pairs shown in US Patent nos. US 11,034,766; US 8,822,652; US 9,676,855; US 11,034,766; US 11 ,034,767 or in PCT publication nos. WO2013/037727 or WO2021/064137.
  • exemplary VH and VL pairs can be selected from the B7-H4-binding VH and VL of alsevalimab or the VH and VL pairs shown in US Patent nos. US 10,626,176; US 9,676,854; US 9,574,000; US 10,150,813; US 10,814,011 or in PCT publication nos. W02009/073533, WO2019/165077, WO2019/169212, WO2019/147670, WO2021/155307, W02022/039490, WO2019/154315 or WO2021/185934.
  • the ABD that binds an antigen of interest binds to a cancer antigen, a viral antigen, a microbial antigen, or an antigen present on an infected cell (e.g. virally infected) or on a pro-inflammatory immune cell.
  • said antigen is a polypeptide selectively expressed or overexpressed on a tumor cell, and infected cell or a pro- inflammatory cell.
  • said antigen is a polypeptide that when inhibited, decreases the proliferation and/or survival of a tumor cell, an infected cell or a pro- inflammatory cell.
  • the ABDs which are incorporated into the polypeptides can be tested for any desired activity prior to inclusion in a multispecific NKp30-binding protein, for example the ABD can be tested in a suitable format (e.g. as conventional IgG antibody, fab, Fab’2 or scFv) for binding to (e.g. binding affinity) for its binding partner.
  • a suitable format e.g. as conventional IgG antibody, fab, Fab’2 or scFv
  • An ABD derived from an antibody will generally comprise at minimum a hypervariable region sufficient to confer binding activity. It will be appreciated that an ABD may comprise other amino acids or functional domains as may be desired, including but not limited to linker elements (e.g. linker peptides, CH1 , CK or CA domains, hinges, or fragments thereof).
  • linker elements e.g. linker peptides, CH1 , CK or CA domains, hinges, or fragments thereof.
  • an ABD comprises an scFv, a VH domain and a VL domain, or a single domain antibody (nanobody or dAb) such as a V-NAR domain or a VHH domain.
  • ABDs can be made of a VH and a VL domain that associate with one another to form the ABD.
  • one or both of the VH and VL pairs that form an ABD for NKp30 and antigen of interest are within a tandem variable region (a VH and VL domain separated by a flexible polypeptide linker), such as an scFv.
  • one or both ABDs for NKp30 and antigen of interest can have a conventional or non-conventional Fab structure.
  • a Fab structure can be characterized as a VH or VL variable domain linked to a CH1 domain and a complementary variable domain (VL or VH, respectively) linked to a complementary CK (or CA) constant domain, wherein the CH1 and CK (or CA) constant domains associate (dimerize).
  • a Fab can be formed from a VH-CH1 unit (VH fused to a CH1) on a first polypeptide chain that dimerizes with a VL-CK unit (VL fused to a CK) on a second chain.
  • a Fab can be formed from a VH-CK unit (VH fused to a CK) on a first polypeptide chain that dimerizes with a VL-CH1 unit (VL fused to a CH1) on a second chain.
  • one of the ABDs for NKp30 and antigen of interest comprises a Fab structure, in which a variable domain is linked to a CH1 domain and a complementary variable domain is linked to a complementary CK (or CA) constant domain, wherein the CH1 and CK (or CA) constant domains associate to form a heterodimeric protein, and the other ABD comprise or consists of an scFv or a single binding domain (e.g. VhH domain, DARPin).
  • the scFv or a single binding domain can optionally be fused to a CK or CA domain or hinge domain.
  • the CH1 and/or CK domains can then be linked to a CH2 domain, optionally in each case via a hinge region (or a suitable domain linker).
  • the CH2 domain(s) is/are then linked to a CH3 domain.
  • the CH2-CH3 domains can thus optionally be embodied as a full-length Fc domain (optionally a full-length Fc domain, except that the CH3 domain that lacks the C- terminal lysine).
  • the CD16 ABD when present, can readily be embodied as a Fc domain dimer that is capable of binding to human CD16A and optionally other Fey receptors, e.g., CD16B, CD32A, CD32B and/or CD64).
  • an Fc moiety may be obtained by production of the polypeptide in a host cell or by a process that yields N297-linked glycosylation, e.g. a mammalian cell.
  • an Fc moiety comprises a human gamma isotype constant region comprising one or more amino acid modifications, e.g. in the CH2 domain, that increases binding to CD16 or CD16A.
  • the cytokine receptor antigen binding domain can readily be embodied as a cytokine, (e.g. a type 1 cytokine such as an IL-2, IL-15, IL-21 , IL-7, IL-27 or IL-12 cytokine, an IL-18 cytokine or a type 1 interferon such as IFN-a or IFN-b).
  • a cytokine e.g. a type 1 cytokine such as an IL-2, IL-15, IL-21 , IL-7, IL-27 or IL-12 cytokine, an IL-18 cytokine or a type 1 interferon such as IFN-a or IFN-b.
  • exemplary cytokine receptor ABDs and modified cytokines are further described herein.
  • nucleic acids encoding each of the or ABD can be separately placed, in suitable arrangements, in an appropriate expression vector or set of vectors, together with DNA encoding any elements such as CH1, CK, CH2 and CH3 domains or portions thereof, mutant IL2 polypeptides and any other optional elements (e.g. DNA encoding a hinge-derived or linker elements) for transfection into an appropriate host.
  • ABDs will be arranged in an expression vector, or in separate vectors as a function of which type of polypeptide is to be produced, so as to produce the Fc-polypeptides having the desired domains operably linked to one another. The host is then used for the recombinant production of the multispecific polypeptide.
  • a polypeptide fusion product can be produced from a vector in which one ABD or a part thereof (e.g. a VH, VL or a VH/VL pair) is operably linked (e.g. directly, or via a CH1 , CK or CA constant region and/or hinge region) to the N-terminus of a CH2 domain, and the CH2 domain is operably linked at its C-terminus to the N-terminus a CH3 domain.
  • Another ABD or part thereof can be on a second polypeptide chain that forms a dimer, e.g. heterodimer, with the polypeptide comprising the first ABD.
  • the multispecific polypeptide can then be produced in an appropriate host cell or by any suitable synthetic process.
  • a host cell chosen for expression of the multispecific polypeptide is an important contributor to the final composition, including, without limitation, the variation in composition of the oligosaccharide moieties decorating the protein in the immunoglobulin CH2 domain.
  • one aspect of the invention involves the selection of appropriate host cells for use and/or development of a production cell expressing the desired therapeutic protein such that the multispecific polypeptide retains FcRn and CD16 binding.
  • the host cell may be of mammalian origin or may be selected from COS-1 , COS-7, HEK293, BHK21, CHO, BSC-1 , Hep G2, 653, SP2/0, 293, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof.
  • the host cell may be any suitable species or organism capable of producing N-linked glycosylated polypeptides, e.g. a mammalian host cell capable of producing human or rodent IgG type N-linked glycosylation.
  • Multimeric, multispecific proteins such as heterodimers, heterotrimers and hetero- tetramers can be produced according to a variety of formats. Different domains onto different polypeptide chain that associate to form a multimeric protein. Accordingly, a wide range of protein formats can be constructed around Fc domain dimers that are capable of binding to human FcRn polypeptide (neonatal Fc receptor), with or without additionally binding to CD16 or CD16A and optionally other Fey receptors, e.g., CD16B, CD32A, CD32B and/or CD64), depending on whether or not the CD16 binding ABD is desired to be present.
  • Fc domain dimers that are capable of binding to human FcRn polypeptide (neonatal Fc receptor), with or without additionally binding to CD16 or CD16A and optionally other Fey receptors, e.g., CD16B, CD32A, CD32B and/or CD64
  • a protein comprises a first and a second polypeptide chain each comprising a variable domain fused to a human Fc domain monomer (i.e.
  • variable domains of each chain can be part of the same or different antigen binding domains.
  • Multispecific proteins can thus be conveniently constructed using VH and VL pairs arranged as scFv or Fab structures, together with CH1 domains, CL domain, Fc domains and cytokines, and domain linkers.
  • the proteins will use minimal non-natural sequences, e.g. minimal use of non-lg linkers, optionally no more than 5, 4, 3, 2 or 1 domain linker(s) that is not an antibody-derived sequence, optionally wherein domain linker(s) are no more than 15, 10 or 5 amino acid residues in length.
  • the CD16 ABD is a Fc domain dimer.
  • NKp30 ABD Fc domain and NKp30 ABD are immediately adjacent to other another within the protein, and NKp30 ABD is immediately adjacent to the cytokine receptor ABD (embodied as a cytokine (Cyt)), and wherein the NKp30 ABD is interposed between the CD16 ABD and the Cyt:
  • NKp30 ABD (Fc domain dimer) (NKp30 ABD) (Cyt) (Structure 1b) wherein the NKp30 ABD and the CD16ABD (e.g. Fc domain dimer) are connected by a domain linker and the NKp30 ABD and Cyt are connected by a domain linker.
  • CD16ABD e.g. Fc domain dimer
  • the multispecific proteins may comprise a domain arrangement of any of the following in which domains can be placed on any of the 2, 3 or 4 polypeptide chains, wherein the NKp30 ABD is interposed between the Fc domain and the cytokine receptor ABD (e.g.
  • the protein has a terminal or distal cytokine receptor ABD at the C-terminal end and a terminal or distal antigen of interest (Antigen) ABD at the topological N-terminal end), wherein the NKp30 ABD is connected to one of the polypeptide chains of the Fc domain dimer via a hinge polypeptide or a flexible linker, and wherein the ABD that binds the cytokine receptor is connected to NKp30 ABD (e.g. to one of the polypeptide chains thereof when the NKp30 ABD is contained on two chains) via a flexible linker (e.g. a linker comprising G and S residues), wherein “n” is 1 or 2:
  • Anti-Antigen ABD n - (Fc domain dimer) - (NKp30 ABD) - (cytokine receptor ABD).
  • the protein comprises an ABD that binds an antigen of interest on a target cell (Antigen ABD) and the ABD that binds NKp30 both placed topologically N- terminal to the Fc domain dimer, as in a heteromultimeric protein having the structure below:
  • NKp30 ABD Fc domain dimer
  • Cyt wherein the Antigen ABD and the Fc domain dimer are connected by a linker or immunoglobulin hinge polypeptide, wherein the NKp30 ABD and the Fc domain dimer are connected by a linker or immunoglobulin hinge polypeptide, and wherein the Fc domain dimer and Cyt are connected by a linker.
  • the cytokine receptor ABD can be an IL2, IL15, IL18, IL21 or IFN-a polypeptide.
  • the Fc domain can be specified to be an Fc domain dimer (e.g. that binds human FcRn and/or Fey receptors).
  • one or both of the antigen of interest (e.g. cancer antigen) ABD and NKp30 ABD is formed from two variable regions present within tandem variable regions, wherein the variable regions that associate to form a particular ABD can be on the same polypeptide chain or on different polypeptide chains.
  • one or both of the antigen of interest ABD and NKp30 ABD comprises a tandem variable region and the other comprises a Fab structure.
  • both of the antigen of interest and NKp30 ABD comprises a Fab structure.
  • one of the antigen of interest and NKp30 ABD comprises a Fab structure and the other comprises an scFv structure.
  • the present disclosure provides advantageous approaches of making multimeric multispecific proteins which bind to the antigen of interest (monovalently or bivalently) and monovalently to each of NKp30, CD16A and cytokine receptor.
  • the approaches readily allow domain configurations where the NKp30 ABD is positioned between the Fc domain and a cytokine polypeptide.
  • These configurations can be achieved through the assembly of different polypeptide chains described herein that each comprise at least one heavy or light chain variable domain fused to a human CH1 or CK constant domain (a V-(CH1/CK) unit), wherein the protein chains undergo CH1-CK dimerization and are bound to one another by non- covalent bonds and optionally further disulfide bonds formed between respective CH1 and CK domains.
  • Exemplary heterodimeric or heterotri meric polypeptides have an NKp30 binding ABD, an antigen of interest binding ABD, a cytokine receptor-binding ABD (e.g. IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b polypeptide) and a Fc domain dimer can optionally be produced as one or more chains that each associate with a central chain, e.g. by CH1-CK heterodimerization and/or by CH3-CH3 dimerization. Different variants can be produced, as illustrated in the Examples herein.
  • an isolated or purified heterodimeric or heterotri meric protein comprises at least two or three polypeptide chains, each comprising a V-(CH1/CK) unit, whereby the chains are bound to one another by non-covalent interactions and optionally further bound via disulfide bonds between CH1 and CK domains, and still further optionally, whereby the chains are bound by non-covalent interactions between the respective variable regions, CH1 and CK domains, and CH3 domains of the Fc portion.
  • the protein comprises a first and a second polypeptide chain each comprising a variable domain fused to a CH1 or CK domain (a V-(CH1/CK) unit), in turn fused at its C-terminus to a human Fc domain monomer comprising a CH2 domain and a CH3 domain capable of undergoing CH3-CH3 dimerization, wherein the first and second chain associate via CH1-CK and CH3-CH3 dimerization such that the protein comprises a Fcdomain dimer.
  • the variable domains of each chain can be part of the same or different antigen binding domains.
  • variable and constant regions can be selected and configured such that each chain will preferentially associate with its desired complementary partner chain.
  • the resulting multimeric protein can be produced reliably and with high productivity using conventional production methods using recombinant host cells.
  • the choice of which VH or VL to associate with a CH1 and CK in a unit is based on affinity between the units to be paired so as to drive the formation of the desired multimer.
  • the resulting multimer will be bound by non-covalent interactions between complementary VH and VL domains, by non-covalent interactions between complementary CH1 and CK domains, and optionally by further disulfide bonding between complementary CH 1 and CK domains (and optionally further disulfide bonds between complementary hinge domains).
  • VH-VL associations are stronger than VH-VH or VL-VL, consequently, as shown herein, one can place a VH or a VL next to either a CH1 or a CK, and the resulting V-C unit will partner preferably with its V-C counterpart.
  • VH-CK will pair with VL-CH1 preferentially over VH-CH1.
  • preferred chain pairing is further improved, as the two Fc monomer-containing chains are bound by non- covalent interactions between CH3 domains of the Fc domain monomers.
  • the different V-C combinations, optionally further combined with Fc pairing thereby provides tools to make heteromultimeric proteins comprising a cytokine (e.g. IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL- 18, IFN-a or IFN-b polypeptide), represented as “Cyt” in the domain arrangements.
  • a cytokine e.g. IL-2, IL-15, IL-21
  • the multispecific protein is a heterodimer comprising a first and a second polypeptide chain each comprising a variable domain fused to a CH1 or CK domain (a V-(CH1/CK) unit), in turn fused at its C-terminus to a human Fc domain monomer, wherein the V-(CH1/CK) unit of the first chain has undergone CH1-CK dimerization with the V-(CH1/CK) unit of the second chain thereby forming a first antigen binding domain (ABDi) and a Fc domain dimer, wherein one of the polypeptide chains further comprises an antigen binding domain that forms a second antigen binding domain (ABD2), and wherein the Fc domain dimer binds to a human CD16 polypeptide, wherein one of ABDi and ABD2 binds NKp30 and the other binds the antigen of interest (e.g. tumor antigen).
  • a V-(CH1/CK) unit a variable domain fused to a CH1 or CK domain
  • the protein has a domain arrangement:
  • ABD antigen binding domain
  • the protein is a heterotrimer and comprises three polypeptide chains, each comprising a variable domain fused to a CH1 or CK domain (a V-(CH1/CK) unit), wherein a first (central) chain comprises two V-(CH1/CK) units and a human Fc domain interposed between the units, the second chain comprises one V-(CH1/CK) unit and a human Fc domain monomer, and the third chain comprises one V-(CH1/CK) unit and a cytokine polypeptide (Cyt), wherein one of the V-(CH1/CK) units of the central chain has undergone CH1-CK dimerization with the V-(CH1/CK) unit of the second chain thereby forming a first antigen binding domain (ABDi) and a Fc domain dimer, and wherein the other of the V- (CH1/CK) units of the central chain has undergone CH1-CK dimerization with the V-(CH1/CK) unit of the third chain thereby forming a second antigen binding domain (ABDi)
  • the protein has a domain arrangement:
  • VK - CK - Fc domain (second polypeptide)
  • VH - CH1 - Fc domain - VH - CK (first polypeptide)
  • VK - CH1 - Cyt (third polypeptide).
  • the protein has a domain arrangement:
  • VK - CK - Fc domain (second polypeptide)
  • VK - CK- Cyt (third polypeptide).
  • the Fab structure interposed between the Fc domain and the cytokine is the NKp30 binding ABD (i.e. the NKp30 binding ABD is interposed between the Fc domain and the C-terminal cytokine).
  • the Fc domain in the first polypeptide is connected (e.g. fused) at its C terminus to the N-terminus of the VH domain via a linker.
  • the constant domain (the CH1 or CK domain in the respective domain arrangements) in the third polypeptide is connected (e.g. fused) at its C terminus to the N-terminus of the cytokine polypeptide via a linker.
  • Each constant domain (the CH1 or CK domain in the respective domain arrangements) that is N-terminal to the Fc domain is fused at the C terminus of the constant domain to the N-terminus of the Fc domain via a hinge region.
  • any of the multispecific proteins of the invention may include CH1, CL or CH3 domains which comprise amino acid modifications (e.g. substitutions) to promote heterodimerization.
  • heterodimerization modifications often involve steric repulsion, charge steering interaction, or interchain disulfide bond formation, wherein the CH3 domain interface of the antibody Fc region is mutated to create altered charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc chains supports favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation.
  • the first (central) polypeptide chain will provide one variable domain that will, together with a complementary variable domain on a second polypeptide chain, form a first antigen binding domain (e.g. the ABD that binds the antigen of interest), and an Fc domain.
  • the first (central) polypeptide chain will also provide a second variable domain (e.g., placed on the opposite end of the interposed Fc domain from the first variable domain, at the C-terminus of the Fc domain) that will be paired with a complementary variable domain to form a second antigen binding domain (e.g. the ABD that binds NKp30); the variable domain that is complementary to the second variable domain can be placed on the central polypeptide (e.g.
  • a tandem variable domain construct such as an scFv
  • a tandem variable domain construct such as an scFv
  • the second (and third, if present) polypeptide chains will associate with the central polypeptide chain by CH1-CK heterodimerization, forming non-covalent interactions and optionally further interchain disulfide bonds between complementary CH1 and CK domains (and optionally interchain disulfide bonds between hinge regions), with a primary multimeric polypeptide being formed so long as CH/CK and VH/VK domains are chosen to give rise to a preferred dimerization configuration that results preferentially in the desired VH-VL pairings.
  • a trimer or when polypeptides are constructed for preparation of a trimer, there will generally be one polypeptide chain that comprises a non-naturally occurring VH-CK or VK-CH1 domain arrangement.
  • a cytokine e.g., IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b
  • the cytokine can be fused via a domain linker, and while not shown in certain domain arrangements herein, any domain arrangement can be specified as comprising a domain linker separating two domains.
  • the cytokine can be placed at the C-terminus of the first (central) polypeptide chain or at the C-terminus of the third polypeptide chain (when such third chain is present).
  • Examples of the domain arrangements (N- to C-terminus, left to right) of central polypeptide chains for use in heterodimeric proteins in which the NKp30 ABD is interposed between the Fc domain and cytokine (Cyt) and wherein the cytokine moiety is placed on a different polypeptide chain (e.g. the second polypeptide chain) include any of the following, wherein each V is a variable domain:
  • V domains when V domains are arranged immediately adjacent to one another in tandem on a chain, one of the V is a light chain and the other V is heavy chain variable domain, and the two V domains are separated by a flexible polypeptide linker and together form a scFv.
  • central polypeptide chains include:
  • ABD e.g. dAb, VHH, DARPin
  • the Fc domain of the central chain may be a full Fc domain or a portion thereof sufficient to confer the desired functionality (e.g. binding to FcRn, binding to CD16, CH3-CH3 dimerization) when it forms a dimeric Fc with the Fc domain of a second polypeptide chain.
  • a second polypeptide chain can then be configured which will comprise an immunoglobulin variable domain and a CH1 or CK constant region, e.g., a (CH1 or CK) b unit, selected so as to permit CH1-CK heterodimerization with the central polypeptide chain; the immunoglobulin variable domain will be selected so as to complement the variable domain of the central chain that is adjacent to the CH1 or CK domain, whereby the complementary variable domains form an antigen binding domain for a first antigen of interest.
  • a CH1 or CK constant region e.g., a (CH1 or CK) b unit
  • a second polypeptide chain for use in a protein in which the NKp30 ABD is interposed between Fc and cytokine can comprise a domain arrangement:
  • V b -i - (CH1 or CK) b - Fc domain such that the (CH1 or CK) b dimerizes with the (CH1 or CK) 3 on the central chain, and the V b -i forms an antigen binding domain together with V a -i of the central chain.
  • V a -i of the central chain is a light chain variable domain
  • Vw will be a heavy chain variable domain
  • Vw will be a light chain variable domain.
  • the second chain can comprise a Cyt fused to its C-terminus (e.g., via a domain linker).
  • the antigen binding domain for the second antigen of interest can then be formed from V a -2 and V b -2 which are configured as tandem variable domains on the central chain forming an ABD (e.g. forming an scFv unit).
  • the resulting heterodimer can, for example, have the following configuration (see further examples of such proteins shown as formats T13 and T13A shown in Figure 2:
  • V b -i - (CH1 or CK) b - Fc domain (second polypeptide chain) wherein one of V a -i of the first polypeptide chain and VM of the second polypeptide chain is a light chain variable domain and the other is a heavy chain variable domain, and wherein one of V a -2 and V b -2 is a light chain variable domain and the other is a heavy chain variable domain.
  • V a -2 and V b -2 can be specified as being separated by a polypeptide linker (V a -2and V b -2 form an scFv).
  • V a -2and V b -2 forms the ABD that binds NKp30 and V a -i and VM forms the ABD that binds the antigen of interest (e.g. cancer antigen).
  • domain arrangements of heteromultimeric proteins include the following, optionally wherein one or both of the hinge domains are replaced by a flexible linker polypeptide, wherein the NKp30 ABD is an scFv or a single domain ABD (e.g. dAb, VHH, DARPin) and the Fc domain is fused to the NKp30 ABD a linker polypeptide, and wherein the NKp30 ABD is fused to the cytokine polypeptide by a domain linker (e.g. a flexible polypeptide linker):
  • a domain linker e.g. a flexible polypeptide linker
  • VK-CK - hinge Fc domain - (NKp30 ABD) - Cyt (VH-CH1- hinge) - Fc domain (V H -CH1- hinge) - Fc domain - (NKp30 ABD) - Cyt (VK-CK- hinge) - Fc domain or
  • Heterotrimeric proteins in which the NKp30 ABD is interposed between the Fc domain and the cytokine polypeptide can for example be formed by using a central (first) polypeptide chain comprising a first variable domain (V) fused to a first CH1 or CK constant region, a second variable domain (V) fused to a second CH1 or CK constant region, and an Fc domain or portion thereof interposed between the first and second variable domains (i.e. the Fc domain is interposed between the first and second (V-(CH1/CK) units.
  • a central polypeptide chain for use in a heterotrimeric protein according to the invention can have the domain arrangements (N- to C- termini) as follows:
  • the first polypeptide chain can optionally further have a Cyt is placed at its C-terminus.
  • a second polypeptide chain can then comprise a domain arrangement (N- to C- termini from left to right):
  • V b -i - (CH1 or CK) c - FC domain such that the (CH1 or CK) C dimerizes with the (CH1 or CK) a on the central chain, and the V a -i and Vn form an antigen binding domain that binds the antigen of interest.
  • a third polypeptide chain can then comprise the following domain arrangement (N- to C- termini from left to right):
  • the third polypeptide chain can then comprise the following domain arrangement (N- to C- termini from left to right):
  • Vb-2 - (CH1 or CK) d Vb-2 - (CH1 or CK) d .
  • Vb-2 - (CH1 or CK) d (third polypeptide).
  • the first polypeptide can have two variable domains that each form an antigen binding domain with a variable domain on a separate polypeptide chain (i.e. the variable domain of the second and third chains), the second polypeptide chain has one variable domain, and the third polypeptide has one variable domain, and one of the polypeptide chains comprises a cytokine polypeptide fused to its C-terminus.
  • a first polypeptide chain comprising from N-terminus to C-terminus: a first variable domain (V) fused to a first CH1 or CK constant region, a hinge domain or portion thereof, an Fc domain or portion thereof, and a second variable domain (V) fused to a second CH1 or CK constant region;
  • a second polypeptide chain comprising from N-terminus to C-terminus: a variable domain fused to a CH1 or CK constant region selected to be complementary to the first CH1 or CK constant region of the first polypeptide chain such that the first and second polypeptides form a CH1-CK heterodimer, a hinge domain or portion thereof, and an Fc domain; and
  • a third polypeptide chain comprising from N-terminus to C-terminus: a variable domain fused to a CH1 or CK constant region, and a cytokine polypeptide (e.g. fused to the constant region via a flexible polypeptide linker), wherein the variable domain and the constant region are selected to be complementary to the second variable domain and second CH1 or CK constant region of the first polypeptide chain such that the first and third polypeptides form a CH1-CK heterodimer bound by non-covalent interactions and optionally further disulfide bond(s) formed between the CH 1 or CK constant region of the third polypeptide and the second CH1 or CK constant region of the first polypeptide, but not between the CH1 or CK constant region of the third polypeptide and the first CH1 or CK constant region of the first polypeptide wherein the first, second and third polypeptides form a heterotrimer, and wherein the first variable domain of the first polypeptide chain and the variable domain of the second polypeptide
  • a trimeric polypeptide can optionally be characterized as comprising three polypeptide chains:
  • a first polypeptide chain comprising from N-terminus to C-terminus: a first variable domain (V) fused to a first CH1 or CK constant region, a hinge domain or portion thereof, an Fc domain or portion thereof, and a second variable domain (V) fused to a second CH1 or CK constant region, and a cytokine polypeptide (e.g. fused to the second CH1 or CK constant region via a flexible polypeptide linker);
  • a second polypeptide chain comprising from N-terminus to C-terminus: a variable domain fused to a CH1 or CK constant region selected to be complementary to the first CH1 or CK constant region of the first polypeptide chain such that the first and second polypeptides form a C -CK heterodimer, a hinge domain or portion thereof, and an Fc domain; and (c) a third polypeptide chain comprising from N-terminus to C-terminus: a variable domain fused to a CH1 or CK constant region, wherein the variable domain and the constant region are selected to be complementary to the second variable domain and second CH1 or CK constant region of the first polypeptide chain such that the first and third polypeptides form a CH1-CK heterodimer bound by non-covalent interactions and optionally further disulfide bond(s) formed between the CH 1 or CK constant region of the third polypeptide and the second CH1 or CK constant region of the first polypeptide, but not between the CH1 or CK constant
  • multispecific proteins can be produced that binds the antigen of interest bivalently, binds NKp30 monovalently and binds the cytokine receptor monovalently, i.e., the multispecific protein has a 2:1:1 configuration. Examples are shown in
  • a heterodimer protein comprises the domain arrangement:
  • the Fc domains of the first and second chains associate via CH3-CH3 dimerization, and (CH1 or Ci ) b on the second chain and the (CH1 or CK) 3 on the first chain undergo CH1- CK dimerization, wherein ABDi that binds NKp30 and ABD2 and ABD3 are each self-contained antigen binding domains that can bind an antigen of interest (e.g. a cancer antigen) without association with a complementary domain on a different polypeptide chain, wherein each (CH1 or CK) b and (CH1 or CK) 3 is fused to the Fc domain via an immunoglobulin hinge amino acid sequence, and wherein Cyt is a cytokine polypeptide (e.g. fused to the ABDi via a flexible polypeptide linker).
  • ABDi that binds NKp30 and ABD2 and ABD3 are each self-contained antigen binding domains that can bind an antigen of interest (e.g. a cancer
  • a heterotrimer protein comprises the domain arrangement:
  • the Fc domains of the first and third chains associate via CH3-CH3 dimerization, (CH1 orCi ) c O n the third chain and the (CH1 orCi ) a O n the central chain undergo CH1- CK dimerization, and the (CH1 or Ci ) b on the first chain and the (CH1 or Ci ) d on the second chain undergo CH1- CK dimerization.
  • the V ai and ⁇ i form a first antigen binding domain that bind NKp30.
  • ABD 2 and ABD 3 are each self-contained antigen binding domains that can bind to an antigen of interest (e.g. a cancer antigen), e.g.
  • ABD 2 and ABDs can for example each comprise a single domain ABD or a VH and a VK pair (in any desired order), placed on a single chain and separated by a flexible peptide linker (e.g. as an scFv), such that a heterotrimer protein can comprise:
  • VK - VH - (CH1 or CK) C - Fc domain (third polypeptide)
  • VH - VK - (CH1 or CK) C - Fc domain (third polypeptide)
  • Examples of possible configurations of a resulting heterotrimer are structures having domain arrangement, from N- to C-terminus: VK - VH - CK - Fc domain (third polypeptide)
  • VKi -CH1 - Cyt (second polypeptide) or
  • VH - VK - CK - Fc domain (third polypeptide)
  • VKi -CH1 - Cyt (second polypeptide) or
  • VK - VH - CK - Fc domain (third polypeptide)
  • VK - VH - CH1 - Fc domain - VKi -CK (first polypeptide)
  • VH - VK - CK - Fc domain (third polypeptide)
  • VH - VK - CK - Fc domain (third polypeptide)
  • VK - VH - CK - Fc domain (third polypeptide)
  • VH - VK - CH1 - Fc domain (third polypeptide)
  • VKi -CH1 - Cyt (second polypeptide) or
  • VK- VH - CH1 - Fc domain (third polypeptide)
  • VKi -CH1 - Cyt (second polypeptide) or
  • VK- VH - CH1 - Fc domain (third polypeptide)
  • VH - VK- CH1 - Fc domain (third polypeptide)
  • VH - VK- CH1 - Fc domain (third polypeptide)
  • VHi -CK - Cyt (second polypeptide).
  • VK- VH - CH1 - Fc domain (third polypeptide)
  • VHi -CK - Cyt (second polypeptide).
  • a heterotetramer protein in which the NKp30 ABD is interposed between the Fc domain and the cytokine, for example molecules having the following domain arrangement: (fourth chain) domain (second chain) domain - V ai - V bi - Cyt (first chain) (Vb-2- (CH1 or C K ) (third chain) wherein the first chain and the second chain associate by CH3-CH3 dimerization and the first chain and the third chain associate by the CH1 or CK dimerization, wherein the domains of the first chain and the third chain are selected to be complementary to permit the first and third chains to associate by CH1-CK dimerization, wherein the domains of the second chain and the fourth chain are selected to be complementary to permit the second and fourth chains to associate by CH1-CK dimerization, and wherein each V domain pair V ai and V bi , V a 2 and V b 2 and V a
  • V a-2 (CH1 or C K ) - (hinge or L) - CH2 - CH3 -L- V a1 - V i - L - Cyt (Chain 1)
  • multimeric proteins with the Fc domain interposed between NKp30 ABD and the cytokine receptor ABD can be constructed.
  • Such proteins may be composed of a central (first) polypeptide chain comprising one or two immunoglobulin variable domains, connected or fused, optionally via a CH1 or CL constant region or via a linker, to the N-terminus of an Fc domain, wherein the Fc domain is connected at its C-terminus to a cytokine polypeptide (e.g. to the C-terminus of the cytokine polypeptide.
  • an additional polypeptide chain e.g.
  • a light chain comprising a V and C domain, optionally a VK and a CK domain
  • V and C domain optionally a VK and a CK domain
  • a further one or two additional chains can then provide at least one further ABD.
  • a further one, two or three polypeptide chains will provide the complementary Fc domain and variable domains depending on whether the ABDs are configured as scFv or Fab.
  • the Fc domain is interposed between the NKp30 binding ABD and the cytokine polypeptide in the multispecific protein. In this way, heterodimeric, heterotrimeric or heterotetrameric multispecific proteins can be constructed.
  • Examples of the domain arrangements (N- to C-termini, left to right) of central polypeptide chains include any of the following, wherein each V is a variable domain:
  • V a -2 - V b -2 (hinge or linker) - CH2 - CH3 - linker - Cyt (first/central chain).
  • V a -i is a light chain or heavy chain variable domain which will form an ABD together with a variable region Vw on a further polypeptide chain (e.g. the further chain comprising the variable region VM fused to a constant region); V a-2 and V b-2 together form an scFv (one of V a-2 and V b-2 is a light chain variable domain and the other is a heavy chain variable domain and they are separated by a flexible polypeptide linker; in any embodiment, a V - V can accordingly be specified as comprising a linker placed between the two V regions); and the CH3 domain is connected or fused to the cytokine via a domain linker (e.g.
  • a second polypeptide chain can then be configured to comprise one or two immunoglobulin variable domains, optionally a constant region, and an Fc domain suitable to undergo CH3-CH3 dimerization with the first/central polypeptide chain.
  • the second polypeptide chain has one immunoglobulin variable domain it can conveniently be fused to a CH1 or CL domain which is in turn fused to the CH2 domain via a hinge region.
  • the two variable domains can together form an scFv and be fused to the CH2 domain via a polypeptide linker.
  • a second polypeptide chain for use in a protein in which the Fc domain is interposed between the NKp30 ABD and IL2v can comprise a domain arrangement:
  • V a -4 - V b -4 (hinge or linker) - CH2 - CH3 (second chain) or
  • V a -3 (CH1 or C K ) - (hinge or linker) - CH2 - CH3 (second chain).
  • V a-3 is a light chain or heavy chain variable domain which will form an ABD together with a variable region V b -3 on a further polypeptide chain (e.g. a chain comprising the variable region V b -3 fused to a light chain constant region); V a-4 and V b-4 together form an scFv (one of V a-4 and V b-4 is a light chain variable domain and the other is a heavy chain variable domain and they are separated by a flexible polypeptide linker).
  • V a-4 and V b-4 together form an scFv (one of V a-4 and V b-4 is a light chain variable domain and the other is a heavy chain variable domain and they are separated by a flexible polypeptide linker).
  • heterodimer When dimeric proteins are desired, the following heterodimer can be configured:
  • V a-4 - V b-4 (hinge or linker) - CH2 - CH3 (second chain).
  • An exemplary heterodimeric protein can have a polypeptide chain 1 and 2:
  • ABD antigen binding domain
  • CH1 is a heavy chain constant domain 1 and CL is a light chain constant domain;
  • Hinge is an immunoglobulin hinge region or portion thereof
  • L1, L2, L3, L4 and L5 are each an amino acid domain linker, wherein L1, L2, L3, L4 and L5 can be different or the same;
  • CH2 and CH3 are human immunoglobulin CH2 and CH3 domains, respectively.
  • Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21 , IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
  • one of the first and second chains will be selected to comprise two variable domains (e.g., as an scFv), and a third polypeptide chain can then be provided, comprising the following domain arrangement:
  • V which can be also be designated VM or V b -3
  • V is a light chain or heavy chain variable domain which will form an ABD together with the variable region V a -i or V a -3 on the first or second polypeptide chain.
  • the CH1 or CK domain in the third chain will be selected so as to undergo CH1-CK dimerization with the respective first or second polypeptide chain with which the third chain is selected to associate (one of the associating chains has CH1 and the other has CK).
  • heterotrimer proteins can then be constructed in which the Fc domain is interposed between the NKp30 ABD and the cytokine, for example molecules having the following domain arrangement:
  • V b -i - (CH1 or CK) (third chain) wherein the first/central chain and the second chain associate by CH3-CH3 dimerization and the first/central chain and the third chain associate by the CH1 or CK dimerization, wherein the domains of the first/central chain and the third chain are selected to be complementary to permit the first and third chains to associate by CH1-CK dimerization, and wherein V a -i, VM, V a -2 and V b -2 are each a VH domain or a VL domain, and wherein one of V a -i and Vw is a VH and the other is a V L such that V a -i and V M form a first antigen binding domain (ABD), wherein one of V a -2 and V b -2 is a VH and the other is a V L such that V a -2 and V b -2 form a second antigen binding domain (e.g.
  • V a -2 and V b -2 are separated by a linker
  • one of the ABD binds NKp30 and the other binds an antigen of interest.
  • V a -2 and V b -2 form an ABD that binds NKp30
  • V a -i and V b -i form an ABD that binds the antigen of interest.
  • Another exemplary structure in which the Fc domain is interposed between the NKp30 ABD and the cytokine has following domain arrangements: (V a -2 - linker- V b -2) - (hinge or linker) - CH2 - CH3 (second chain)
  • V b -i - (CH1 or C K ) (third chain)
  • V a -i and Vw form an ABD that binds NKp30 and V a -2and
  • amino acid sequences of the polypeptide chains of such a protein are provided in Table 4 below.
  • both of the first and second chains can be selected to comprise one variable domain fused to a CH1 or CK constant domain, and a third polypeptide chain as shown above can be provided, as well as a fourth polypeptide chain comprising the following domain arrangement:
  • V which can be also be designated ⁇ - 1 or V b -3
  • V is a light chain or heavy chain variable domain which will form an ABD together with the variable region V a -i or V a -3 on the first or second polypeptide chain.
  • V of the third chain is designated VM and the third chain associates with the first chain such that Vw and V a -i form an ABD
  • the V of the fourth chain will be V b -3 and the fourth chain will associate with the second chain such that V b -3 associates with V a -3 to form an ABD.
  • the CH1 or CK domain in the fourth chain will be selected so as to undergo CH1-CK dimerization with the respective first or second polypeptide chain with which the fourth chain is selected to associate (one of the associating chains has CH1 and the other has CK).
  • heterotetramer proteins can then be constructed in which the Fc domain is interposed between the NKp30 ABD and the cytokine, for example molecules having the following domain arrangements:
  • V b -i - (CH1 or CK) (third chain) wherein the first/central chain and the second chain associate by CH3-CH3 dimerization and the first/central chain and the third chain associate by the CH1 or CK dimerization, wherein the domains of the first/central chain and the third chain are selected to be complementary to permit the first and third chains to associate by CH1-CK dimerization, wherein the domains of the second chain and the fourth chain are selected to be complementary to permit the second and fourth chains to associate by CH1-CK dimerization, and wherein V a -i, VM, V a -3 and V b -3 are each a VH domain or a VL domain, and wherein one of V a -i and Vn is a VH and the other is a VL such that V a -i and Vn form a first antigen binding domain (ABD), wherein one of V a -3 and V b -3 is a VH and the other is a VL such that
  • V a -3 and V b -3 an ABD that binds NKp30 and V a -i and VM form an ABD that binds the antigen of interest.
  • V a -3 and V b -3 an ABD that binds the antigen of interest and V a -i and Vn form an ABD that binds NKp30.
  • multispecific heterotetramer proteins can be generated on the natural immunoglobulin architecture containing two pairs of heavy chain and light chain combination with each pair having distinct binding specificity, with the cytokine polypeptide bound to the C-terminus of the Fc domain of one of the two heavy chains (one of the first and second chains), e.g. via a domain linker.
  • Other multispecific heterotrimer and heterotetramer proteins can be constructed in which VH and VK domains are substituted by one another and/or in which CH1 and CK are domains are substituted by one another compared to the natural immunoglobulin architecture.
  • CH1 and/or CL domains can be engineered to promote or enhance the desired heterodimerization through steric repulsion, or charge steering interaction.
  • the correct dimerization of the light chains (third and where present fourth chains) can be enhanced through the introduction of amino acid substitutions that give rise to attractive/repulsive charge pairs into the CH1 and CK domains.
  • Homodimerization of the two heavy chains is mediated by the CH3 interaction.
  • amino acid substitutions can be introduced to the two respective CH3 regions.
  • a multispecific protein can be generated by post-production assembly from structures based on half-antibodies, wherein one of the heavy chains bears at its C-terminus a cytokine fused via a domain linker (e.g., a ( - linker - Cyt) moiety fused to the C-terminus of the Fc domain monomer), thereby solving the issues of heavy and light chain mispairing.
  • a domain linker e.g., a ( - linker - Cyt) moiety fused to the C-terminus of the Fc domain monomer
  • Such multispecific protein will advantageously contain modification to favor heterodimerization of the half-antibodies, for example comprising a F405L mutation in one Fc monomer and a K409R mutation in the other Fc monomer, see, e.g., Labrijn et al., (2013) PNAS 110 (13) 5145-515.
  • Each half-antibody-type structure is individually produced in separate cell line and purified. The
  • multispecific proteins can be produced using similar architecture that binds the antigen of interest bivalently, binds NKp30 monovalently and binds the cytokine receptor monovalently, i.e., the multispecific protein has a 2:1:1 configuration. Examples are shown in
  • heterotetramer protein made from three different polypeptides which has two Fab structures and one scFv, where the two Fabs bind to the antigen of interest.
  • the protein can be constructed in which the Fc domain is interposed between the NKp30 ABD and the cytokine, having the following domain arrangements:
  • V a -i and VM is a VH and the other is a VL such that V a -i and VM form a first antigen binding domain (ABD), wherein one of V a -2 and V b -2 is a VH and the other is a VL such that V a -2 and V b -
  • VM and V form a second antigen binding domain, wherein VM and V an ABD that binds NKp30 and
  • V a -2 and V b -2 form the ABDs that binds the antigen of interest.
  • heteropentamer protein constructed from four different chains which has three Fab structures, two of which bind to the antigen of interest.
  • the protein can be constructed in which the Fc domain is interposed between the NKp30 ABD and the cytokine, having the following domain arrangements:
  • Vb-2 - (CH1 or CK) (VM - (CH1 or C K )
  • the protein has a Fc domain dimer comprised of a first and second Fc domain monomer placed on separate chains that dimerize via CH3-CH3 association, wherein one of the Fc domain monomers is connected to the both the anti-NKp30 ABD and the cytokine, and the other (second) Fc domain monomer has a free C-terminus (no anti-NKp30 ABD or cytokine fused to its C-terminus).
  • fusions or linkages on the same polypeptide chain between different domains may occur via intervening amino acid sequences, for example via a hinge region or linker peptide.
  • domain arrangements or structures herein are depicted without showing domain linkers, and it will be appreciated that the domain arrangements can be specified as having domain linkers between specified domain(s).
  • the cytokine can be specified as being fused to an adjacent domain via a domain linker, and a domain linker can be inserted in the relevant domain arrangement or structure.
  • tandem variable domains e.g. in an scFv
  • tandem variable domains can be specified as being fused to one another via a domain linker, and a domain linker can be inserted between the two V regions in the relevant domain arrangement or structure.
  • a CH1 or CL (or CK) constant region can be fused to an Fc domain or CH2 domain thereof via a domain linker or hinge domain or portion thereof, and accordingly a domain linker or hinge domain or portion thereof can be inserted between CH1 or CL domain and the Fc domain or CH2 domain in the relevant domain arrangement or structure.
  • An example of the domain arrangement of a multispecific protein with linkers shown is shown in Figure 2A for the representative heterotrimer in format “T5”, shows domain linkers such as hinge and glycine-serine linkers, and interchain disulfide bridges.
  • a polypeptide chain (e.g., chain 1, 2, 3 or 4) can be specified as having a free N and/or C terminus (no other protein domains at the terminus of the polypeptide chain).
  • the proteins domains described herein can optionally be specified as being indicated from N- to C- termini. Protein arrangements of the disclosure for purposes of illustration are shown from N-terminus (on the left) to C-terminus (on the right). Adjacent domains on a polypeptide chain can be referred to as being fused to one another (e.g.
  • a domain can be said to be fused to the C-terminus of the domain on its left, and/or a domain can be said to be fused to the N-terminus of the domain on its right).
  • the proteins domains described herein can be fused to one another directly (e.g. V domains fused directly to CH1 or CL domains) or via linkers or short intervening amino acid sequences that serve to connect the domains on a polypeptide chain (e.g. they may optionally be specified to lack other predetermined functionality, or to lack specific binding to a predetermined ligand).
  • Two polypeptide chains will be bound to one another (indicated by “”), by non-covalent interactions, and optionally can further be attached via interchain disulfide bonds, formed between cysteine residues within complementary CH1 and CK domains.
  • linker is a "domain linker”, used to link any two domains as outlined herein together. Adjacent protein domains can be specified as being connected or fused to one another by a domain linker.
  • An exemplary domain linker is a (poly)peptide linker, optionally a flexible (poly)peptide linker.
  • Peptide linkers or polypeptide linkers may have a subsequence derived from a particular domain such as a hinge, CH1 or CL domain, or may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr.
  • the linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity.
  • the linker is from about 1 to 50 amino acids in length, preferably about 2 to 30 amino acids in length.
  • linkers of 4 to 20 amino acids in length may be used, with from about 5 to about 15 amino acids finding use in some embodiments.
  • linkers can utilize a glycine-serine polypeptide or polymer, including for example comprising (GS)n, (GSG 2 S)n, (G4S) n , (GSs)n, (GS ⁇ n and (GsS)n, where n is an integer of at least one (optionally n is 1 , 2, 3 or 4), glycine-alanine polypeptide, alanine-serine polypeptide, and other flexible linkers.
  • Linkers comprising glycine and serine residues generally provides protease resistance.
  • a (GS)i linker is a linker having the amino acid sequence STGS; such a linker can be useful to fuse a domain to the C-terminus of an Fc domain (or a CH3 domain thereof).
  • a domain linker comprises a (G4S) n peptide, wherein, for example, n is an integer from 1-10, optionally 1-6, optionally 1-4.
  • a domain linker comprises a (GS4) n peptide, wherein, for example, n is an integer from 1-10, optionally 1-6, optionally 1-4.
  • a domain linker comprises a C-terminal GS dipeptide, e.g., a (GS4) comprising linker has the sequence (GS4)GS.
  • any of the peptide or domain linkers may be specified to comprise a length of at least 2 residues, 3 residues, 4 residues, at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, or more.
  • the linkers comprise a length of between 2-4 residues, between 2-4 residues, between 2-6 residues, between 2-8 residues, between 2-10 residues, between 2-12 residues, between 2-14 residues, between 3-15 residues, between 4-15 residues, between 2-16 residues, between 2-18 residues, between 2- 20 residues, between 2-22 residues, between 2-24 residues, between 2-26 residues, between 2-28 residues, between 2-30 residues, between 2 and 50 residues, or between 10 and 50 residues.
  • polypeptide linkers may include sequence fragments from CH1 or CL domains; for example the first 4-12 or 5-12 amino acid residues of the CL/CH1 domains are particularly useful for use in linkages of scFv moieties.
  • Linkers can be derived from immunoglobulin light chains, for example CK or CA.
  • Linkers can be derived from immunoglobulin heavy chains of any isotype, including for example Cy1, Cy2, Cy3, Cy4 and Cp.
  • Linker sequences may also be derived from other proteins such as Ig-like proteins (e.g. TCR, FcR, KIR), hinge region-derived sequences, and other natural sequences from other proteins.
  • V H and VL domains are linked to another in tandem separated by a linker peptide (e.g. a scFv) and in turn be fused to the N- or C-terminus of an Fc domain (or CH2 domain thereof).
  • a linker peptide e.g. a scFv
  • Such tandem variable regions or scFv can be connected to the Fc domain via a hinge region or a portion thereof, an N-terminal fragment of a CH1 or CL domain, or a glycine- and serine-containing flexible polypeptide linker.
  • Fc domains can be connected to other domains via immunoglobulin-derived sequence or via non-immunoglobulin sequences, including any suitable linking amino acid sequence.
  • immunoglobulin-derived sequences can be readily used between CH1 or CL domains and Fc domains, in particular, where a CH1 or CL domain is fused at its C-terminus to the N-terminus of an Fc domain (or CH2 domain).
  • An immunoglobulin hinge region or portion of a hinge region can and generally will be present on a polypeptide chain between a CH1 domain and a CH2 domain.
  • a hinge or portion thereof can also be placed on a polypeptide chain between a CL (e.g.
  • CK CK domain and the CH2 domain of an Fc domain when a CL is adjacent to an Fc domain on the polypeptide chain.
  • a hinge region can optionally be replaced for example by a suitable linker peptide, e.g. a flexible polypeptide linker.
  • the NKp30 ABD and cytokine receptor ABD are advantageously linked to the rest of the multispecific protein (e.g. or to a constant domain or Fc domain thereof) via a flexible linker (e.g. polypeptide linker) that leads to less structural rigidity or stiffness (e.g. between or amongst the ABD and Fc domain) compared to a conventional (e.g. wild-type full length human IgG) antibody.
  • the multispecific protein may have a structure or a flexible linker between the NKp30 ABD and constant domain or Fc domain that permits an increased range of domain motion compared to the two ABDs in a conventional (e.g.
  • the structure or a flexible linker can be configured to confer on the antigen binding sites greater intrachain domain movement compared to antigen binding sites in a conventional human lgG1 antibody.
  • Rigidity or domain motion/interchain domain movement can be determined, e.g., by computer modeling, electron microscopy, spectroscopy such as Nuclear Magnetic Resonance (NMR), X-ray crystallography, or Sedimentation Velocity Analytical ultracentrifugation (AUC) to measure or compare the radius of gyration of proteins comprising the linker or hinge.
  • a test protein or linker may have lower rigidity relative to a comparator protein if the test protein has a value obtained from one of the tests described in the previous sentence differs from the value of the comparator, e.g., an lgG1 antibody ora hinge, by at least 5%, 10%, 25%, 50%, 75%, or 100%.
  • a cytokine can for example be fused to the C-terminus of a CH3 domain by a linker selected from GSSSS (SEQ ID NO: 41), GSSSSGSSSS (SEQ ID NO: 42), GSSSSGSSSSGS (SEQ ID NO: 43) or GSSSSGSSSSGSSSS (SEQ ID NO: 44).
  • the multispecific protein may have a structure or a flexible linker between the NKp30 ABD and Fc domain that permits the NKp30 ABD and the ABD which binds an antigen of interest to have a spacing between said ABDs comprising less than about 80 angstroms, less than about 60 angstroms or ranges from about 40-60 angstroms.
  • an Fc domain (or a CH3 domain thereof) can be connected to the N- terminus of a NKp30 ABD or a cytokine polypeptide via a polypeptide linker, for example a glycine-serine-containing linker, optionally a linker having the amino acid sequence STGS.
  • a polypeptide linker for example a glycine-serine-containing linker, optionally a linker having the amino acid sequence STGS.
  • a CH1 or CL domain of a Fab is fused at its C-terminus to the N-terminus of the cytokine via a flexible polypeptide linker, for example a glycine-serine-containing linker.
  • the linker will have a chain length of at least 4 amino acid residues, optionally the linker has a length of 5, 6, 7, 8, 9 or 10 amino acid residues.
  • the NKp30 ABD is placed C-terminal to the Fc domain, and the NKp30 is positioned between an Fc domain and the cytokine polypeptide in the multispecific protein.
  • the NKp30 ABD will be connected or fused at its N-terminus (at the N- terminus of a VH or a VL domain) to the C-terminus of the Fc domain via a linker (e.g. a glycine and serine containing linker, a linker having the sequence STGS, a flexible polypeptide linker) of sufficient length to enable the NKp30 binding ABD to fold and/or adopt an orientation in such a way as to permit binding to NKp30 at the surface of an NK cell, while at the same time possesses a sufficient distance and range of motion relative to the adjacent Fc domain (or more generally to rest of the multispecific protein) such that the Fc domain can also simultaneously be found by CD16 expressed at the surface of the same NK cell.
  • a linker e.g. a glycine and serine containing linker, a linker having the sequence STGS, a flexible polypeptide linker
  • the C-terminus of a VH or VL of an scFv NKp30 ABD, or the CH1 or CL domain of a Fab NKp30 ABD will be connected or fused to the N-terminus of the cytokine polypeptide via a flexible linker (e.g.
  • the linker will have a chain length of at least 4 amino acid residues, optionally the linker has a length of 5, 6, 7, 8, 9 or 10 amino acid residues.
  • two V domains e.g. a VH domain and VL domains are generally linked together by a linker of sufficient length to enable the ABD to fold in such a way as to permit binding to the antigen for which the ABD is intended to bind.
  • linkers include linkers comprising glycine and serine residues, e.g., the amino acid sequence GEGTSTGSGGSGGSGGAD (SEQ ID NO : 260).
  • the VH domain and VL domains of an scFv are linked together by the amino acid sequence (G4S)3.
  • a (poly)peptide linker used to link a VH or VL domain of an scFv to a CH2 domain of an Fc domain comprises a fragment of a CH 1 domain or CL domain and/or hinge region.
  • an N-terminal amino acid sequence of CH1 can be fused to a variable domain in order to mimic as closely as possible the natural structure of a wild-type antibody.
  • the linker comprises an amino acid sequence from a hinge domain or an N-terminal CH1 amino acid.
  • the linker peptide mimics the regular VK-CK elbow junction, e.g., the linker comprises or consists of the amino acid sequence RTVA.
  • the hinge region used to connect the C-terminal end of a CH1 or CK domain (e.g. of a Fab) with the N-terminal end of a CH2 domain will be a fragment of a hinge region (e.g. a truncated hinge region without cysteine residues) or may comprise one or more amino acid modifications which remove (e.g. substitute by another amino acid, or delete) a cysteine residue, optionally both cysteine residues in a hinge region. Removing cysteines can be useful to prevent undesired disulfide bond formation, e.g., the formation of disulfide bridges in a monomeric polypeptide.
  • a “hinge” or “hinge region” or “antibody hinge region” herein refers to the flexible polypeptide or linker between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237. Thus for an IgG the hinge generally includes positions 221 (D221 in lgG1) to 236 (G236 in lgG1), wherein the numbering is according to the EU index as in Kabat. References to specific amino acid residues within constant region domains found within the polypeptides shall be, unless otherwise indicated or as otherwise dictated by context, be defined according to Kabat, in the context of an IgG antibody.
  • the hinge region (or fragment thereof) is derived form a hinge domain of a human lgG1 antibody.
  • a hinge domain may comprise the amino acid sequence: THTCPPCPAPELL (SEQ ID NO: 36), or a fragment comprising the first 8 resides thereof, or an amino acid sequence at least 60%, 70%, 80% or 90% identical to any of the foregoing, optionally wherein one or both cysteines are deleted or substituted by a different amino acid residue, optionally a serine.
  • the hinge region (or fragment thereof) is derived from a Cp2-C Cp3 hinge domain of a human IgM antibody.
  • a hinge domain may comprise the amino acid sequence: NASSMCVPSPAPELL (SEQ ID NO: 37), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto, optionally wherein one or both cysteines are deleted or substituted by a different amino acid residue.
  • Polypeptide chains that dimerize and associate with one another via non-covalent bonds or interactions may or may not additionally be bound by an interchain disulfide bond formed between respective CH1 and CK domains, and/or between respective hinge domains on the chains.
  • CH1 , CK and/or hinge domains can optionally be configured such that interchain disulfide bonds are formed between chains such that the desired pairing of chains is favored and undesired or incorrect disulfide bond formation is avoided.
  • the polypeptide chains when two polypeptide chains to be paired each possess a CH1 or CK adjacent to a hinge domain, the polypeptide chains can be configured such that the number of available cysteines for interchain disulfide bond formation between respective CH1/CK-hinge segments is reduced (or is entirely eliminated).
  • the amino acid sequences of respective CH1 , CK and/or hinge domains can be modified to remove cysteine residues in both the CH1/CK and the hinge domain of a polypeptide; thereby the CH1 and CK domains of the two chains that dimerize will associate via non-covalent interaction(s).
  • the CH1 or CK domain adjacent to (e.g., N-terminal to) a hinge domain comprises a cysteine capable of interchain disulfide bond formation
  • the hinge domain which is placed at the C-terminus of the CH1 or CK comprises a deletion or substitution of one or both cysteines of the hinge (e.g. Cys 239 and Cys 242, as numbered for human lgG1 hinge according to Kabat).
  • the hinge region (or fragment thereof) comprise the amino acid sequence: THTSPPSPAPELL (SEQ ID NO: 38), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto.
  • the CH1 or CK domain adjacent (e.g., N-terminal to) a hinge domain comprises a deletion or substitution at a cysteine residue capable of interchain disulfide bond formation
  • the hinge domain placed at the C-terminus of the CH1 or CK comprises one or both cysteines of the hinge (e.g. Cys 239 and Cys 242, as numbered for human lgG1 hinge according to Kabat).
  • the hinge region (or fragment thereof) comprises the amino acid sequence: THTCSSCPAPELL (SEQ ID NO: 39), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto.
  • a hinge region is derived from an IgM antibody.
  • the CH1/CK pairing mimics the Cp2 domain homodimerization in IgM antibodies.
  • the CH1 or CK domain adjacent (e.g., N-terminal to) a hinge domain comprises a deletion or substitution at a cysteine capable of interchain disulfide bond formation, and an IgM hinge domain which is placed at the C-terminus of the CH1 or CK comprises one or both cysteines of the hinge.
  • the hinge region (or fragment thereof) comprises the amino acid sequence: THTCSSCPAPELL (SEQ ID NO: 40), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto.
  • nonproteinaceous polymer or chemical linkers may find use in the multispecific proteins.
  • nonproteinaceous polymers including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers.
  • an amino acid sequence in a polypeptide chain of a multispecific protein may be modified to introduce a reactive group, optionally a protected reactive group, and the so-modified protein or chain is then reacted with a linker or a polypeptide comprising a complementary reactive group.
  • an amino acid residue in a polypeptide chain of a multispecific protein can be bound to a linker comprising a reactive group (for further reaction with a second polypeptide functionalized with a linker with a complementary reactive group) or directly a second polypeptide via an enzyme catalyzed reaction.
  • a polypeptide comprising an acceptor glutamine or lysine can reacted with linker comprising a primary amine in the presence of a transglutamine enzyme (e.g.
  • BTG Bacterial Transglutaminase, BTG
  • the transglutaminase enzyme catalyzes the conjugation of the linker to an acceptor glutamine residue within the primary structure of the polypeptide, for example within an immunoglobulin constant domain or within a TGase recognition tag inserted or appended to (e.g., fused to) a constant region.
  • a second polypeptide can also be functionalized with a linker in a similar manner, and when the conjugated linkers each bear complementary reactive groups (e.g.
  • reactive group pairs R and R’ include a range of groups capable of biorthogonal reaction, for example 1,3-dipolar cycloaddition between azides and cyclooctynes (copper-free click chemistry), between nitrones and cyclooctynes, oxime/hydrazone formation from aldehydes and ketones and the tetrazine ligation (see also WO2013/092983).
  • the resulting linker and functionalized antibody, or the Y element thereof, can thus comprise a RR’ group resulting from the reaction of R and R’, for example a triazole.
  • Methods and linkers for use in BTG-mediated conjugation to antibodies is described in PCT publication no. WO2014/202773, the disclosure of which is incorporated by reference.
  • Transglutaminase used interchangeably with “TGase” or “TG”, refers to an enzyme capable of cross-linking proteins through an acyl-transfer reaction between the y-carboxamide group of peptide-bound glutamine and the e-amino group of a lysine or a structurally related primary amine such as amino pentyl group, e.g.
  • TGases include, inter alia, bacterial transglutaminase (BTG) such as the enzyme having EC reference EC 2.3.2.13 (protein-glutamine-Y-glutamyltransferase).
  • BCG bacterial transglutaminase
  • acceptor glutamine residue, when referring to a glutamine residue of an antibody, means a glutamine residue that is recognized by a TGase and can be cross-linked by a TGase through a reaction between the glutamine and a lysine or a structurally related primary amine such as amino pentyl group.
  • the acceptor glutamine residue is a surface-exposed glutamine residue.
  • TGase recognition tag refers to a sequence of amino acids comprising an acceptor glutamine residue and that when incorporated into (e.g. appended to) a polypeptide sequence, under suitable conditions, is recognized by a TGase and leads to cross-linking by the TGase through a reaction between an amino acid side chain within the sequence of amino acids and a reaction partner.
  • the recognition tag may be a peptide sequence that is not naturally present in the polypeptide comprising the enzyme recognition tag. Examples of TGase recognition tags include the amino acid sequences disclosed in WO2012/059882 and WO2014/072482, the disclosure of which sequences are incorporated herein by reference.
  • Constant region domains can be derived from any suitable human antibody, particularly human antibodies of gamma isotype, including, the constant heavy (CH1) and light (CL, CK or Cl) domains, hinge domains, CH2 and CH3 domains.
  • CH1 and light CL, CK or Cl
  • hinge domains CH2 and CH3 domains.
  • CH1 generally refers to positions l ie- 220 according to the EU index as in Kabat.
  • a CH1 domain e.g. as shown in the domain arrangements
  • the CH1 domain when positioned C-terminal on a polypeptide chain and/or or C-terminal to the Fc domain, and/or within a Fab structure that is or C-terminal to the Fc domain, can optionally comprise at least part of a hinge region, for example CH1 domains can comprise at least an upper hinge region, for example an upper hinge region of a human lgG1 hinge, optionally further in which the terminal threonine of the upper hinge can be replaced by a serine.
  • a CH2 domain can therefore comprise at its C-terminus the amino acid sequence: EPKSCDKTHS. (SEQ ID NO : 261).
  • Exemplary human CH1 domain amino acid sequences include: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (SEQ ID NO: 26) or
  • Exemplary human CK domain amino acid sequences include: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29).
  • the multispecific protein can be a heterodimer, a heterotrimer or a heterotetramer comprising one or two Fabs (e.g. one Fab binding NKp30 and the other binding the antigen of interest), in which variable regions, CH1 and/or CL domains are engineered by introducing amino acid substitutions in a knob-into-holes or electrostatic steering approach to promote the desired chain pairings of CH1 domains with CK domains.
  • the multispecific protein can be a heterodimer, a heterotrimer or a heterotetramer comprising one or two Fabs (e.g.
  • a Fab has a VH/VL crossover (VH and VL replace one another) or a CH1/CL crossover (the CH1 and CL replace one another), and wherein the CH1 and/or CL domains comprise amino acid substitutions to promote correct chain association by knob-into-holes or electrostatic steering.
  • CH2 and CH3 domains can be derived from any suitable antibody. Such CH2 and CH3 domains can be used as wild-type domains or may serve as the basis for a modified CH2 or CH3 domain.
  • the CH2 and/or CH3 domain is of human origin or may comprise that of another species (e.g., rodent, rabbit, non-human primate) or may comprise a modified or chimeric CH2 and/or CH3 domain, e.g., one comprising portions or residues from different CH2 or CH3 domains, e.g., from different antibody isotypes or species antibodies.
  • another species e.g., rodent, rabbit, non-human primate
  • a modified or chimeric CH2 and/or CH3 domain e.g., one comprising portions or residues from different CH2 or CH3 domains, e.g., from different antibody isotypes or species antibodies.
  • the Fc domain monomer may comprise a CH2- CH3 unit (a full length CH2 and CH3 domain or a fragment thereof).
  • the CH3 domain will be capable of CH3-CH3 dimerization (e.g. it will comprise a wild-type CH3 domain or a CH3 domain with modifications to promote a desired CH3-CH3 dimerization).
  • Exemplary human lgG1 CH2-CH3 (Fc) domain amino acid sequences include: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 30).
  • an Fc domain may optionally further comprise a C-terminal lysine (K).
  • the multispecific protein can be a heterodimer, a heterotrimer or a heterotetramer, wherein the polypeptide chains are engineered for heterodimerization among each other so as to produce the desired protein.
  • the chains may comprise constant or Fc domains with amino acid modifications (e.g., substitutions) that favor the preferential chain pairing, e.g. favor a desired hetero-dimerization of the two different chains over the homo-dimerization of two identical chains.
  • a “knob-into-holes” approach is used in which the domain interfaces (e.g. CH3 domain interface of the antibody Fc region) are mutated so that the antibodies preferentially heterodimerize. Mutations can be introduced to create altered charge polarity across the interface (e.g. Fc dimer interface) such that co-expression of electrostatically matched chains (e.g. Fc-containing chains) support favorable attractive interactions thereby promoting desired heterodimer (e.g. Fc heterodimer) formation, whereas unfavorable repulsive charge interactions suppress unwanted heterodimer (e.g., Fc homodimer) formation.
  • the domain interfaces e.g. CH3 domain interface of the antibody Fc region
  • Mutations can be introduced to create altered charge polarity across the interface (e.g. Fc dimer interface) such that co-expression of electrostatically matched chains (e.g. Fc-containing chains) support favorable attractive interactions thereby promoting desired heterodimer (e.g.
  • one heavy chain comprises a T366W substitution and the second heavy chain comprises a T366S, L368A and Y407V substitution, see, e.g. Ridgway et al (1996) Protein Eng., 9, pp. 617-621; Atwell (1997) J. Mol. Biol., 270, pp. 26-35; and W02009/089004, the disclosures of which are incorporated herein by reference.
  • the “Hole” mutations on a first Fc monomer can comprise Y349C/T366S/L368A/Y407V and the complementary “Knob” mutations on the second Fc monomer can comprise S354C/T366W (Kabat EU numbering).
  • one heavy chain comprises a F405L substitution and the second heavy chain comprises a K409R substitution, see, e.g., Labrijn et al. (2013) Proc. Natl. Acad. Sci. U.S.A., 110, pp. 5145-5150.
  • one heavy chain comprises T350V, L351Y, F405A, and Y407V substitutions and the second heavy chain comprises T350V, T366S, K392L, and T394W substitutions, see, e.g. Von Kreudenstein et al., (2013) mAbs 5:646-654.
  • one heavy chain comprises both K409D and K392D substitutions and the second heavy chain comprises both D399K and E356K substitutions, see, e.g. Gunasekaran et al. , (2010) J. Biol. Chem. 285:19637-19646.
  • one heavy chain comprises D221E, P228E and L368E substitutions and the second heavy chain comprises D221 R, P228R, and K409R substitutions, see, e.g. Strop et al., (2012) J. Mol. Biol. 420: 204-219.
  • one heavy chain comprises S364H and F405A substitutions and the second heavy chain comprises Y349T and T394F substitutions, see, e.g. Moore et al., (2011) mAbs 3: 546-557.
  • one heavy chain comprises a H435R substitution and the second heavy chain optionally may or may not comprise a substitution, see, e.g. US Patent no. 8,586,713.
  • the Fc regions of these antibodies can be engineered to contain amino acid modifications that permit CD16 binding.
  • the antibody may comprise mammalian antibody-type N-linked glycosylation at residue N297 (Kabat EU numbering).
  • a multispecific protein comprises one or more amino acid modifications (e.g. substitutions) in a CH3 domain that affect binding to an affinity purification medium, e.g. Protein A.
  • an affinity purification medium e.g. Protein A.
  • Introduction into one of the CH3 domains of mutations that diminish binding to Protein A can be used to distinsguish unwanted chain pairings from the desired protein.
  • mutations can be introduced at amino acids H435 and Y436 (Kabat EU numbering), for example H435R and Y436F.
  • one or more pairs of disulfide bonds such as A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C are introduced into the Fc region to increase stability, for example further to a loss of stability caused by other Fc modifications. Additional example includes introducing K338I, A339K, and K340S mutations to enhance Fc stability and aggregation resistance (Gao et al, 2019 Mol Pharm. 2019; 16:3647).
  • one of the Fc monomers that make up the dimeric domain can comprise mutations in the CH3 domain (H435R and Y436F, according to EU numbering, disclosed in Jendeberg et al. (1997) Journal of Immunological Methods 201: 25-34) to reduce binding to Protein A; these mutations which are optional do not affect the activity of the protein but can potentially improve efficiency of purification by permitting the elimination of homodimers.
  • the Fc domain is a human lgG4 Fc domain, optionally further wherein the Fc domain comprises a S228P mutation to stabilize the hinge disulfide.
  • the Fc domain has an amino acid sequence at least 90%, 95% or 99% identical to a human lgG4 Fc domain, optionally further comprising a Kabat S228P mutation.
  • a CH2 and/or CH3 domain may comprise a modification to decrease or abolish binding to FcyRIIIA (CD16).
  • CD16 FcyRIIIA
  • CH2 mutations in a Fc domain dimer proteins at reside N297 can substantially eliminate CD16A binding.
  • the asparagine (N) at Kabat heavy chain residue 297 can be substituted by a residue other than an asparagine (e.g. a glutamine, a residue other than glutamine, for example a serine).
  • an Fc domain modified to reduce binding to CD16A comprises a substitution in the Fc domain at Kabat residues 234, 235 and 322.
  • the protein comprises a substitution in the Fc domain at Kabat residues 234, 235 and 331.
  • the protein comprises a substitution in the Fc domain at Kabat residues 234, 235, 237 and 331.
  • the protein comprises a substitution in the Fc domain at Kabat residues 234, 235, 237, 330 and 331.
  • the Fc domain is of human lgG1 subtype. Amino acid residues are indicated according to EU numbering according to Kabat.
  • an Fc domain modified to reduce binding to CD16A comprises an amino acid modification (e.g. substitution) at one or more of Kabat residue(s) 233-236, optionally one or more of residues 233-237, or at one, two or three of residues 234, 235 and/or 237, and an amino acid modification (e.g. substitution) at Kabat residue(s) 330 and/or 331.
  • an Fc domain comprises substitutions at Kabat residues L234, L235 and P331 (e.g., L234A/L235E/P331S or (L234F/L235E/P331S).
  • Fc domain comprises substitutions at Kabat residues L234, L235, G237 and P331 (e.g., L234A/L235E/G237A/P331S).
  • Fc domain comprises substitutions at Kabat residues L234, L235, G237, A330 and P331 (e.g.,
  • an antibody comprises an human lgG1 Fc domain comprising L234A/L235E/N297X/P331S substitutions, L234F/L235E/N297X/P331S substitutions, L234A/L235E/G237A/N297X/P331S substitutions, or L234A/L235E/G237A/ N297X/A330S/P331S substitutions, wherein X can be any amino acid other than an asparagine.
  • X is a glutamine; in another embodiment, X is a residue other than a glutamine (e.g. a serine).
  • an Fc domain that has low or reduced binding to CD16A comprises a human lgG4 Fc domain, wherein the Fc domain has the amino acid sequence below (human lgG4 with S228P substitution), or an amino acid sequence at least 90%, 95% or 99% identical thereto.
  • an Fc domain modified to reduce binding to CD16A comprises the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235 and 331 (underlined):
  • an Fc domain modified to reduce binding to CD16A comprises the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235 and 331 (underlined):
  • an Fc domain modified to reduce binding to CD16A comprises the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235, 237, 330 and 331
  • an Fc domain modified to reduce binding to CD16A comprises the amino acid sequence below, or a sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235, 237 and 331 (underlined):
  • any of the above Fc domain sequences can optionally further comprise a C-terminal lysine (K), i.e. as in the naturally occurring sequence.
  • a CH2 and/or CH3 domain may be a wild-type domain or a domain having CD16 interface residues from the wild-type human lgG1 domain, or may comprise one or more amino acid modifications (e.g. amino acid substitutions) which increase binding to human CD16 and optionally another receptor such as FcRn.
  • the modifications will not substantially decrease or abolish the ability of the Fc-derived polypeptide to bind to neonatal Fc receptor (FcRn), e.g. human FcRn.
  • Typical modifications include modified human lgG1 -derived constant regions comprising at least one amino acid modification (e.g.
  • FcyRI CD64
  • FcyRII CD32
  • FcyRIII CD16
  • FcyRI CD64
  • FcyRIIA CD32A
  • FcyRIII CD 16
  • a modification may, for example, increase binding of the Fc domain to FcyRII la on effector (e.g. NK) cells and/or decrease binding to FcyRIIB.
  • the multispecific protein comprises a variant Fc region comprise at least one amino acid modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 and/or CH3 domain of the Fc region, wherein the modification enhances binding to a human CD16 polypeptide.
  • the multispecific protein comprises at least one amino acid modification (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 domain of the Fc region from amino acids 237-341 , or within the lower hinge-CH2 region that comprises residues 231-341.
  • the multispecific protein comprises at least two amino acid modifications (for example, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications), wherein at least one of such modifications is within the CH3 region and at least one such modifications is within the CH2 region.
  • amino acid modifications in the hinge region encompassed are amino acid modifications in the CH1 domain, optionally in the upper hinge region that comprises residues 216-230 (Kabat EU numbering). Any suitable functional combination of Fc modifications can be made, for example any combination of the different Fc modifications which are disclosed in any of United States Patents Nos.
  • WO2011/109400 WO 2008/105886; WO 2008/002933; WO 2007/021841; WO 2007/106707; WO 06/088494; WO 05/115452; WO 05/110474; WO 04/1032269; WO 00/42072; WO 06/088494; WO 07/024249; WO 05/047327; WO 04/099249 and WO 04/063351; and/or in Lazar et al. (2006) Proc. Nat. Acad. Sci. USA 103(11): 405-410; Presta, L.G. et al. (2002) Biochem. Soc. Trans. 30(4):487-490; Shields, R.L.
  • the multispecific protein comprises an Fc domain comprising at least one amino acid modification (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region, such that the molecule has an enhanced binding affinity for human CD16 relative to the same molecule comprising a wild-type Fc region, optionally wherein the variant Fc region comprises a substitution at any one or more of positions 221 , 239, 243, 247, 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 308, 309, 310, 311 , 312, 316, 320, 322, 326, 329, 330, 332, 331 , 332, 333, 334, 335, 337, 338, 339, 340, 359, 360, 370, 373, 3
  • the multispecific protein comprises an Fc domain comprising at least one amino acid modification (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region, such that the molecule has enhanced binding affinity for human CD16 relative to a molecule comprising a wild-type Fc region, optionally wherein the variant Fc region comprises a substitution at any one or more of positions 239, 298, 330, 332, 333 and/or 334 (e.g. S239D, S298A, A330L, I332E, E333A and/or K334A substitutions), optionally wherein the variant Fc region comprises a substitution at residues S239 and I332, e.g. a S239D and I332E substitution (Kabat EU numbering).
  • amino acid modification for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications
  • the multispecific protein comprises an Fc domain comprising N-linked glycosylation at Kabat residue N297. In some embodiments, the multispecific protein comprises an Fc domain comprising altered glycosylation patterns that increase binding affinity for human CD16.
  • carbohydrate modifications can be accomplished by, for example, by expressing a nucleic acid encoding the multispecific protein in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery are known in the art and can be used as host cells in which to express recombinant antibodies to thereby produce an antibody with altered glycosylation. See, for example, Shields, R.L. et al. (2002) J. Biol. Chem.
  • the multispecific protein contains one or more hypofucosylated constant regions.
  • Such multispecific protein may comprise an amino acid alteration or may not comprise an amino acid alteration and/or may be expressed or synthesized or treated under conditions that result in hypofucosylation.
  • a multispecific protein composition comprises a multispecific protein described herein, wherein at least 20, 30, 40, 50, 60, 75, 85, 90, 95% or substantially all of the antibody species in the composition have a constant region comprising a core carbohydrate structure (e.g. complex, hybrid and high mannose structures) which lacks fucose.
  • a multispecific protein composition which is free of N- linked glycans comprising a core carbohydrate structure having fucose.
  • the core carbohydrate will preferably be a sugar chain at Asn297.
  • a multispecific protein comprising a Fc domain dimer can be characterized by having a binding affinity to a human CD16A polypeptide that is within 1-log of that of a conventional human lgG1 antibody, e.g., as assessed by surface plasmon resonance.
  • the multispecific protein comprising a Fc domain dimer in which an Fc domain is engineered to enhance Fc receptor binding can be characterized by having a binding affinity to a human CD16A polypeptide that is at least 1-log greater than that of a conventional or wild-type human lgG1 antibody, e.g., as assessed by surface plasmon resonance.
  • a multispecific protein comprising a Fc domain dimer can be characterized by having a binding affinity to a human FcRn (neonatal Fc receptor) polypeptide that is within 1-log of that of a conventional human lgG1 antibody, e.g., as assessed by surface plasmon resonance.
  • a multispecific protein comprising a Fc domain dimer can be characterized by a Kd for binding (monovalent) to a human Fc receptor polypeptide (e.g., CD16A) of less than 10 5 M (10 pmolar), optionally less than 10 -6 M (1 pmolar), as assessed by surface plasmon resonance (e.g. as in the Examples herein, SPR measurements performed on a Biacore T100 apparatus (Biacore GE Healthcare), with bispecific antibodies immobilized on a Sensor Chip CM5 and serial dilutions of soluble CD16 polypeptide injected over the immobilized bispecific antibodies.
  • a human Fc receptor polypeptide e.g., CD16A
  • surface plasmon resonance e.g. as in the Examples herein, SPR measurements performed on a Biacore T100 apparatus (Biacore GE Healthcare), with bispecific antibodies immobilized on a Sensor Chip CM5 and serial dilutions of soluble CD16 polypeptide injected over the immobilized bi
  • the antigen binding domain that binds to a cytokine receptor on NK cells can advantageously comprise a suitable cytokine polypeptide or polypeptide fragment such that the cytokine receptor ABD binds the cytokine receptor on the surface of an NK cell.
  • the cytokine can for example be a full-length wild-type IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b polypeptide, a fragment thereof sufficient to bind to the NK cell receptor for such cytokine, or a variant of any of the foregoing.
  • the cytokine molecule can be a fragment comprising at least 20, 30, 40, 50, 60, 70, 80 or 100 contiguous amino acids of a human cytokine, wherein the cytokine retains the ability to bind its cytokine receptor present on the surface of an NK cell.
  • the cytokine is a variant of a human cytokine comprising one or more amino acid modifications (e.g. amino acid substitutions) compared to the wild-type human cytokine, for example to decrease binding affinity to a receptor present on non-NK cells, for example Treg cells, CD4 T cells, CD8 T cells.
  • the cytokine can for example be a type I cytokine and a member of the common cytokine receptor gamma-chain (cg-chain) cytokine family, that signals via a heteromultimeric or heterdimeric receptor complex comprised of a receptor subunit (e.g., I L-2R /l L-15Rb or IL-21 R) subunit that associates with the common gamma-chain (CD132).
  • a receptor subunit e.g., I L-2R /l L-15Rb or IL-21 R
  • the multispecific proteins that binds to NKp30 and optionally further CD16A incorporates a cytokine (or fragment of variant thereof) which is modified to attenuate (reduce) binding affinity at the cytokine receptor expressed on NK cells, in comparison to the human wild-type cytokine counterpart.
  • the modified cytokine (or fragment of variant thereof) retains partial activity and/or binding affinity at the cytokine receptor expressed on NK cells, in comparison to the human wild-type cytokine counterpart.
  • the cytokine retains at least 5%, 10%, 20% or 50% of the ability of a wild-type cytokine counterpart to induce signaling through its receptor on NK cells.
  • the multispecific proteins that binds to NKp30 and optionally further CD16A permits the incorporation of a wild-type cytokine (or fragment of variant thereof) that retain substantially full activity and/or binding affinity at the cytokine receptor expressed on NK cells, in comparison to the human wild-type cytokine counterpart.
  • the cytokine is a wild-type cytokine or fragment thereof, or is a modified cytokine, wherein the cytokine is does not have a substantially reduced ability to induce signaling and/or does not have a substantially reduced binding affinity at its receptor on NK cells (e.g. CD122, IL-21 R, IL-7Ra, IL-27Ra, IL-12R, IL-18R).
  • the cytokine does not comprise a modification (e.g., substitution, deletion, etc.) that substantially reduces its ability to induce signaling through its receptor on NK cells (e.g. CD122, IL-21 R, IL-7Ra, IL-27Ra, IL-12R, IL- 18R).
  • the cytokine retains at least 80%, 90% of the ability of a wild-type cytokine counterpart to induce signaling through its receptor on NK cells (e.g. CD122, IL-21R, IL-7Ra, IL-27Ra, IL-12R, IL-18R).
  • signaling is assessed by bringing the cytokine (e.g. as a recombinant protein domain or within a multispecific protein of the disclosure) into contact with an NK cell and measuring signaling, e.g. measuring ST AT phosphorylation in the NK cells.
  • the cytokine or cytokine receptor ABD can be specified as binding its receptor, as determined by SPR, with a binding affinity (KD) of 200 nM or less, 100 nM or less, 50 nM or less or 25 nM or less.
  • the cytokine or cytokine receptor ABD binds its receptor, as determined by SPR, with a binding affinity (KD) that is 1 nM or higher than 1 nM, optionally that is higher than 10 nM optionally that is higher than 15 nM. In one embodiment, the cytokine or cytokine receptor ABD binds its receptor, as determined by SPR, with a binding affinity (KD) between about 1 nm and about 200 nm, optionally) between about 1 nm and about 100 nm optionally between about 10 nM and about 200 nM, optionally between about 10 nM and about 100 nM optionally between about 15 nM and about 100 nM.
  • KD binding affinity
  • the ABD can be or comprise a suitable interleukin-2 (IL-2) polypeptide such that the CD122 ABD binds CD122.
  • IL-2 interleukin-2
  • the ABD is advantageously a variant or modified IL-2 polypeptide that has reduced binding to CD25 (IL-2Ra) (e.g. reduced or abolished binding affinity, for example as determined by SPR) compared to a wild-type human interleukin-2.
  • IL-2Ra CD25
  • Such a variant or modified IL-2 polypeptide is also referred herein to as an “IL2v” ora “not-alpha IL-2”.
  • the CD122-binding ABD can optionally be specified to have a binding affinity for human CD122 that is substantially equivalent to that of wild-type human IL-2, or that is reduced compared to wild-type human IL- 2.
  • the CD122-binding ABD can optionally be specified to have an ability to induce CD122 signaling and/or binding affinity for CD122 that is substantially equivalent to that of wild-type human IL-2.
  • the CD122-binding ABD has a reduction in binding affinity for CD25 that is greater than the reduction in binding affinity for CD122, for example a reduction of at least 1-log, 2-log or 3-log in binding affinity for CD25 and a reduction in binding affinity for CD122 that is less than 1-log.
  • a heteromultimeric multispecific protein can be specified as being capable of binding to NKp30 and CD122, and optionally further CD16A, on an NK cell, and which is capable of potentiating NK cell cytotoxicity toward a target cell expressing an antigen of interest, and comprising:
  • an ABD that binds a human CD122 polypeptide wherein the ABD is placed at the C-terminus of the polypeptide chain which comprises such ABD, optionally wherein such ABD comprises an IL-2 polypeptide or portion thereof that binds CD122 and displays reduced binding affinity for human CD25 respectively, compared to a human wild-type IL-2 polypeptide.
  • the ABD that binds CD122 can thus be placed for example C-terminal and/or adjacent to the Fc domain and/or ABD that binds NKp30 on a polypeptide chain.
  • IL-2 is believed to bind II_-2Rb (CD122) in its form as a monomeric IL-2 receptor (IL- 2R), followed by recruitment of the IL-2Ry (CD132; also termed common g chain) subunit.
  • binding e.g. reduced binding
  • CD122 can therefore optionally be specified as being binding in or to a CD122:CD132 complex.
  • the CD122 can optionally be specified as being present at the surface of an NK cell.
  • IL-2 is believed to bind CD25 (IL- 2Ra) in its form as a monomeric IL-2 receptor, followed by association of the subunits IL-2Rb and IL-2RY. Binding (e.g. reduced binding, partially reduced binding) to CD25 can therefore optionally be specified as being binding in or to a CD25:CD122 complex or a CD25:CD122:CD132 complex.
  • the multispecific protein can optionally be specified as being configured and/or in a conformation (or capable of adopting a conformation) in which the CD122 ABD (e.g. IL2v) is capable of binding to CD122 at the surface of a cell (e.g. an NK cell, a CD122+CD25- cell) when the multispecific protein is bound to NKp30 (and optionally further to CD16) at the surface of said cell.
  • the multispecific protein:CD122 complex is capable of binding to CD132 at the surface of said cell.
  • the CD122 ABD or IL2v can be a modified IL-2 polypeptide, for example a monomeric IL-2 polypeptide modified by introducing one more amino acid substitutions, insertions or deletions that decrease binding to CD25, with or without a decrease in binding to CD122.
  • a IL-2 polypeptide can be modified by binding or associating it with one or more other additional molecules such as polymers or (poly)peptides that result in a further decrease of or abolished binding to CD25.
  • additional molecules such as polymers or (poly)peptides that result in a further decrease of or abolished binding to CD25.
  • a wild-type or mutated IL-2 polypeptide can be modified or further modified by binding to it another moiety that shields, masks, binds or interacts with CD25-binding site of human IL-2, thereby decreasing binding to CD25.
  • molecules such as polymers (e.g.
  • PEG polymers are conjugated to an IL-2 polypeptide to shield or mask the epitope on IL-2 that is bound by CD25, for example by introduction (e.g. substitution) to install an amino acid containing a dedicated chemical hook at a specific site on the IL-2 polypeptide.
  • a wild-type or variant IL-2 polypeptide is bound to anti-IL-2 monoclonal antibody or antibody fragment that binds or interacts with CD25-binding site of human IL-2, thereby decreasing binding to CD25.
  • an IL2 polypeptide can be a full-length IL-2 polypeptide or it can be an IL-2 polypeptide fragment, so long as the fragment or IL2v that comprises it retains the specified activity (e.g. retaining at least partial CD122 binding, compared to wild-type IL-2 polypeptide).
  • an IL2v polypeptide can advantageously comprise an IL-2 polypeptide comprising one or more amino acid mutations designed to reduce its ability to bind to human CD25 (IL-2Ra), while retaining at least at least some, or optionally substantially full, ability to bind human CD122.
  • IL2v or not-alpha IL-2 moieties have been described which reduce the activation bias of IL-2 on CD25+ cells. Such IL2v reduce binding to IL-2Ra and maintain at least partial binding to IL-2Rb.
  • IL2v polypeptides have been described, many having mutations in amino acid residue regions 35-72 and/or 79-92 of the IL-2 polypeptide.
  • decreased affinity to IL-2Ra may be obtained by substituting one or more of the following residues in the sequence of a wild-type IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69, and L72 (amino acid residue numbering is with reference to the mature IL-2 polypeptide shown in SEQ ID NO: 213).
  • the wild-type mature human IL-2 protein and a wild-type mature IL-2p protein fragment lacking the three first residues APT are shown below in SEQ ID NOS: 213 and 214, respectively:
  • An exemplary IL2v (also referred to herein as IL2v in the Examples) can have the amino acid of wild-type IL-2 with the five amino acid substitutions T3A, F42A, Y45A, L72G and C125A, as shown below, optionally further with deletion of the three N-terminal residues APA:
  • IL2v As few as one or two mutations can reduce binding to IL-2Ra and E-2Rb.
  • the IL2v polypeptide having two amino acid substitutions R38A and F42K in the wild-type IL-2p amino acid sequence displayed suitable reduced binding to IL-2Ra, with retention of binding to II_-2Rb resulting in highly active multispecific proteins, referred to herein as IL2v2.
  • an IL2v polypeptide has the wild-type IL-2p amino acid sequence with the three amino acid substitutions R38A, F42K and T41A, as shown below, referred to herein as IL2v3:
  • IL2v3 (R38A/T41A/F42K substitutions):
  • an IL2 variant comprises at least one or at least two amino acid modifications (e.g. substitution, insertion, deletion) compared to a human wild type IL-2 polypeptide.
  • an IL2v comprises a R38 substitution (e.g. R38A) and an F42 substitution (e.g., F42K), compared to a human wild type IL-2 polypeptide.
  • an IL2v comprises a R38 substitution (e.g. R38A), an F42 substitution (e.g., F42K) and a T41 substitution (e.g. T41A), compared to a human wild type IL-2 polypeptide.
  • an IL2v comprises a T3 substitution (e.g.
  • the IL2v comprises an amino acid sequence identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to the polypeptide of SEQ ID NOS: 213-217.
  • the IL2v comprises a fragment of a human IL-2 polypeptide, wherein the fragment has an amino sequence identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NOS: 213-217.
  • the IL-2 variant comprises two or more modification. In some embodiments, the IL-2 variant comprises three or more modification. In some embodiments, the IL-2 variant comprises four, five, or six or more modifications.
  • IL2 variant polypeptides can for example comprise two, three, four, five, six or seven amino acid modifications (e.g. substitutions).
  • US Patent No. 5,229,109 the disclosure of which is incorporated herein by reference, provides a human IL2 polypeptide having a R38A and F42K substitution. US Patent No.
  • residue Y45 e.g., Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R and Y45K
  • W02020/057646 relates to amino acid sequence of IL-2v polypeptides comprising amino acid substitutions in various combinations among amino acid residues K35, T37, R38, F42, Y45, E61 and E68.
  • WO2020252418 relates to amino acid sequence of IL-2v polypeptides having at least one amino acid residues position R38, T41 , F42, F44, E62, P65, E68, Y107, or S125 substituted with another amino acid, for example wherein the amino acid substitution is selected from the group consisting of: the substitution of L19D, L19H, L19N, L19P, L19Q, L19R, L19S, L19Y at position 19, the substitution of R38A, R38F, R38G at position 38, the substitution of T41A, T41G, and T41V at position 41 , the substitution of F42A at position 42, the substitution of F44G and F44V at position 44, the substitution of E62A, E62F, E62H, and E62L at position 62, the substitution of P65A, P65E, P65G, P65H, P65K, P65N, P65Q, P65
  • a modified IL-2 can optionally be specified as exhibiting a KD for binding to CD25 or to a CD25:CD122:CD132 complex that is decreased by at least 1-log, optionally at least 2-log, optionally at least 3-log, compared to a wild-type human IL-2 polypeptide (e.g. comprising the amino acid sequence of SEQ ID NO: 213).
  • a modified IL-2 can optionally be specified as exhibiting less than 20%, 30%, 40% or 50% of binding affinity to CD25 or to a CD25:CD122:CD132 complex compared to a wild-type human IL-2 polypeptide.
  • An IL2 can optionally be specified as exhibiting at least 50%, 70%, 80% or 90% of binding affinity to CD122 or to a CD122:CD132 complex compared to a wild-type human IL-2 polypeptide. In some embodiments, an IL2 exhibits at least 50%, 60%, 70% or 80% but less than 100% of binding affinity to CD122 or to a CD122:CD132 complex compared to a wild-type human IL-2 polypeptide. In some embodiments, an IL2v exhibits less than 50% of binding affinity to CD25 and at least 50%, 60%, 70% or 80% of binding affinity to CD122, compared to wild-type IL-2 polypeptide.
  • Differences in binding affinity of wild-type and disclosed mutant polypeptide for CD25 and CD122 and complexes thereof can be measured, e.g., in standard surface plasmon resonance (SPR) assays that measure affinity of protein-protein interactions familiar to those skilled in the art.
  • SPR surface plasmon resonance
  • Exemplary IL2 variant polypeptides have one or more, two or more, or three or more CD25-affinity-reducing amino acid substitutions relative to the wild-type mature IL-2 polypeptide having an amino acid sequence of SEQ ID NO: 213.
  • the exemplary IL2v polypeptides comprise one or more, two or more, or three or more substituted residues selected from the following group: Q11, H16, L18, L19, D20, D84, S87, Q22, R38, T41, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91 , I92, T123, Q126, S127, 1129, and S130.
  • the exemplary IL2 variant polypeptide has one, two, three, four, five or more of amino acid residues position R38, T41, F42, F44, E62, P65, E68, Y107, or S125 substituted with another amino acid.
  • decreased affinity to CD25 or a protein complex comprising such may be obtained by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69, and L72.
  • a CD122 ABD or IL-2 polypeptide is an IL-2 mimetic polypeptide.
  • Synthetic IL-2/IL-15 polypeptide mimics can be computationally designed to bind to CD122, but not to CD25, for example as described in Silva et al, (2019) Nature 565(7738): 186-191 and W02020/005819, the disclosures of which are incorporated herein by reference, also provides IL-2 and IL-15 mimetic polypeptides that bind CD122 but not CD25.
  • an IL-2 mimetic polypeptide can be characterized as a non-naturally occurring polypeptide comprising domains Xi, X 2 , X 3 , and X 4 , wherein:
  • Xi is a peptide comprising the amino acid sequence at least 85% identical to EHALYDAL (SEQ ID NO: 218);
  • X 2 is a helical -peptide of at least 8 amino acids in length
  • X 3 is a peptide comprising the amino acid sequence at least 85% identical to YAFNFELI (SEQ ID NO: 219);
  • X 4 is a peptide comprising the amino acid sequence at least 85% identical to ITILQSWIF (SEQ ID NO: 220); wherein Xi, X2, X3, and X* may be in any order in the polypeptide, wherein amino acid linkers may be present between any of the domains, and wherein the polypeptide binds to CD122 (or to the CD122:CD132 heterodimer).
  • the polypeptides bind the CD122:CD132 heterodimer with a binding affinity of 200 nM or less, 100 nM or less, 50 nM or less or 25 nM or less.
  • the invention provides non-naturally occurring polypeptides comprising domains Xi, X2, X3, and X4, wherein:
  • Xi is a peptide comprising the ammo acid sequence EHALYDAL (SEQ ID NO: 218);
  • X 2 is a helical-peptide of at least 8 amino acids in length
  • X3 is a peptide comprising the amino acid sequence YAFNFELI (SEQ ID NO: 219);
  • X 4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ ID NO: 220); wherein Xi, X2, X3, and X 4 may be in any order in the polypeptide, wherein amino acid linkers may be present between any of the domains, and wherein the polypeptide binds to CD122 (or to the CD122:CD132 heterodimer).
  • the polypeptides bind the CD122:CD132 heterodimer with a binding affinity of 200 nM or less, 100 nM or less, 50 nM or less or 25 nM or less, optionally between about 1 nm and about 100 nm, optionally between about 10 nM and about 200 nM, optionally between about 10 nM and about 100 nM optionally between about 15 nM and about 100 nM.
  • Xi, X3, and X4 may be any suitable length, meaning each domain may contain any suitable number of additional amino acids other than the peptides of SEQ I D NOS: 218, 219 and 220, respectively.
  • Xi is a peptide comprising the amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along its length to the peptide
  • PKKKIQLHAEHALYDALMILNI (SEQ ID NO: 221);
  • X 3 is a peptide comprising the amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along its length the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO: 222);
  • X 4 is a peptide comprising the amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along its length to the amino acid sequence EDEQEEMANAIITILQSWIFS (SEQ ID NO: 223).
  • a computationally designed synthetic IL-2 polypeptide or mimetic comprises the amino acid sequence (neoleukin) shown below (with or without a (08 4 ) 3 domain linker:
  • an IL-2 polypeptide is modified by connecting, fusing, binding or associating it with one or more other additional compounds, chemical compounds, polymer (e.g. PEG), or polypeptides or polypeptide chains that result in a decrease of binding to CD25.
  • a wild-type IL-2 polypeptide or fragment thereof can be modified by binding to it a CD25 binding peptide or polypeptide, including but not limited to an anti-IL-2 monoclonal antibody or antibody fragment thereof that binds or interacts with CD25-binding site of human IL-2, thereby decreasing binding to CD25.
  • an IL-2 further comprises a receptor domain, e.g., a cytokine receptor domain.
  • the cytokine molecule comprises an IL-2 receptor, or a fragment thereof (e.g., an IL-2 binding domain of an IL-2 receptor alpha).
  • a CD25- derived polypeptide is fused to an IL-2 polypeptide, as described in Lopes et al, J Immunother Cancer. 2020; 8(1), the disclosure of which is incorporated herein by reference.
  • the IL-2 is a variant fusion protein comprising a circularly permuted (cp) IL-2 fused to a CD25 polypeptide (see e.g., PCT publication no.
  • the CD122 ABD comprises a circularly permuted (cp) IL-2 fused to a CD25 polypeptide
  • the ABD can comprise a cplL-2:IL-2Ra polypeptide or protein of described in PCT publication no. WO2013/184942, the disclosure of which is incorporated herein by reference.
  • the permuted (cp) IL-2 variant fused to a CD25 polypeptide can have the amino acid sequence:
  • IL-2 is associated with a specific anti-IL-2 monoclonal antibody (mAb), thus forming an I L-2/anti-l L-2 mAb complex (IL-2cx).
  • mAb monoclonal antibody
  • IL-2cx I L-2/anti-l L-2 mAb complex
  • Such complexes have been shown to overcome the CD25 binding (Boyman et al., Science 311, 1924-1927 (2006).
  • An exemplary anti-IL2 antibody is antibody NARA1.
  • PCT publication no. W02017/122130 the disclosure of which is incorporated herein by reference, describes fusion proteins in which flexible linkers are used to connect IL-2 to the variable region of the light or heavy chain of NARA1. Sahin et al.
  • an IL-2 polypeptide or fragment thereof can be modified by binding to it a moiety of interest (e.g. a compound, chemical compounds, polymer, linear or branched PEG polymer), covalently attached to a natural amino acid or to an unnatural amino acid installed at a selected position.
  • a modified interleukin 2 (IL-2) polypeptide can comprise at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-2 polypeptide and CD25 but retains significant binding to the CD122:CD132 signaling complex, wherein the reduced binding to CD25 is compared to binding between a wild-type IL-2 polypeptide and CD25.
  • An unnatural amino acid can be positioned at any one or more of residues K35, T37, R38, T41 , F42, K43, F44, Y45, E60, E61 , E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107 of IL-2.
  • the unnatural amino acid can be incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNA pair.
  • the unnatural amino acid can for example comprise a lysine analogue, an aromatic side chain, an azido group, an alkyne group, or an aldehyde or ketone group.
  • the modified IL-2 polypeptide can then be covalently attached to a water-soluble polymer, a lipid, a protein, or a peptide through the unnatural amino acid.
  • suitable polymers include polyethylene glycol (PEG), polypropylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), polyplefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly (a- hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), or a combination thereof, or a polysaccharide such as dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • an exemplary IL2v/not-alpha IL-2 conjugate can comprise a full- length or fragment of an IL-2 polypeptide in which an amino acid residue in the IL-2 polypeptide (e.g. a residue at position selected from K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, and L72) is replaced by a natural or non-natural amino acid residue attached to a polymer via a chemical linker.
  • the polymer can be a PEG polymer, e.g.
  • a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa.
  • a modified IL2 polypeptide can comprising at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-2 polypeptide and CD25 but retains significant binding with CD122:CD132 signaling complex to form a CD122:CD132 complex, wherein the reduced binding to CD25 is compared to binding between a wild-type IL-2 polypeptide and CD25
  • An exemplary N2v/not-alpha IL-2 conjugate is THOR-707 (Synthorx inc).
  • Y is CH2 and Z is or
  • Y is CH2 and Z is and wherein, W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa; and X has the structure:
  • an IL-2 comprises a releasable polymer (e.g. a releasable PEG polymer), e.g. the IL-2 is conjugated, linked or bound to a releasable polymer that results in a decrease in CD25 binding in vivo and/or in vitro.
  • a releasable polymer e.g. a releasable PEG polymer
  • the IL-2 is conjugated, linked or bound to a releasable polymer that results in a decrease in CD25 binding in vivo and/or in vitro.
  • modified IL-2 include bempegaldesleukin or RSLAIL-2 (Nektar Therapeutics inc.), which exhibits about a 60-fold decrease in affinity to CD25 relative to IL-2, but only about a 5-fold decrease in affinity CD122 relative to IL-2.
  • Bempegaldesleukin (CAS No.1939126-74-5) is an IL-2 in which human interleukin-2 (des-1-alanine, 125-serine), is N-substituted with an average of six [(2,7- bis ⁇ [methylpoly(oxyethylene)iokD]carbamoyl ⁇ -9H-fluoren-9-yl)methoxy]carbonyl moieties at its amino residues.
  • human interleukin-2 des-1-alanine, 125-serine
  • the releasable PEG comprised can be based upon a 2,7,9-substituted fluorene, with poly(ethylene glycol) chains extending from the 2- and 7- positions on the fluorene ring via amide linkages (fluorene-C(O)-NH-), and having releasable covalent attachment to IL-2 via attachment to a carbamate nitrogen atom attached via a methylene group (-CH2-) to the 9-position of the fluorene ring.
  • the modified IL-2 can comprise compounds encompassed by the following formula: or wherein each “n”, the number of CH2CH2O units) is an integer from about 3 to about 4000, or more preferably is an integer from about 200-300. “m”, referring to the number of polyethylene glycol moieties attached to IL-2 is an integer selected from the group consisting of 1, 2, 3, 7 and more than 7. In some embodiments, each “n” is approximately the same, i.e. , the weight average molecular weight of each polyethylene glycol “arm” covalently attached to the fluorenyl core is about the same. Optionally, the weight average molecular weight of each PEG arm is about 10,000 daltons, such that the weight average molecular weight of the overall branched polymer moiety is about 20,000 daltons.
  • the ABD can be or comprise a suitable interleukin-15 (IL-15) polypeptide such that the CD122 ABD binds CD122.
  • the cytokine molecule is an IL-15 molecule, e.g., a full length, a fragment or a variant (I L-15v) of IL-15, e.g., human IL-15.
  • the IL-15 molecule comprises a wild-type human IL-15 amino acid sequence, e.g., having the amino acid sequence of SEQ ID NO: 227.
  • the IL-15 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-15 amino acid sequence of SEQ ID NO: 227.
  • the IL-15 molecule is a variant of human IL-15, e.g., having one or more amino acid modifications.
  • the IL-15 comprises a fragment of a human IL-15 polypeptide, wherein the fragment has an amino sequence is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the wild-type mature human IL-15 polypeptide of SEQ ID NO: 227.
  • an IL-15 variant comprises a modification (e.g. substitution) at position 45, 51 , 52, or 72 (with reference to the sequence of human IL-15, SEQ ID NO: 227), e.g., as described in US 2016/0184399.
  • the IL-15 variant comprises four, five, or six or more modifications.
  • the IL-15 variant comprises one or more modification at amino acid position 8, 10, 61, 64, 65, 72, 101, or 108 (with reference to the sequence of human IL-15, SEQ ID NO: 227).
  • the IL-15 variant possesses increased affinity for CD122 as compared with wild-type IL-15.
  • the IL-15 variant possesses decreased affinity for CD122 as compared with wild-type IL-15.
  • the mutation is chosen from D8N, K10Q, D61 N, D61 H, E64H, N65H, N72A, N72H, Q101N, Q108N, or Q108H (with reference to the sequence of human IL-15, SEQ ID NO: 227). Any combination of the positions can be mutated.
  • the IL-15 variant comprises two or more mutations.
  • the IL-15 variant comprises three or more mutations.
  • the IL-15 variant comprises four, five, or six or more mutations.
  • the IL-15 variant comprises mutations at positions 61 and 64.
  • the mutations at positions 61 and 64 are D61 N or D61 H and E64Q or E64H.
  • the IL-15 variant comprises mutations at positions 61 and 108.
  • the mutations at positions 61 and 108 are D61 N or D61 H and Q108N or Q108H.
  • the extracellular domain of IL-15Ra comprises a domain referred to as the sushi domain, which binds IL-15.
  • the general sushi domain also referred to as complement control protein (CCP) modules or short consensus repeats (SCR), is a protein domain found in several proteins, including multiple members of the complement system.
  • CCP complement control protein
  • SCR short consensus repeats
  • the sushi domain adopts a beta-sandwich fold, which is bounded by the first and fourth cysteine of four highly conserved cysteine residues, comprising a sequence stretch of approximately 60 amino acids (Norman, Barlow, et al. J Mol Biol. 1991 ;219(4):717-25).
  • the amino acid residues bounded by the first and fourth cysteines of the sushi domain IL-15Ra comprise a 62 amino acid polypeptide referred to as the minimal domain.
  • Including additional amino acids of IL-15Ra at the N- and C-terminus of the minimal sushi domain, such as inclusion of N-terminal lie and Thr and C- terminal lie and Arg residues result in a 65 amino acid extended sushi domain.
  • the CD122 ABD can further comprise a receptor domain, e.g., a cytokine receptor domain.
  • a receptor domain e.g., a cytokine receptor domain.
  • the cytokine molecule comprises an IL-15 receptor, or a fragment thereof (e.g., an IL-15 binding domain of an IL-15 receptor alpha).
  • the CD122 ABD binds an IL-15 receptor alpha (IL-15Ra) sushi domain, a first domain linker, and an IL-15 polypeptide, e.g. from N- to C-terminus, a IL-15Ra sushi domain fused to a domain linker, in turn fused to an IL-15 polypeptide.
  • the IL- 15 polypeptide is a variant IL-15 polypeptide, e.g., comprising one or more amino acid substitutions.
  • the variant IL-15 domain comprises the amino acid sequence of SEQ ID NO: 227 and amino acid substitutions selected from the group consisting of N4D/N65D, D30N/N65D, and D30N/E64Q/N65D.
  • a sushi domain as described herein may comprise one or more mutations relative to a wild-type sushi domain.
  • the IL-l5Ra sushi domain comprises the amino acid sequence:
  • An IL-15 polypeptide can be modified by connecting, fusing, binding or associating it with one or more other additional compounds using any of several known techniques, for example by conjugation or binding to chemical compounds, polymer (e.g. PEG), or polypeptides or polypeptide chains that result in a decrease of binding to IL-15Ra.
  • a wild-type IL-15 polypeptide or fragment thereof can be modified by binding to it a IL-15Ra binding peptide or polypeptide, including but not limited to an anti-IL-15 monoclonal antibody or antibody fragment thereof that binds or interacts with IL-15Ra -binding site of human IL-15, thereby decreasing binding to IL-15Ra.
  • an IL-15 polypeptide or fragment thereof can be modified by binding to it a moiety of interest (e.g. a compound, chemical compounds, polymer, linear or branched PEG polymer), covalently attached to a novel amino acid installed at a selected position.
  • a modified IL-15 polypeptide can comprise at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-15 polypeptide and IL-15Ra but retains significant binding with CD122:CD132 signaling complex to form an CD122:CD132 complex, wherein the reduced binding to IL-15Ra is compared to binding between a wild-type IL-15 polypeptide and IL-15Ra.
  • the unnatural amino acid can be positioned at any one or more of residues N1, W2, V3, N4, 16, S7, D8, K10, K11, E13, D14, L15, Q17, S18, M19, H20, 121, D22, A23, T24, L25, Y26, E28, S29, D30, V31, H32, P33, S34, C35, K36, V37, T38, K41, L44, E46, Q48, V49, S51, L52, E53, S54, G55, D56, A57, S58, H60, D61 , T62, V63, E64, N65, I67, I68, L69, N71, N72, S73, L74, S75, S76, N77, G78, N79, V80, T81 , E82, S83, G84, C85, K86, E87, C88, E89, E90, L91 , E92, E93, K94, N95,
  • the unnatural amino acid can be incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNA pair.
  • the unnatural amino acid can for example comprise a lysine analogue, an aromatic side chain, an azido group, an alkyne group, or an aldehyde or ketone group.
  • the modified IL-15 polypeptide can then be covalently attached to a water-soluble polymer, a lipid, a protein, or a peptide through the unnatural amino acid.
  • suitable polymers include polyethylene glycol (PEG), polypropylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, polypxyethylated polyol), polyplefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly (a- hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), or a combination thereof, or a polysaccharide such as dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • HA hyaluronic acid
  • Y is CH2 and Z is or
  • Y is CH2 and Z is and wherein, W is a PEG group having an average molecular weight selected from
  • an IL-15 comprises a releasable polymer (e.g. a releasable PEG polymer), e.g. the IL-15 is conjugated, linked or bound to a releasable polymer that results in a decrease in IL-15R binding in vivo and/or in vitro.
  • a releasable polymer e.g. a releasable PEG polymer
  • the IL-15 is conjugated, linked or bound to a releasable polymer that results in a decrease in IL-15R binding in vivo and/or in vitro.
  • Examples include compounds disclosed in PCT publication no. W02020/097556, the disclosure of which is incorporated herein by reference.
  • the modified IL-15 can comprise comprising compound having the structure: wherein (n) is an integer from about 150 to about 3,000, (m) is an integer selected from 2, 3, 4, and 5, (n’) is 1, and ⁇ NH ⁇ represents an amino group of the IL-15 polypeptide.
  • the ABD can be or comprise a suitable interleukin-21 (IL-21) polypeptide such that the IL-21 R ABD binds IL-21 R on the surface of NK cells.
  • IL-21 R is similar in structure to the IL-2 receptor and the IL-15 receptor, in that each of these cytokine receptors comprises a common gamma chain (yc).
  • the cytokine molecule is an IL-21 molecule, e.g., a full length, a fragment or a variant of IL-21, e.g., human IL-21.
  • the IL-21 molecule is a wild-type, human IL-21 , e.g., having the amino acid sequence of SEQ ID NO: 229.
  • the IL-15 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-21 amino acid sequence of SEQ ID NO: 229.
  • the IL-21 molecule is a variant of human IL-21, e.g., having one or more amino acid modifications.
  • the IL-21 comprises a fragment of a human IL-21 polypeptide, wherein the fragment has an amino sequence is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 229.
  • the IL-21 variant can comprise an IL-21 polypeptide comprising one or more amino acid mutations designed to reduce its ability to bind to human IL-21 R, while retaining substantial ability to bind human IL-21 R.
  • the IL-21 can be characterized as binding to human IL-21 R with a KD that is greater than or is about 0.04 nM, as determined by SPR.
  • the amino acid substitutions are located at two amino acid positions selected from the group consisting of 10, 14, 20, 75, 76, 77, 78 and 81 according to numbering of SEQ ID NO: 229 or at two amino acid positions selected from the group consisting of 5, 9, 15, 70, 71 , 72, 73, and 76, according to the amino acid position numbering of SEQ ID NO: 230.
  • the IL-21 variant comprises the amino acid sequence:
  • the IL-21 variant comprises the sequence of SEQ ID NO: 230, wherein SEQ ID NO: 230 differs from SEQ ID NO: 229 by at least one amino acid at a position designated by X in SEQ ID NO: 230.
  • the IL-21 variant has at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 229.
  • the IL-21 variant comprises an amino acid substitution relative to the wild-type IL-21 amino acid sequence within the N-terminal half of the amino acid sequence, e.g. at a position within positions 10-30 or 13-28 (both inclusive), according to the amino acid position numbering of SEQ ID NO: 229.
  • the IL- 21 variant comprises an amino acid substitution relative to the wild-type IL-21 amino acid sequence within the C-terminal half of the amino acid sequence, e.g., at a position within positions 105-138 or 114-128 (both inclusive), according to the amino acid position numbering of SEQ ID NO: 229.
  • the IL-21 variant comprises an amino acid substitution relative to the wild-type IL-21 amino acid sequence in the middle third of the amino acid sequence, e.g., at a position within positions 60-90 or 70-85 (both inclusive), according to the amino acid position numbering of SEQ ID NO: 229.
  • the IL-21 variant comprises only one amino acid substitution, relative to the wild-type IL-21 amino acid sequence.
  • the amino acid substitution is located at an amino acid position selected from the group consisting of: 10, 13, 14, 16, 17, 18, 19, 20, 21, 24, 28, 70, 71, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 114, 115, 117, 118, 121, 122, 124, 125, or 128, according to the amino acid position numbering of SEQ ID NO: 229.
  • the ABD can be or comprise a suitable interleukin-18 (IL-18) polypeptide such that the IL-18R ABD binds IL-18Ra on the surface of NK cells.
  • the cytokine molecule is an IL-18 molecule, e.g., a full length, a fragment or a variant of IL-18, e.g., human IL-18.
  • the IL-18 molecule is a wild-type, human IL-18, e.g., having the amino acid sequence of SEQ ID NO: 422.
  • the IL-18 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-18 amino acid sequence of SEQ ID NO: 231.
  • the IL-18 molecule is a variant of human IL-18, e.g., having one or more amino acid modifications.
  • the IL-18 comprises a fragment of a human IL-18 polypeptide, wherein the fragment has an amino sequence is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 231.
  • an IL-18 is modified to decrease its binding affinity for IL-18BP while not substantially decreasing affinity for IL-18Ra.
  • an IL-18 may comprise of a modification such as amino acid substitutions at positions M51 , S55, R104 and/or N 110 that are not involved in IL-18Ra binding, optionally further in combination with a substitution at K53 and/or M60 (positions are with reference to the wild-type mature IL-18 amino acid sequence).
  • the IL-18 has a M51S, S55A, R104Q, R104K or R104S and/or N110A substitution.
  • the IL-18 comprises a K53S or K53A substitution.
  • the IL-18 comprises a M60S or M60K substitution.
  • the cytokine-binding ABD binds a type I interferon receptor, for example interferon-a receptor (IFN-aR).
  • the ABD can be or comprise a suitable type I interferon, for example an interferon-a (IFN-a) or interferon-b (IFN-b) polypeptide such that the IFN-a ABD binds IFN-aR on the surface of NK cells.
  • the interferon-a receptor is also known as the interferon a/b receptor (IFNAR), a heterodimeric transmembrane receptor that is composed of the two subunits IFNAR1 and IFNAR2.
  • the main STAT signaling complex is formed by IFN-stimulated gene factor 3 consisting of STAT 1 , STAT2, and IFN regulatory factor (IRF)-9.
  • the cytokine molecule is an IFN-a or IFN- b molecule, e.g., a full length, a fragment or a variant of IFN-a or IFN-b, e.g., human IFN-a or IFN-b, for example a human IFN-a1, IFN-a2, IFN-a4, IFN-a5, IFN-a6, IFN-a7, IFN-a8, IFN- a10, IFN-a12, IFN-a14, IFN-a16 or IFN-a17 polypeptide.
  • the IFN-a or IFN-b molecule is a wild-type, human IFN-a or IFN-b, e.g., having the amino acid sequence of any of SEQ ID NOS: 232-243.
  • the IFN-a or IFN-b molecule is a variant of human IFN-a or IFN-b, e.g., having one or more amino acid modifications.
  • the IFN-a or IFN-b molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IFN-a or IFN-b amino acid sequence of SEQ ID NOS: 232-243, respectively.
  • the IFN-a or IFN-b molecule is a variant of human IFN-a or IFN-b, e.g., having one or more amino acid modifications.
  • the IFN-a or IFN-b comprises a fragment of a human IFN-a or IFN-b polypeptide, wherein the fragment has an amino sequence is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NOS: 232-243.
  • the IFN-a or IFN-b variant polypeptide has an amino acid sequence that has at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NOS: 232-243, respectively.
  • the wild type or modified signaling agent is a modified interferon-a having decreased binding affinity for its receptor, particularly IFNAR2.
  • the modified IFNal , IFNa2, IFNa4, IFNa5, IFNa6, IFNa7, IFNa8, IFNa10, IFNa12, IFNa14, IFNa16 or IFNa17 agent has reduced affinity for and/or induction of signaling at IFNAR (IFNAR1 and/or IFNAR2 chains).
  • wild-type IFNs bind to IFNAR2 at affinities (KD), e.g., as determined by microcal or SPR, between 0.1 nM and 5nM and to IFNAR1 at an affinity of about 1 mM.
  • IFNal binds to IFNAR2 with a KD of about 200 nM.
  • an IFN is modified so as to have an affinity for IFNAR1 and/or IFNAR2 that is equal or less than that of the NKp30 ABD for NKp30.
  • an IFN is modified so as to have an affinity for IFNAR1 and/or IFNAR2 that is at least 1-log less than that of the NKp30 ABD for NKp30.
  • the NKp30 ABD has a KD for NKp30 binding of about 15 nM.
  • An IFN can thus be modified by introduction of a modification that causes a reduction of binding affinity of between 10-fold (1-log) and 1000- fold (3-log) (an increase in KD of between 1-log and 3-log).
  • An IFN can include any of the amino acid substitutions shown in the table below.
  • the table below shows exemplary single amino acid substitutions that decrease binding affinity of IFN-a polypeptides to IFNAR2, with a cut-off of a decrease in affinity (higher KD) of at least 1 log compared to the wild-type counterpart and no more than 3-log compared to the wild-type counterpart.
  • the table below shows the relative affinity based on KD values for IFNAR2 of the mutated cytokine compared to the wild-type cytokine.
  • the table below shows exemplary single amino acid substitutions decreasing binding affinity of IFN-a polypeptides to IFNAR1, with an at least 2-fold decrease in affinity.
  • the table shows the relative affinity based on KD values for IFNAR1 of the mutated cytokine compared to the wild-type cytokine.
  • said IFNa2 mutant (IFNa2a or IFNa2b) is mutated at one or more amino acids at positions 144-154, such as amino acid positions 148, 149 and/or 153.
  • the I FNa2 mutant comprises one or more mutations selected from L153A, R149A, and M148A, described in WO2013/107791.
  • the IFNa2 mutants have reduced affinity and/or activity for IFNAR1.
  • the IFNa2 mutant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A, as described in WO2010/030671.
  • the IFNa2 mutant comprises one or more mutations selected from K133A, R144A, R149A, and L153A as described in W02008/124086. In some embodiments, the IFNa2 mutant comprises one or more mutations selected from R120E and R120E/K121E, as described in W02015/007520 and WO20 10/030671.
  • the mutant human IFNa2 comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 232, wherein the mutant IFNa2 has one or more mutations at positions L15, A19, R22, R23, L26, F27, L30, L30, K31, D32, R33, H34, D35, Q40, H57, E58, Q61 , F64, N65, T69, L80, Y85, Y89, D 114, L117, R120, R125, K 133, K 134, R144, A145, M 148, R149, S 152, L153, and N156 with respect to SEQ ID NO: 232.
  • the human IFNa2 mutant comprises one or more mutations selected from, L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, T106A, T106E, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A as disclosed in WO 2013/059885, for example in some embodiments, the human IFNa2 mutant comprises the mutations H57Y, E58N, Q61S, and/or L30A; the mutations H57Y, E58N, Q61S, and/or R33A; the mutations H57Y, E58N, Q61S, and/or M148A; the mutations H57Y, E58N, Q61S, and/or
  • the wild type or modified signaling agent is a modified interferon-a having decreased binding affinity for its receptor, particularly IFNAR2.
  • the modified IFNa2 agent has reduced affinity for and/or induction of signaling at IFNAR (IFNAR1 and/or IFNAR2 chains).
  • the IFNal interferon is modified to have a mutation at one or more amino acids at positions L15, A19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154, and N157 with reference to SEQ ID NO: 233.
  • the mutations can optionally be a hydrophobic mutation and can be, e.g., selected from alanine, valine, leucine, and isoleucine.
  • the FNa1 interferon is modified to have a one or more mutations selected from L15A, A19W, R23A, S25A, L30A, L30V, D32A, R33K, R33A, R33Q, H34A, Q40A, C86S, C86A, D115R, L118A, K121A, K121 E, R126A, R126E, E133A, K134A, K135A, R145A, R145D, R145E,
  • the FNa1 mutant comprises one or more multiple mutations selected from L30A/H58Y/E59N/Q62S, R33A/H58Y/E59N/Q62S,
  • the IFN-a1 is a variant that comprises one or more mutations which reduce undesired disulphide pairings wherein the one or more mutations are, e.g., at amino acid positions C1, C29, C86, C99, or C139 with reference to SEQ ID NO: 233.
  • the mutation at position C86 can be, e.g., C86S or C86A or C86Y.
  • the wild type or modified signaling agent is IFN-b.
  • the IFN-b is human having a sequence as shown below:
  • the human IFN-b is a non-glycosylated form of human IFN-b that has a Met-1 deletion and a Cys-17 to Ser mutation.
  • the modified IFN-b has one or more mutations that reduce its binding to or its affinity for the IFNAR1 subunit of IFNAR. In one embodiment, the modified IFN-b has reduced affinity and/or activity at IFNAR1.
  • the modified IFN-b is human IFN-b and has one or more mutations at positions F67, R71, L88, Y92, 195, N96, K123, and R124.
  • the one or more mutations are substitutions selected from F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G, and R124G.
  • the modified IFN-b has one or more mutations that reduce its binding to or its affinity for the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFN-b has reduced affinity and/or activity at IFNAR2. In various embodiments, the modified IFN-b is human IFN-b and has one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155. In some embodiments, the one or more mutations are substitutions selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G.
  • IFN-b mutations are described in PCT publication nos. W02020/198661 , W02000/023114 and US20150011732 the disclosures of which are incorporated hereby by reference.
  • the ABD can be or comprise a suitable interleukin-7 (IL-7) polypeptide such that the IL-7R ABD binds IL-7Ra on the surface of NK cells.
  • the cytokine molecule is an IL-7 molecule, e.g., a full length, a fragment or a variant of IL-7, e.g., human IL-7.
  • the IL-7 molecule is a wild-type, human IL-7, e.g., having the amino acid sequence of SEQ ID NO: 244.
  • the IL-7 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild- type human IL-7 amino acid sequence of SEQ ID NO: 244.
  • the IL-7 molecule is a variant of human IL-7, e.g., having one or more amino acid modifications.
  • the IL-7 comprises a fragment of a human IL-7 polypeptide, wherein the fragment has an amino sequence is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 244.
  • Wild-type IL-7 bind to IL-7Ra with an affinity (KD), e.g., as determined by microcal or SPR, of between about 50-100 nM.
  • an IL-7 is modified so as to have an affinity for IL-7Ra that is equal or less than that of the NKp30 ABD for NKp30.
  • an IL-7 is modified so as to have an affinity for IL-7Ra that is at least 1-log less than that of the NKp30 ABD for NKp30.
  • an IL-7 is modified so as to have an affinity for IL-7Ra that is at least 1-log less than that of the NKp30 ABD for NKp30, but no more than 3-log, or optionally 2-log, less than that of the NKp30 ABD for NKp30.
  • the NKp30 ABD has a KD for NKp30 binding of about 15 nM.
  • An IL-7 can thus be modified by introduction of a modification such as amino acid substitutions Q22A, D74A and/or K81A (with reference to the wild-type mature IL-7 amino acid sequence) that causes a reduction of affinity between IL-7 and IL-7Ra.
  • the ABD can be or comprise a suitable interleukin-27 (IL-27) polypeptide such that the IL-27R ABD binds IL-27R (WSX-1 and/or gp130) on the surface of NK cells.
  • IL-27 interleukin-27
  • the cytokine molecule is an IL-27 molecule, e.g., a full length, a fragment or a variant comprising the P28 and EBI3 subunits, e.g., human single chain or heterodimeric IL-27 comprising the P28 and EBI3 subunits, optionally wherein the EBI3 and p28 subunits of IL-27 are linked by a domain linker (e.g., a flexible polypeptide linker, a linker containing glycine a serine residues, a (G4S)2 or (G4S)3 linker) into a single-chain format.
  • a domain linker e.g., a flexible polypeptide linker, a linker containing glycine a serine residues, a (G4S)2 or (G4S)3 linker
  • Single-chain forms of IL-27 can be generated consisting of the p28 subunit linked to the EBI3 subunit by a flexible linker, either through the C-terminus of p28 linked to the N-terminus of EBI3 or vice versa.
  • the IL-27 molecule is a wild-type, human IL-27, e.g., a single chain fusion product or a heterodimer comprising the amino acid sequences of SEQ ID NOS: 245 and 246 or a IL27R-binding fragment of any of the SEQ ID NOS: 245 and 246.
  • the IL-27 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-27 p28 subunit amino acid sequence of SEQ ID NO: 245 and/or an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-27 EBI3 subunit amino acid sequence of SEQ ID NO: 246.
  • the IL-27 molecule is a variant of human IL-27, e.g., having one or more amino acid modifications.
  • the IL-27 comprises a fragment of a human IL-27 p28 subunit polypeptide, wherein the fragment has an amino sequence that is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 245, and/or a fragment of a human IL-27 EBI3 subunit polypeptide, wherein the fragment has an amino sequence that is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 246.
  • the p28 subunit can be specified as being linked at its N-terminus to the multispecific protein (or to the NKp30 ABD thereof).
  • the EBI3 subunit can be specified as being linked, at its N-terminus, to the C- terminus of the p28 subunit, optionally via a domain linker, or can be specified as being placed on a separate polypeptide that associates with the p28 subunit.
  • an IL-27 is modified so as to have an affinity for WSX-1 and/or gp130 that is equal or less than that of the NKp30 ABD for NKp30. In some embodiments, an IL-27 is modified so as to have an affinity for WSX-1 and/or gp130 that is at least 1-log less than that of the NKp30 ABD for NKp30. In some embodiments, an IL-27 is modified so as to have an affinity for WSX-1 and/or gp130 that is at least 1-log less than that of the NKp30 ABD for NKp30, but no more than 3-log, or optionally 2-log, less than that of the NKp30 ABD for NKp30.
  • the ABD can be or comprise a suitable interleukin-12 (IL-12) polypeptide such that the IL-12R ABD binds IL-12R (II_- ⁇ b1 and/or II_- ⁇ b2) on the surface of NK cells.
  • IL-12 interleukin-12
  • the cytokine molecule is an IL-12 molecule, e.g., a full length, a fragment or a variant comprising the P35 and P40 subunits, e.g., human single chain or heterodimeric IL-12 comprising the P35 and P40 subunits, optionally wherein the p40 and p35 subunits of IL-12 are linked by a domain linker (e.g., a flexible polypeptide linker, a linker containing glycine a serine residues, a (G4S)2 or (G4S)3 linker) into a single-chain format.
  • a domain linker e.g., a flexible polypeptide linker, a linker containing glycine a serine residues, a (G4S)2 or (G4S)3 linker
  • Single-chain forms of IL-12 can be generated consisting of the p35 subunit linked to the p40 subunit by a flexible linker, either through the C-terminus of p35 linked to the N-terminus of p40 or vice versa.
  • the IL-12 molecule is a wild-type, human IL-12, e.g., a single chain fusion product or a heterodimer comprising the amino acid sequences of SEQ ID NOS: 247 and 248 or a IL12R-binding fragment of any of the SEQ ID NOS: 247 or 248.
  • the IL-12 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-12 p35 subunit amino acid sequence of SEQ ID NO: 247 and/or an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-12 p40 subunit amino acid sequence of SEQ ID NO: 248.
  • the IL-12 molecule is a variant of human IL-12, e.g., having one or more amino acid modifications.
  • the IL-12 comprises a fragment of a human IL-12 p35 subunit polypeptide, wherein the fragment has an amino sequence that is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 247, and/or a fragment of a human IL-12 p40 subunit polypeptide, wherein the fragment has an amino sequence that is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 248.
  • the p35 (alpha) and P40 (beta) can be specified as being linked by a disulphide bridge between Cys74 of the P35 subunit and the Cys177 of the P40 subunit.
  • the p35 subunit can be specified as being linked at its N-terminus to the multispecific protein (or to the NKp30 ABD thereof).
  • the p40 subunit can be specified as being linked, at its N-terminus, to the C-terminus of the p35 subunit, optionally via a domain linker, or can be specified as being placed on a separate polypeptide that associates with the p35 subunit.
  • Wild-type mature human IL-12 p40 subunit IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTC HKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKS SRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSS FFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT SATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 248).
  • Wild-type IL-12 dimer binds to II_-12Eb1 and ⁇ LA2R ⁇ 2 with an affinity (KD), e.g., as determined by microcal or SPR, of about 5-7 nM and 5 nM, respectively, and IL-12 dimer binds to I ⁇ 12 b1 :I ⁇ -12 b2 dimers with a KD of about 50 pM.
  • KD affinity
  • an IL-12 is modified so as to have an affinity for I ⁇ -12 b1 and/or I ⁇ -12 b2 that is equal or less than that of the NKp30 ABD for NKp30.
  • an IL-12 is modified so as to have an affinity for I L-12 b1 and/or ⁇ L 2R ⁇ > 2 that is at least 1-log less than that of the NKp30 ABD for NKp30. In some embodiments, an IL-12 is modified so as to have an affinity for I ⁇ -12 b1 and/or I ⁇ -12 b2 that is at least 1-log lower (1-log higher KD) than that of the NKp30 ABD for NKp30, but no more than 3-log, or optionally 2-log, lower than that of the NKp30 ABD for NKp30.
  • a multispecific protein can be assessed for biological activity, e.g., antigen binding, ability to elicit proliferation of NK cells, ability to elicit target cell lysis by NK and/or elicit activation of NK cells, including any specific signaling activities elicited thereby, for example cytokine production or cell surface expression of markers of activation.
  • biological activity e.g., antigen binding, ability to elicit target cell lysis and/or specific signaling activities elicited thereby, of a multispecific protein of the disclosure. It will be appreciated that when the specific contribution or activity of one of the components of the multispecific protein is to be assessed (e.g.
  • the multispecific format can be produced in a suitable format which allows for assessment of the component (e.g. domain) of interest.
  • the present disclosure also provides such methods, for use in testing, assessing, making and/or producing a multispecific protein.
  • the multispecific protein can be produced as a protein having the cytokine and another protein in which the cytokine is modified to delete it or otherwise modulate its activity (e.g., wherein the two multispecific proteins otherwise have the same or comparable structure), and tested in an assay of interest.
  • the multispecific protein can be produced as a protein having the ABD and another protein in which the ABD is absent or is replaced by an ABD that does not bind NKp30 (e.g., an ABD that binds an antigen not present in the assay system), wherein the two multispecific proteins otherwise have the same or comparable structure, and the two multispecific proteins are tested in an assay of interest.
  • an anti-antigen of interest e.g.
  • the multispecific protein can be produced as a protein having the ABD and another protein in which the ABD is absent or is replaced by an ABD that does not bind the tumor antigen or anti-antigen of interest (e.g., an ABD that binds an antigen not present in the assay system, an ABD that bind to a different tumor antigen), wherein the two multispecific proteins otherwise have the same or comparable structure, and the two multispecific proteins are tested in an assay of interest.
  • the multispecific protein is capable of inducing activation of an NKp30-expressing cell (e.g. an NK cell, a reporter cell) when the protein is incubated in the presence of the NKp30-expressing cell (e.g. purified NK cells) and a target cell (e.g. tumor cell) that expresses the antigen of interest (e.g. tumor antigen).
  • an NKp30-expressing cell e.g. an NK cell, a reporter cell
  • a target cell e.g. tumor cell
  • the antigen of interest e.g. tumor antigen
  • the multispecific protein is capable of inducing NKp30 signaling in an NKp30-expressing cell (e.g. an NK cell, a reporter cell) when the protein is incubated in the presence of an NKp30-expressing cell (e.g. purified NK cells) and a target cell that expresses the antigen of interest).
  • the multispecific protein is capable of inducing CD16A signaling in an CD16A and NKp30-expressing cell (e.g. an NK cell, a reporter cell) when the protein is incubated in the presence of a CD16A and NKp30-expressing cell (e.g. purified NK cells) and a target cell that expresses the antigen of interest).
  • NK cell activation or signaling in characterized by the increased expression of a cell surface marker of activation, e.g. CD107, CD69, Sca-1 or Ly-6A/E, KLRG1, etc.
  • a cell surface marker of activation e.g. CD107, CD69, Sca-1 or Ly-6A/E, KLRG1, etc.
  • the multispecific protein is capable of inducing an increase of CD137 present on the cell surface of an NKp30- and/or a CD16- expressing cell (e.g. an NK cell, a reporter cell) when the protein is incubated in the presence of the NKp30- and/or a CD16-expressing cell (e.g. purified NK cells), optionally in the absence of target cells.
  • a CD16-expressing cell e.g. an NK cell, a reporter cell
  • the multispecific protein is capable of activating or enhancing the proliferation of NK cells by at least 10-fold, at least 50-fold, or at least 100-fold compared to the same multispecific protein lacking the cytokine receptor ABD (e.g. the CD122 ABD).
  • the multispecific protein displays an EC50 for activation or enhancing the proliferation of NK cells that is at least 10-fold, 50-fold or 100-fold lower than its EC50 for activation or enhancing the proliferation of CD25-expressing T cells.
  • the multispecific protein is capable of activating or enhancing the proliferation of NK cells over CD25-expressing T cells, by at least 10-fold, at least 50-fold, or at least 100-fold.
  • the CD25 expressing T cells are CD4 T cells, optionally Treg cells, or CD8 T cells.
  • Activation or enhancement of proliferation via cytokine receptor in cells e.g. NK cells, CD4 T cells, CD8 T cells or T reg cells
  • cytokine receptor ABD-containing protein can be determined by measuring the expression of pSTAT or the cell proliferation markers (e.g. Ki67) in said cells following the treatment with the multispecific protein.
  • Activation or enhancement of proliferation via the IL-2R pathway in cells e.g. NK cells, CD4 T cells, CD8 Tcells or Treg cells
  • Activation or enhancement of proliferation via the IL-2R pathway in cells e.g. NK cells, CD4 T cells, CD8 Tcells or Treg cells
  • IL-2R pathway in cells e.g. NK cells, CD4 T cells, CD8 Tcells or Treg cells
  • IL-2 and IL-15 lead to the phosphorylation of the STAT5 protein, which is involved in cell proliferation, survival, differentiation and apoptosis.
  • Phosphorylated STAT5 (pSTAT5) translocates into the nucleus to regulate transcription of the target genes including the CD25.
  • STAT5 is also required for NK cell survival and NK cells are tightly regulated by the JAK-STAT signaling pathway.
  • the multispecific protein is capable of inducing STAT5 signaling in an NKp30-expressing cell (e.g. an NK cell) when the protein is incubated in the presence of an NKp30-expressing cell (e.g. purified NK cells).
  • the multispecific protein is capable of causing an increase of expression of pSTAT5 in NK cells over CD25-expressing T cells, by at least 10-fold, at least 50-fold, or at least 100-fold.
  • the multispecific protein displays an EC50 for induction of expression of pSTAT5 in NK cells that is at least 10- fold, 50-fold or 100-fold lower than its EC50 for induction of expression of pSTAT5 in CD25- expressing T cells.
  • cytokine receptor signal transduction can also be assessed for other cytokine/cytokine receptor pairs, such as IL-15 (STAT5), IL-21 (STAT3), IL-27 (STAT1), IL-12 (STAT4), etc.
  • Activity can be measured for example by bringing NKp30-expressing cells (or CD25- expressing cells, depending on the assay) into contact with the multispecific polypeptide, optionally further in presence of target cells (e.g. tumor cells).
  • activity is measured for example by bringing target cells and NK cells (i.e. NKp30-expressing cells) into contact with one another, in presence of the multispecific polypeptide.
  • the NKp30- expressing cells may be employed either as purified NK cells or NKp30-expressing cells, or as NKp30-expressing cells within a population of peripheral blood mononuclear cell (PBMC).
  • the target cells can be cells expressing the antigen of interest, optionally tumor cells.
  • the multispecific protein can be assessed for the ability to cause a measurable increase in any property or activity known in the art as associated with NK cell activity, respectively, such as marker of cytotoxicity (CD107) or cytokine production (for example IFN-g orTNF-a), increases in intracellular free calcium levels, the ability to lyse target cells, for example in a redirected killing assay, etc.
  • marker of cytotoxicity CD107
  • cytokine production for example IFN-g orTNF-a
  • the multispecific protein will be capable of causing an increase in a property or activity associated with NK cell activity (e.g. activation of NK cell cytotoxicity, CD107 expression, IFNy production, killing of target cells) in vitro.
  • a multispecific protein according to the invention can be selected based on its ability to increase an NK cell activity by more than about 20%, preferably by least about 30%, at least about 40%, at least about 50%, or more compared to that achieved with the same effector: target cell ratio with the same NK cells and target cells that are not brought into contact with the multispecific protein, as measured by an assay that detects NK cell activity, e.g., an assay which detects the expression of an NK activation marker or which detects NK cell cytotoxicity, e.g., an assay that detects CD107 or CD69 expression, IFNy production, or a classical in vitro chromium release test of cytotoxicity.
  • an assay that detects NK cell activity e.g., an assay which detects the expression of an NK activation marker or which detects NK cell cytotoxicity, e.g., an assay that detects CD107 or CD69 expression, IFNy production, or a classical in vitro chromium release test of
  • a multispecific protein according to the invention can be selected for or characterized by its ability to have greater ability to induce NK cell activity towards target cells, i.e., lysis of target cells compared to a conventional human lgG1 antibody that binds to the same antigen of interest, as measured by an assay of NK cell activity (e.g. an assay that detects NK cell-mediated lysis of target cells that express the antigen of interest).
  • a multispecific protein the different ABDs contribute to the overall activity of the multispecific protein that ultimately manifests itself in potent anti-tumor activity in vivo.
  • Testing methods exemplified herein allow the in vitro assessment of the activities of the different individual ABDs of the multispecific protein by making variants of the multispecific protein that lack a particular ABD and/or using cells that lack receptors for the particular ABD.
  • a multispecific protein according to the disclosure when it does not comprise the cytokine receptor ABD (e.g.
  • the CD122 ABD when it possesses an Fc domain that does not bind CD16, does not, substantially induce NKp30 signaling (and/or NK activation that results therefrom) of NK cells when the protein is not bound to the antigen of interest on target cells (e.g. in the absence of the antigen of interest and/or target cells).
  • the monovalent NKp30 binding component of the multispecific protein does not itself cause NKp30 signaling.
  • such multispecific protein can be produced in a configuration where the cytokine receptor ABD (e.g. CD122 ABD) is inactivated (e.g.
  • the multispecific protein can optionally be characterized as not substantially causing (or increasing) NKp30 signaling by an NKp30- expressing, CD16-negative cell (e.g. a NKp30 + CD16 _ NK cell, a reporter cell) when the multispecific protein is incubated with such NKp30-expressing, CD16-negative cells (e.g., purified NK cells or purified reporter cells) in the absence of target cells.
  • a multispecific protein can for example be characterized by:
  • cytokine receptor e.g. CD122
  • signaling e.g., as determined by assessing ST AT signaling, for example assessing ST AT phosphoylation
  • an NKp30-expressing cell e.g. an NK cell
  • an NKp30-expressing cell e.g. purified NK cells
  • NK cells (c) lack of NK cell activation or cytotoxicity and/or lack of agonist activity at NKp30 when incubated with NK cells (optionally CD16-negative NK cells, NKp30- expressing NK cells that do not express CD16), in the absence of target cells, optionally wherein the NK cells are purified NK cells, when the multispecific protein is modified to lack the cytokine receptor ABD (e.g. CD122 ABD) or comprises an inactivated cytokine receptor ABD.
  • cytokine receptor ABD e.g. CD122 ABD
  • any of the multispecific proteins and/or cells which express the proteins (or a polypeptide chain thereof) for the manufacture of a pharmaceutical preparation for the treatment, prevention or diagnosis of a disease in a mammal in need thereof.
  • the invention provides methods for preparing a pharmaceutical composition containing a compound as defined herein, to provide a solid or a liquid formulation for administration (e.g., by subcutaneous or intravenous injection). Such a method or process at least comprises the step of mixing the compound with a pharmaceutically acceptable carrier.
  • a method to treat, prevent or more generally affect a predefined condition in an individual or to detect a certain condition by using or administering a multispecific protein or antibody described herein, or a (pharmaceutical) composition comprising same.
  • the invention provides a method of restoring or potentiating the activity and/or proliferation of NKp30-expressing cells, particularly NKp30 + NK cells (e.g. NKp30 + CD16 + NK cells, NKp30 + CD16 _ NK cells) in a patient in need thereof (e.g. a patient having a cancer, or a viral or bacterial infection), comprising the step of administering a multispecific protein described herein to said patient.
  • the invention provides a method of selectively restoring or potentiating the activity and/or proliferation of NK cells of over CD25-expressing lymphocytes, e.g. CD4 T cells, CD8 T cells, Treg cells.
  • the method is directed at increasing the activity of NKp30 + lymphocytes (e.g. NKp30 + CD16 + NK cells, NKp30 + CD16 NK cells) in patients having a disease in which increased lymphocyte (e.g. NK cell) activity is beneficial or which is caused or characterized by insufficient NK cell activity, such as a cancer, or a viral or microbial/bacterial infection.
  • NKp30 + lymphocytes e.g. NKp30 + CD16 + NK cells, NKp30 + CD16 NK cells
  • the invention provides a method of restoring or potentiating the activity and/or proliferation of tumor-infiltrating NK cells or intra-tumoral NKp30-expressing cells, particularly NKp30 + NK cells (e.g. NKp30 + CD16 + NK cells, NKp30 + CD16- NK cells) in a patient in need thereof (e.g. a patient having a solid tumor), comprising the step of administering a multispecific protein described herein to said patient.
  • NKp30 + NK cells e.g. NKp30 + CD16 + NK cells, NKp30 + CD16- NK cells
  • the invention provides a method of increasing the number of tumor- infiltrating NK cells or intra-tumoral NKp30-expressing cells, particularly activated NKp30- expressing cells, particularly NKp30 + NK cells (e.g. NKp30 + CD16 + NK cells, NKp30 + CD16- NK cells) in a patient in need thereof (e.g. a patient having a solid tumor), comprising the step of administering a multispecific protein described herein to said patient.
  • NKp30 + NK cells e.g. NKp30 + CD16 + NK cells, NKp30 + CD16- NK cells
  • the invention provides a method of restoring or potentiating the activity and/or proliferation of NKp30 + NK cells (e.g. NKp30 + CD16 + NK cells, NKp30 + CD16- NK cells) in a patient in need thereof (e.g. a patient having a cancer, or a viral, parasite or bacterial infection), comprising the step of contacting cells derived from the patient, e.g., immune cells and optionally target cells expressing an antigen of interest with a multispecific protein according to the invention and reinfusing the multispecific protein treated cells into the patient.
  • this method is directed at increasing the activity of NKp30+ lymphocytes (e.g.
  • NKp30 + CD16 + NK cells in patients having a disease in which increased lymphocyte (e.g. NK cell) activity is beneficial or which is caused or characterized by insufficient NK cell activity, such as a cancer, or a viral or microbial, e.g., bacterial or parasite infection.
  • lymphocyte e.g. NK cell
  • a viral or microbial e.g., bacterial or parasite infection.
  • the subject multispecific proteins may be used or administered in combination with immune cells, particularly NK cells, derived from a patient who is to be treated or from a different donor, and these NK cells administered to a patient in need thereof such as a patient having a disease in which increased lymphocyte (e.g. NK cell) activity is beneficial or which is caused or characterized by insufficient NK cell activity, such as a cancer, or a viral or microbial, e.g., bacterial or parasite infection.
  • immune cells particularly NK cells, derived from a patient who is to be treated or from a different donor
  • these NK cells administered to a patient in need thereof such as a patient having a disease in which increased lymphocyte (e.g. NK cell) activity is beneficial or which is caused or characterized by insufficient NK cell activity, such as a cancer, or a viral or microbial, e.g., bacterial or parasite infection.
  • NK cells unlike CAR-T cells
  • TCRs TCRs
  • these NK cells even those derived from different donors will not induce a GVHD reaction (see e.g., Glienke et al., “Advantages and applications of CAR-expressing natural killer cells”, Front. Pharmacol. 6, Art. 21:1-6 (2015); Hermanson and Kaufman, Front. Immunol. 6, Art. 195:1-6 (2015)).
  • the multispecific protein disclosed herein that mediates NK cell activation, proliferation, tumor infiltration and/or target cell lysis via multiple activating receptors of effector cells can be used advantageously for treatment of individuals whose effector cells or tumor-infiltrating effector cells (e.g. NKp30 + NK cells) cells are hypoactive, exhausted or suppressed, for example a patient who has a significant population of effector cells characterized by the expression and/or upregulation of one or multiple inhibitory receptors (e.g. TIM-3, PD1 , CD96, TIGIT, etc.), or the downregulation or low level of expression of CD16 (e.g., presence of elevated proportion of NKp30 + CD16 _ NK cells).
  • the multispecific polypeptides described herein can be used to prevent or treat disorders that can be treated with antibodies, such as cancers, solid and non-solid tumors, hematological malignancies, infections such as viral infections, and inflammatory or autoimmune disorders.
  • the antigen of interest is an antigen expressed on the surface of a malignant cell of a type of cancer selected from the group consisting of: carcinoma, including that of the bladder, head and neck, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, hairy cell lymphoma and Burkett’s lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma
  • a multispecific protein is used to prevent or treat a cancer selected from the group consisting of: carcinoma, including that of the bladder, head and neck, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, hairy cell lymphoma and Burkett’s lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including neuroblastom
  • T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including of the small cell and cerebriform cell type; large granular lymphocyte leukemia (LGL) preferably of the T-cell type; Sezary syndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal) T- cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinal T-cell lymphoma; T- lymphoblastic; and lymphoma/leukaemia (T-Lbly/T-ALL).
  • T-PLL T-prolymphocytic leukemia
  • LGL large granular lymphocyte leukemia
  • SS Sezary syndrome
  • ATLL Adult T-cell
  • the tumor antigen is an antigen expressed on the surface of a lymphoma cell or a leukemia cell, and the multispecific protein is administered to, and/or used for the treatment of, an individual having a lymphoma or a leukemia.
  • the tumor antigen is selected from HER2, CD19, CD20, CD22, CD30 or CD33.
  • the inventive multispecific polypeptides described herein can be used to prevent or treat a cancer characterized by tumor cells that express the antigen of interest (e.g. tumor antigen) to which the multispecific protein of the disclosure specifically binds.
  • a cancer characterized by tumor cells that express the antigen of interest (e.g. tumor antigen) to which the multispecific protein of the disclosure specifically binds.
  • the methods of treatment comprise administering to an individual a multispecific protein described herein in a therapeutically effective amount, e.g., for the treatment of a disease as disclosed herein, for example any of the cancers identified above.
  • a therapeutically effective amount may be any amount that has a therapeutic effect in a patient having a disease or disorder (or promotes, enhances, and/or induces such an effect in at least a substantial proportion of patients with the disease or disorder and substantially similar characteristics as the patient).
  • the multispecific protein may be used with our without a prior step of detecting the expression of the antigen of interest (e.g. tumor antigen) on target cells in a biological sample obtained from an individual (e.g. a biological sample comprising cancer cells, cancer tissue or cancer-adjacent tissue).
  • the disclosure provides a method for the treatment or prevention of a cancer in an individual in need thereof, the method comprising: a) detecting cells (e.g. tumor cells) in a sample from the individual that express an antigen of interest (e.g.
  • the antigen of interest to which the multispecific protein specifically binds via its antigen of interest ABD and b) upon a determination that cells which express an antigen of interest are comprised in the sample, optionally at a level corresponding at least to a reference level (e.g. corresponding to an individual deriving substantial benefit from a multispecific protein, or optionally at a level that is increased compared to a reference level (e.g. corresponding to a healthy individual or an individual not deriving substantial benefit from a protein described herein), administering to the individual a multispecific protein of the disclosure that binds to an antigen of interest, to NKp30, to cytokine receptor (e.g. CD122), and optionally to CD16A (e.g., via its Fc domain).
  • a reference level e.g. corresponding to an individual deriving substantial benefit from a multispecific protein, or optionally at a level that is increased compared to a reference level (e.g. corresponding to a healthy individual or an individual not
  • the multispecific proteins are used to treat a tumor characterized by low levels of surface expression of the antigen of interest.
  • the tumor or cancer can be characterized by cells expressing a low level of tumor antigen.
  • the level of the tumor antigen is less than 100,000 tumor antigen copies per cancer cell.
  • the level of the tumor antigen is less than 90,000, less than 75,000, less than 50,000, or less than 40,000 tumor antigen copies per cancer cell.
  • the uses optionally further comprise detecting the level of tumor antigen of one or more cancer cells of the subject.
  • the multispecific protein may be used with our without a prior step of detecting or characterizing NK cells from an individual to be treated.
  • the invention provides a method for the treatment or prevention of a cancer in an individual in need thereof, the method comprising: a) detecting NK cells (e.g. tumor-infiltrating NK cells) in a tumor sample from an individual (or within the tumor and/or within adjacent tissue), and b) upon a determination that the tumor or tumor sample is characterized by a low number or activity of NK cells, optionally at a level or number that is decreased compared to a reference level (e.g.
  • NK cells e.g. tumor-infiltrating NK cells
  • a conventional IgG antibody therapy such a conventional lgG1 antibody that binds to the same cancer antigen
  • administering to the individual a multispecific protein that binds to a cancer antigen, to NKp30 (e.g., monovalently), to cytokine receptor (e.g., CD122) and optionally to CD16A.
  • an individual has a tumor characterized by a CD16 (e.g. CD16A) deficient tumor microenvironment.
  • the methods of treatment using a multispecific protein comprise a step of detecting the expression level of CD16 in a sample (e.g. a tumor sample) from the individual. Detecting the CD16 optionally comprises detecting the level of CD16A or CD16B.
  • the CD16 deficient microenvironment is assessed in a patient having undergone a hematopoietic stem cell transplantation.
  • the CD16 deficient microenvironment comprises a population of infiltrating NK cells, and the infiltrating NK cells have less than 50% expression of CD16 as compared to a control NK cell.
  • the infiltrating NK cells have less than 30%, less than 20%, or less than 10% expression of CD16 as compared to a control NK cell.
  • the CD16 deficient microenvironment comprises a population of infiltrating NK cells, and at least 10% of the infiltrating NK cells have reduced expression of CD16 as compared to a control NK cell. In some aspects, at least 20%, at least 30%, or at least 40% of the infiltrating NK cells have reduced expression of CD16 as compared to a control NK cell.
  • a method for the treatment or prevention of a cancer in an individual in need thereof comprising: a) detecting CD16 expression in cells (e.g. in tumor-infiltrating NK cells) from a tumor or tumor sample (e.g., tumor and/or within adjacent tissue) from an individual, and b) upon a determination that the tumor or tumor sample is characterized by a CD16 deficient microenvironment, administering to the individual a multispecific protein that binds to a cancer antigen, to NKp30, and to the cytokine receptor (e.g. CD122) (and optionally further to CD16A).
  • a multispecific protein that binds to a cancer antigen, to NKp30, and to the cytokine receptor (e.g. CD122) (and optionally further to CD16A).
  • a method for the treatment or prevention of a cancer in an individual in need thereof comprising: a) detecting CD16 expression at the surface of NK cells (e.g. tumor-infiltrating NK cells) in a tumor sample from an individual (or within the tumor and/or within adjacent tissue), and b) upon a determination that the tumor or tumor sample is characterized by an elevated proportion of CD16- NK cells, optionally at a level or number that is increased compared to a reference level, administering to the individual a multispecific protein that binds to a cancer antigen, to NKp30, and to the cytokine receptor (e.g. CD122) (and optionally further to CD16A).
  • NK cells e.g. tumor-infiltrating NK cells
  • the disclosure provides a method for the treatment or prevention of a disease (e.g. a cancer) in an individual in need thereof, the method comprising: a) detecting cell surface expression of one or a plurality inhibitory receptors on immune effector cells (e.g. NK cells, T cells) in a sample from the individual (e.g. in circulation or in the tumor environment), and b) upon a determination of cell surface expression of one or a plurality inhibitory receptors on immune effector cells, optionally at a level that is increased compared to a reference level (e.g.
  • a disease e.g. a cancer
  • a multispecific protein e.g. a multispecific protein
  • an antigen of interest e.g. a cancer antigen
  • NKp30 e.g., monovalently
  • the cytokine receptor e.g. CD122
  • the multispecific proteins are used to treat an individual having a gastric cancer or a prostate cancer. Decreased cell surface expression of NKG2D on immune effector cells has been observed in gastric cancer and prostate cancer.
  • an individual has NK cells and/or T cells characterized by decreased expression of NKG2D, e.g. decreased cell surface expression.
  • the level of expression can be for example compared to a reference value, for example a reference value corresponding to NKG2D levels observed on NK and/or T cells in healthy individuals.
  • an individual has NK and/or T cell characterized by decreased expression of NKG2D on NK and/or T cells in the tumor microenvironment.
  • an individual has NK and/or T cell characterized by presence (e.g. at increased levels) of soluble ligands of NKG2D in the tumor microenvironment, for example soluble MICA, MICB or ULBP proteins.
  • the disclosure provides a method for the treatment or prevention of a disease (e.g. a cancer) in an individual in need thereof, the method comprising: a) detecting cell surface expression of NKG2D polypeptides on immune effector cells (e.g. NK cells, T cells) in a sample from the individual (e.g. in circulation or in the tumor environment), and b) upon a determination of decreased cell surface expression of one or a plurality inhibitory receptors on immune effector cells, optionally at a level that is decreased compared to a reference level (e.g.
  • a disease e.g. a cancer
  • a multispecific protein e.g. a multispecific protein
  • an antigen of interest e.g. a cancer antigen
  • NKp30 e.g., monovalently
  • the cytokine receptor e.g. CD122
  • a multispecific protein may be used as a monotherapy (without other therapeutic agents), or in combined treatments with one or more other therapeutic agents.
  • a multispecific protein is administered in the absence of combined treatment with an IL-2, IL-15, IL-21 , IL-7, IL-27, IL-12, IL-18, IFN-a or lFN-b polypeptide.
  • kits may optionally further contain any number of polypeptides and/or other compounds, e.g., 1, 2, 3, 4, or any other number of multispecific proteins and/or other compounds. It will be appreciated that this description of the contents of the kits is not limiting in any way.
  • the kit may contain other types of therapeutic compounds.
  • the kits also include instructions for using the polypeptides, e.g., detailing the herein-described methods such as in the detection or treatment of specific disease conditions.
  • compositions comprising the subject multispecific proteins and optionally other compounds as defined above.
  • a multispecific protein and optionally another compound may be administered in purified form together with a pharmaceutical carrier as a pharmaceutical composition.
  • the form depends on the intended mode of administration and therapeutic or diagnostic application.
  • the pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver the compounds to the patient.
  • Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as (sterile) water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters, alcohol, fats, waxes, and inert solids.
  • a pharmaceutically acceptable carrier may further contain physiologically acceptable compounds that act for example to stabilize or to increase the absorption of the compounds
  • physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients
  • carbohydrates such as glucose, sucrose or dextrans
  • antioxidants such as ascorbic acid or glutathione
  • chelating agents such as ascorbic acid or glutathione
  • low molecular weight proteins or other stabilizers or excipients include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients
  • chelating agents such ascorbic acid or glutathione
  • Non-limiting examples of such adjuvants include by way of example inorganic and organic adjuvants such as alum, aluminum phosphate and aluminum hydroxide, squalene, liposomes, lipopolysaccharides, double stranded (ds) RNAs, single stranded(s-s) DNAs, and TLR agonists such as unmethylated CpG’s.
  • inorganic and organic adjuvants such as alum, aluminum phosphate and aluminum hydroxide, squalene, liposomes, lipopolysaccharides, double stranded (ds) RNAs, single stranded(s-s) DNAs, and TLR agonists such as unmethylated CpG’s.
  • Multispecific proteins according to the invention can be administered parenterally.
  • Preparations of the compounds for parenteral administration must be sterile. Sterilization is readily accomplished by filtration through sterile filtration membranes, optionally prior to or following lyophilization and reconstitution.
  • the parenteral route for administration of compounds is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial, or intralesional routes.
  • the compounds may be administered continuously by infusion or by bolus injection.
  • a typical composition for intravenous infusion could be made up to contain 100 to 500 ml of sterile 0.9% NaCI or 5% glucose optionally supplemented with a 20% albumin solution and 1 mg to 10 g of the compound, depending on the particular type of compound and its required dosing regimen.
  • Methods for preparing parenterally administrable compositions are well known in the art.
  • the amino acid sequences of the three chains of the GA101-T5-Com_mAb8-IL2v protein which bears an Fc domain capable of binding to CD16A are shown in SEQ ID NOS: 251 , 252 and 253.
  • the amino acid sequences of the three chains of the GA101-T6- Com_mAb8-IL2v protein which bears an Fc domain that substantially lacks binding to CD16A are shown in SEQ ID NOS: 254, 255 and 256.
  • FIG. 2B shows the topology of the exemplary “T5” format multispecific proteins used in the Examples; the T6 format share the structure of the T5 format but differs from the T5 format by introduction of amino acid substitution in the Fc domain that reduces binding to CD16A (and other Fc gamma receptors).
  • the T5 and T6 formats have one Fab structure located topologically N-terminal, and a cytokine topologically C-terminal, with the dimeric Fc domain interposed between Fab on the N-terminal side and the NKp30-binding Fab on the C-terminal side of the Fc dimer, and the cytokine on the C- terminal side of the NKp30 binding Fab.
  • FIG. 2A The domain structure of the exemplary “T5” format protein used in the Examples is shown in Figure 2A.
  • Figure 2A shows domain linkers (such as hinge and glycine-serine linkers) of different lengths, and interchain disulfide bridges.
  • the Fc domain has Fc gamma receptor binding site amino acid sequences of wild-type human lgG1 and thus retains binding to CD16A.
  • the sequences encoding each polypeptide chain for each multispecific antigen binding protein were inserted into the pTT-5 vector between the Hindlll and BamHI restriction sites.
  • the three vectors (prepared as endotoxin-free midipreps or maxipreps) were used to cotransfect EXPI-293F cells (Life Technologies) in the presence of PEI (37°C, 5% CO2, 150 rpm).
  • the cells were used to seed culture flasks at a density of 1 x 10 6 cells per ml (EXPI293 medium, Gibco).
  • Valproic Acid final concentration 0.5 mM
  • glucose (4 g/L)
  • tryptone N1 0.5%) were added.
  • the supernatant was harvested after six days after and passed through a Stericup filter with 0.22 pm pores.
  • the multispecific antigen-binding proteins were purified from the supernatant following harvesting using rProtein A Sepharose Fast Flow (GE Healthcare, reference 17- 1279-03). Size Exclusion Chromatography (SEC) purifications were then performed and the proteins eluted at the expected size were finally filtered on a 0.22 pm device.
  • SEC Size Exclusion Chromatography
  • Example 1 IL2v limits IL2R activation on Treg
  • a heterotrimeric Fc-domain-containing protein containing one C-terminal moiety of mutant IL-2 was assessed for its ability to activate Treg cells.
  • the protein incorporates a variant IL-2 polypeptide (IL-2v) in which a human IL-2 polypeptide is modified by introducing the mutations T3A, F42A, Y45A, L72G and C125A, conferring decreased binding affinity for CD25 compared to wild-type human IL-2.
  • the heterotrimeric had an IL2v fused to the C-terminus of one of the chains of an isotype control (IC) Fab, which in turn was fused to the C-terminus of an Fc domain mutated to substantially eliminate CD16A binding, in turn fused to another IC Fab.
  • the IC Fabs have a VH/VL pair which does not bind to any protein in the test system.
  • This heterotrimeric protein (IL2v immunoconjugate) was compared to an identical heterotrimeric protein in which IL2v was replaced by a wild-type human IL-2 polypeptide (IL2pWT immunoconjugate), and to recombinantly produced full-length wild-type IL-2 (rec hulL-2).
  • PBMC peripheral blood mononuclear cells
  • IC-T6-IC-IL2 IL2pWT immunoconjugate
  • IC-T6- IC-IL2v IL2v immunoconjugate
  • results are shown in Figure 3.
  • the IL2v immunoconjugate resulted in an approximately 3-log decrease in percent of pSTAT5+ cells among the Treg, compared to IL2pWT immunoconjugate and rec hulL-2.
  • the IL2v immunoconjugate protein incorporating a mutated human IL-2 therefore displays a strongly decreased ability to activate Treg cells compared to wild-type IL-2.
  • Example 2 CD20 x NKp30 binding NKCE-IL2v promotes potent NK cell-mediated cytotoxicity
  • NK cells were co-cultured with Raji tumor cells previously loaded with calcein, in a 10 to 1 ratio.
  • Cells were incubated with test proteins described above (doses from 61 nM to 0.00000061 nM) for 4h at 37°C, 5.5% CO2 in incubator.
  • Results are shown in Figure 4, showing % specific lysis induced by NK cells on the y- axis and concentration of test protein on the x-axis. All proteins were highly potent in ability to mediate NK cell cytotoxicity toward tumor target cells. However, at concentrations consistent with use in treatment, the GA101-T5-Com_mAb8-IL2v provided a significant improvement in potency compared to the gold-standard Fc-engineered antibody obinutuzmab. GA101-T6- Com_mAb8-IL2v which engages NKp30 but not CD16A was as potent as conventional full- length human lgG1 antibody having GA101 VH/VL domains despite the ability of the latter to bind CD20 bivalently.
  • GA101-T5-Com_mAb8-IL2v was more potent that all other proteins, including the gold-standard Fc-engineered antibody obinutuzmab, despite the ability of obinutuzumab to bind CD20 bivalently and to bind CD16 with greater affinity.
  • Monovalent co-engagement of NKp30, CD16A and cytokine receptor (CD122) on NK cells therefore provides an important advantage in mediating NK cell lysis of tumor cells.

Abstract

The invention provides multispecific proteins that bind the human NKp30 polypeptide and specifically redirect NK cells to lyse a target cell of interest via multiple receptors. The proteins have utility in the treatment of disease, notably cancer.

Description

MULTISPECIFIC PROTEINS BINDING TO NKP30, A CYTOKINE RECEPTOR,
A TUMOUR ANTIGEN AND CD16A
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 63/208,512 filed on 9 June 2021, the disclosure of which is incorporated herein by reference in its entirety; including any drawings and sequence listings.
REFERENCE TO THE SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “NKp46-15 PCT_ST25 txt”, created May 20, 2022, which is 281 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
Multispecific proteins that bind and specifically redirect effector cells to lyse a target cell of interest via multiple receptors are provided. The proteins have utility in the treatment of disease, notably cancer or infectious disease.
BACKGROUND
Interleukin 2 (IL2 or IL-2) is one example of a pluripotent cytokine that acts on a cytokine receptor expressed by NK cells. IL-2 is mainly produced by activated T cells, especially CD4+ T helper cells, and functions in aiding the proliferation and differentiation of B cells, T cells and NK cells. IL-2 is also essential for Treg function and survival. In eukaryotic cells, human IL-2 (uniprot: P60568) is synthesized as a precursor peptide of 153 amino acids with a 20 residue signal sequence, that gives rise to a mature secreted IL-2 having the amino acid sequence of SEQ ID NO: 213. Interleukin 2 has four antiparallel, amphiphilic alpha helices. These four alpha helices form a quaternary structure that is essential for its function. In most cases, IL-2 works through three different receptors: interleukin 2 receptor alpha (IL- 2Ra; CD25), interleukin 2 receptor beta (IL-2Rb; CD122), and interleukin 2 receptor gamma (IL-2Ry; CD132). \L-2R and IL-2Ry are essential for IL-2 signaling, while IL-2Ra (CD25) is not necessary for signaling, but can confer high affinity binding of IL-2 to receptors. The trimer receptor (Iί-2abg) formed by the combination of IL-2Ra, b, and g is the IL-2 high affinity receptor (KD about 10 pM), and the dimer receptor (IL- 2bg) is an intermediate affinity receptor (KD about 1 nM).
Immune cells express dimer or trimer IL-2 receptors. Dimer receptors are expressed on cytotoxic CD8 + T cells and natural killer cells (NK), while trimer receptors are mainly expressed on activated lymphocytes and CD4 + CD25 + FoxP3 + inhibitory regulatory T cells (Treg). Because resting effector T cells and NK cells do not have CD25 on the cell surface, they are relatively insensitive to IL-2. Treg cells consistently express the highest level of CD25 in the body. Due to the low concentrations of IL-2 that typically exists in tissues, IL-2 preferentially activates cells that express the high affinity receptor complex (CD25:CD122:CD132), and therefore under normal circumstances, IL-2 will preferentially stimulate Treg cell proliferation.
IL-15, IL-12, IL-7, IL-27, IL-18, IL-21, and IFN-a share many aspects of receptor binding, complex assembly and signaling with IL-2. For example IL-15, IL-21 and IL-7 like IL- 2 both act on NK cells via the common-g chain receptor (CD132). IL-15 binds to the IL-15 receptor (IL-15R) which is composed of three subunits: IL-15Ra, CD122, and CD132. Two of these subunits, CD122 and CD132, are shared with the receptor for IL-2, but IL-2 receptor has an additional subunit (CD25). IL-15Ra (CD215) specifically binds IL15 with very high affinity, and is capable of binding IL-15 independently of other subunits. IL-21 is another example of a type I cytokine, and its IL-21 receptor (IL-21 R) has been shown to form a heterodimeric receptor complex with the IL-2/IL-15 receptor common gamma chain (CD132).
NK cells have the potential to mediate anti-tumor immunity. However, NK cells have been shown to cause toxicity in mice through their hyper-activation and secretion of multiple inflammatory cytokines when IL-2 was administered together with IFN-a (Rothschilds et al, Oncoimmunology. 2019;8(5):). In addition, NK cells were also shown to cause toxicity of the cytokine IL-15 that also signals through IL-2Rby (see WO2020247843 citing Guo et al, J Immunol. 2015;195(5):2353-64).
One potential solution to the immune toxicity mediated by cytokines such as IL-2 was to fuse it to or associate it with a tumor-specific antibody. However, it was found that that while IL-2 indeed synergized with antitumor antibody in anti-tumor effect in vivo, the inclusion of IL- 2 and anti-tumor antigen antibody in the same molecule presented no efficacy or toxicity advantage. The IL-2 moiety entirely governed biodistribution, explaining the observation that immunocytokines recognizing irrelevant antigen performed equivalently to tumor-specific immunocytokines when combined with antibody (Tzeng et al. Proc Natl Acad Sci USA. 2015 Mar 17; 112(11): 3320-332).
Studies focusing on the effect of cytokines on NK cells have generally focused on single cytokines or simple combinations. More recently, it has been reported that IL-15, IL-18, IL-21, and IFN-a, alone and in combination, and their potential to synergize with IL-2, and that very low concentrations of both innate and adaptive common g chain cytokines synergize with equally low concentrations of IL-18 to drive rapid and potent NK cell CD25 and IFN-g expression (Nielsen et al. Front Immunol. 2016; 7: 101). However, administration of cytokines to humans has involved toxicity, which makes combination treatment with cytokines challenging. Furthermore, little remains known on potential synergies or interaction between cytokine receptor signaling pathways and other activating receptors in NK cells. There is therefore a need for new ways to mobilize NK cells in the treatment of disease, particularly cancer.
SUMMARY OF THE INVENTION
The present provides multi-specific proteins that bind to NKp30 and a cytokine receptor (e.g. CD122 and/or CD132) on NK cells, and optionally that further bind CD16A on NK cells, and that also bind to an antigen of interest (e.g. a cancer antigen) on a target cell, where in the multi-specific proteins are capable of increasing NK cell cytotoxicity toward a target cell that expresses the antigen of interest (e.g., a cell that contributes to disease, a cancer cell). The multi-specific protein’s ability to bind, in cis, to NKp30 and to the cytokine receptor (and optionally further through CD16A) at the surface of an NK cell is believed to lead to a particularly advantageous cell surface receptor signalling, in turn resulting a potent anti-tumor response by NK cells. The examples made use of a variant IL-2 cytokine (IL-2v) which was modified to reduce affinity for its receptor(s) on T cells (CD25) but retained substantially full affinity (comparable to wild-type IL-2) for its receptor on NK cells (CD122 and/or CD132). The NKp30 binding domain (exemplified as a VH/VL pair comprised in a Fab or scFv), the CD16A- binding Fc domain and the cytokine were placed adjacent to one another in series within the protein, each separated from the adjacent element (i.e. NKp30 ABD, Fc domain or cytokine) solely by a short peptide linker. These configurations of multispecific proteins were designed to present the respective antigen binding domains so as to permit co-engagement of NKp30 and cytokine receptor on the same cell surface plane (i.e. in NKp30, cytokine receptor (and further CD16A) are bound in cis). Further, the examples used a Fc domain that binds CD16A, showing that binding to CD16A does not negatively affect the tumor and NK-targeted biodistribution and instead led to triple co-engagement of NKp30, CD16A and cytokine receptor, and in turn permitted the incorporation of a cytokine that retained binding affinity for its receptor on NK cells. By incorporating into the multispecific protein anti-NKp30 VH/VL domains that conferred a binding affinity for NKp30 in the low nanomolar range for the KD (e.g. KD of about 1-20 nM), cytokines can be used that retain good affinity, optionally substantially full binding affinity, for their receptor on NK cells sufficient to mediate potent signaling in NK cells. Typically, cytokines such as the ones described herein generally have an affinity for binding to their receptor on NK cells that is no stronger than that of the affinity of the multispecific protein for NKp30 (affinity can be determined as the KD). The multispecific proteins tested herein all bear an Fc domain that bound FcRn with a long in vivo half-life, with or without CD16A binding. At concentrations consistent with use in treatment, the multispecific proteins mediated considerably higher NK cell cytotoxicity compared to a conventional human lgG1 antibody bearing the same variable regions of model anti-CD20 antibody GA101, despite the latter lgG1 binding to CD20 bivalently. Yet further, the multispecific proteins mediated considerably higher NK cell cytotoxicity compared to the gold standard antibody obinutuzumab (GA101) in glycoengineered form (Fc enhanced for binding to CD16A).
It is believed, in view of these results, that targeting the cytokine, e.g. a type 1 cytokine such as an IL-2, IL-15, IL-21, IL-7, IL-27 or IL-12 cytokine, an IL-18 cytokine or a type 1 interferon (e.g. IFN-a, IFN-b), to NKp30 promotes cis-presentation to the cytokine’s receptor (e.g. II_2/15bg, IL-21R, IL-7Ra, IL-27Ra, IL-12R, IL-18R, IFNAR), as shown in Figure 1 for the cytokine IL2 and cytokine receptor complex II_2bg). As shown herein, IL2v placed immediately adjacent to (and on the C-terminal side of) the NKp30-binding ABD permitted the triple receptor cis-presentation to occur (the IL2v was connected to the NKp30-binding ABD by a linker peptide of as little as five amino acid residues). Advantageously, use of a dimeric Fc domain as the CD16A-binding ABD provides FcRn, in turn conferring a half-life sufficiently long to induce NK cell tumor infiltration and proliferation of NK cells in vivo.
The multispecific proteins directed to NKp30 on NK cells have the advantage that they permit a range of cytokines to be used and/or tested without a requirement for reduced binding affinity for their receptor on NK cells (e.g. CD122). The cytokine therefore may or may not be modified to attenuate or decrease binding affinity for its receptor. The multispecific proteins directed to NKp30 on NK cells can thus make use of ayn one of several cytokines in their wild- type form or where the cytokine retains binding affinity for its receptor on NK cells that is not substantially diminished compared to the wild-type cytokine, particularly where the cytokine does not have substantially reduced activity at its receptor on NK cells, and/or where the cytokine’s affinity for its receptor is no stronger than the affinity of the NKp30 ABD for NKp30. Accordingly, in any embodiment, the cytokine ABD (e.g. cytokine moiety within the multispecific protein) can be specified as having a binding affinity and/or an activity (e.g. induction of signalling) on its receptor on NK cells that is not substantially reduced compared to the wild-type form of the cytokine. Optionally, the cytokine moiety induces signalling at its receptor on NK cells (e.g. CD122) that is at least 70% or 80% of that observed with the wild- type form of the cytokine. Accordingly, in any embodiment, the cytokine ABD (e.g. cytokine moiety within the multispecific protein) can be specified as having an affinity for its receptor on NK cells that is not substantially reduced compared to the wild-type form of the cytokine. In some embodiments, the cytokine moiety has a binding affinity for its receptor on NK cells (e.g. CD122) that is within 3-log, 2-log or 1-log of that of the wild-type form of the cytokine (e.g. the cytokine moiety has a KD for binding to the cytokine receptor that is not more than 3-, 2- or 1- log higher than that observed for the wild-type form of the cytokine). Affinity can be KD for binding to recombinant receptor protein, as determined using SPR. Signaling or receptor binding affinity of cytokines can be specified as being when incorporated into an otherwise equivalent multispecific protein.
High efficacy combined with low effect on Treg cells and/or low immune toxicity is a particular advantage of a therapeutic molecule that combines the ability to bind each of NKp30 and cytokine receptor (e.g. CD122), and further CD16A, on an individual NK cell, particularly for a therapeutic agent having a long in vivo half-life.
The multispecific proteins are particularly advantageous due to high potency in enhancing NK cell activity (e.g. NK cell proliferation, activation, cytotoxicity and/or cytokine release, including by tumor-infiltrating NK cells), yet with low immune toxicity (e.g. low systemic increase or release of cytokines IL-6 and TNF-a). The present disclosure provides examples using protein formats that permit sufficient distance between NKp30 and cytokine receptor (e.g. CD122) and CD16A binding domains to permit all three receptors to be bound by a single NK cell, thereby providing combined NK cell receptor activation. Importantly, the combined binding on a single cell may account for the minimal off-target immune toxicity and lack of fratricidal killing of NKp30-expressing and/or CD16-expressing cells (e.g., NK cells) because the multispecific protein is bound by at least one activating receptor in addition to cytokine receptor (e.g. CD122) at the surface of the NKp30 and/or CD16+ effector cell.
The multispecific proteins are further advantageous due to their ability to potentiate the activity and/or proliferation of both NKp30+CD16+ and NKp30+CD16A_ NK cells. As shown herein, combined dual binding to NKp30 and CD122, in the absence of binding to CD16A, demonstrates strong potentiation of NK cell activity. In healthy individuals, the CD16- population represents 5-15% of the total NK cell population, while in some cancer patients the proportion of CD16- NK cells is greatly increased, making up as much as 50% of the total NK cell population. Further, the tumor micro-environment has been shown to affect the phenotype of CD16A+ NK cells by either inducing shedding of CD16A from the surface of the cells or promoting conversion from CD16A+ to CD16 NK cells. In addition, due to CD16A polymorphism, some individuals have mutations in CD16A (e.g. at residue 158 of CD16A) that result in reduced ability to mediate ADCC. Overcoming CD16A deficiencies, particular as may occur in the tumor environment, while increasing both the number of activated NKp30+ NK cells in the tumor, is particularly advantageous. Yet further, multispecific proteins do not require binding or signaling via NKG2D and can be used to potentiate NK cell activity in patients having NK and/or T cells characterized by relatively low levels of surface expression of the activating receptor NKG2D, for example as is known to be a general or common feature in gastric and prostate cancer.
Provided, inter alia, is a multispecific protein comprising: a NKp30-binding domain that binds to a human NKp30 polypeptide, (b) a binding domain that binds an antigen of interest (e.g. a tumor-associated or cancer antigen; an antigen of interest present expressed by a target cell), (c) an optional CD16A-binding domain (e.g. an Fc dimer) that binds to a human CD16A polypeptide, and (c) an antigen binding domain that binds to a human cytokine receptor polypeptide expressed on NK cells (e.g. a receptor such as CD122 (I L2/15Rb), IL- 21R, IL-7Ra, IL-27Ra, IL-12R, IL-18R, IFNAR (IFNAR1 and/or IFNAR2). Provided also, is a multispecific protein comprising a NKp30-binding domain that binds to a human NKp30 polypeptide, a binding domain that binds an antigen of interest (e.g. a tumor-associated or cancer antigen; an antigen of interest present expressed by a target cell), an Fc domain (e.g. an Fc domain dimer) that is bound by human FcRn (and optionally that is further bound by a human CD16A polypeptide), and an antigen binding domain that binds to a human cytokine receptor polypeptide (e.g. CD122 (IL2/15R ), IL-21 R, IL-7Ra, IL-27Ra, IL-12R, IL-18R, IFNAR (IFNAR1 and/or IFNAR2). The antigen binding domain that binds a cytokine receptor can be a variant cytokine having a modification that reduces binding to a receptor counterpart found on non-NK cells (e.g. T cells, Treg cells) compared to its wild-type form.
In any embodiment, the ABD that binds to a human NKp30 polypeptide and the ABD that binds a human cytokine receptor can be specified as being configured to be capable of adopting a membrane planar binding confirmation.
In any embodiment, the multispecific protein can be specified as being capable of interacting with, binding to or co-engaging NKp30 and the cytokine receptor, and optionally further CD16A, on the surface of an NK cell.
In any embodiment, optionally the ABD that binds to a human NKp30 polypeptide and the ABD that binds a human cytokine receptor, and optionally further the Fc domain, are specified as being positioned or connected within the multispecific protein in series (e.g. with respect to the N- and C-termini of the multispecific protein).
The ABD that binds NKp30 (e.g. a Fab, single variable domain or scFv) can optionally be specified as being connected to the CD16A- binding domain (e.g. an Fc domain) by an Ig- derived (e.g. a peptide from a hinge domain or heavy or light chain constant domain) or non- Ig-derived domain linker, optionally wherein the domain linker is a flexible polypeptide linker. The ABD that binds a cytokine receptor can optionally be specified as comprising a wild-type or variant cytokine connected to the rest of the multispecific protein or to the NKp30 ABD by a domain linker, optionally a flexible polypeptide linker.The cytokine can be specified as being positioned C-terminal to both the NKp30- and CD16A- binding domains on the multispecific protein, and optionally further the cytokine is connected to the rest of the multispecific protein (or e.g., a domain thereof, the NKp30 ABD) via a peptide linker of 15, 10 or 5 residues or less. The NKp30- and CD16A- binding domains can optionally be specified as being placed adjacent to one another on the multispecific protein and optionally connected to one another by a peptide linker (e.g. an immunoglobulin-derived linker such as a hinge-derived linker, a non-immunoglobulin-derived linker, a flexible linker) having a length of 15, 10 or 5 residues or less.
In one embodiment, the ABD that binds to a human NKp30 polypeptide is positioned adjacent to the Fc domain within the protein (or on a polypeptide chain thereof), and wherein either the ABD that binds to a human NKp30 polypeptide or the Fc domain are positioned adjacent to the ABD that binds a human cytokine receptor, optionally further wherein the ABD that binds a human cytokine is connected to the ABD that binds to a human NKp30 polypeptide or the Fc domain by a linker peptide having 20 or less than 20 amino acid residues, optionally less than 15 amino acid residues, optionally less than 10 amino acid residues, optionally between 5 and 15 residues, optinally between 5 and 10 residues, optionally between 3 and 5 residues.
In any aspect, the ABD that binds a cytokine receptor can be a human cytokine polypeptide, for example CD122 (IL2/15RP), IL-21R, IL-7Ra, IL-27Ra, IL-12R, IL-18R, IFNAR (IFNAR1 and/or IFNAR2). The ABD that binds a cytokine receptor can optionally be a human cytokine polypeptide (e.g. IL-2, IL-15, IL-21) that is modified (e.g. by introducing amino acid modifications) to reduce the binding affinity for a cytokine receptor to which it binds, optionally wherein binding affinity is selectively reduced for a receptor not expressed at the surface of NK cells or a receptor also expressed at the surface of non-NK cells (e.g. T cells, Treg cells). For example, the ABD that binds a human cytokine receptor can be a variant cytokine that displays reduced binding affinity for cytokine receptor present on T cells compared to the non- modified or wild-type cytokine polypeptide.
As further described herein, where a cytokine has more than one receptor as its natural binding partner and one of the receptors is expressed on non-NK cells, the cytokine polypeptide can be modified to reduce binding to such receptor that is expressed on non-NK cells (e.g. Treg cells, T cells) compared to its wild-type cytokine counterpart. In one embodiment, the multispecific protein comprises an NKp30-binding domain or portion thereof fused, optionally via a domain linker, to a cytokine receptor-binding domain, e.g. a cytokine that binds a receptor expressed at the surface of an NK cell. In one embodiment, the portion of the NKp30-binding domain comprises a single variable domain (e.g. a first variable domain fused to a first constant domain) that, together with a complementary variable domain (e.g. a second variable domain fused to a second constant region), forms an ABD (e.g., a Fab) that binds NKp30.
In one embodiment, the multispecific protein comprises: (i) a first polypeptide chain comprising, from N- to C-terminal, an NKp30-binding domain or portion thereof comprising a variable domain, a human CH1 or CL constant domain, optionally a domain linker, and a wild- type or variant I L-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or lFN-b polypeptide, and
(ii) a second polypeptide chain comprising, from N- to C-terminal, a variable domain that associates with the variable domain of (i) to form a NKp30-binding domain, and a human CH1 or CL constant domain; wherein one of the constant domains of (i) and (ii) is a CH1 and the other is a CL such that the constants domains of (i) and (ii) associate by CH1-CL dimerization. In one embodiment, the protein further comprises a dimeric Fc domain and an ABD that binds an antigen of interest.
In one embodiment, provided is a protein comprising a NKp30 ABD-cytokine unit. In one embodiment, the protein is a multispecific protein comprising a VHand a VL that associate to form an ABD that binds a cancer antigen or other antigen of interest, and a NKp30 ABD- cytokine unit (and optionally further a CD16A ABD (e.g. a dimeric Fc domain)). The NKp30 ABD-cytokine unit comprises an NKp30 ABD fused, optionally via a domain linker, to a cytokine that binds a receptor expressed at the surface of an NK cell.
In any embodiment herein, a multispecific protein can thus comprise a NKp30 ABD- cytokine unit that is formed from the association of two polypeptide chains and has one of the following structures:
Va-2 — (CH 1 or CL)a (chain 1)
V b-2 - (CH1 or CL) - L- Cyt (chain 2) or
- L1 — Va-2 — (CH 1 or CL)a (chain 1)
V b-2 - (CH1 or CL)b- L2- Cyt (chain 2) or
Va-2 — (CH 1 or CL)a (chain 1)
- L1 - V b-2 - (CH1 or CL) - L2- Cyt (chain 2) wherein : Va-2 and Vb-2 are each a VH domain or a VL domain, wherein one of Va-2 and Vb-2 is a VH and the other is a VL and wherein Va-2 and Vb-2 form an ABD that binds NKp30;
CH1 is a human immunoglobulin CH1 domain and CL is a human light chain constant domain; one of (CH1 or CL)a and (CH1 or CL)b is a CH1 and the other is a CL such that a (CH1/CL) pair is formed;
L, L1 and L2 are each an amino acid domain linker, wherein L, L1 and L2 can be different or the same, wherein L1 is a linker connecting the NKp30 ABD-cytokine unit to the rest of the multispecific protein (e.g., a protein comprising a VH and a VL and that associate to form an ABD that binds a cancer antigen); and Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21 , IL-7, IL-27, IL-12, IL-18, IFN-a or lFN-b polypeptide. The Cyt can be specified as having a free C-terminus (no further domains or amino acid sequences fused to the Cyt at its free terminus). Optionally, chain 1 can be specified as having a free C- terminus. can be specified to indicate connection via covalent bond (e.g. peptide bond) to other amino acid residues.
In one embodiment, the NKp30 ABD-cytokine unit comprises: (i) a first polypeptide chain comprising, from N- to C-terminal, an NKp30-binding domain or portion thereof comprising a variable domain comprising an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 123-212 or 250, a human CH1 or CL constant domain, optionally a domain linker, and a wild-type or variant IL- 2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b polypeptide comprising an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 213-248 or to a fragment thereof of at least 40, 50, 60, 80 or 100 contiguous amino acids thereof; and
(ii) a second polypeptide chain comprising, from N- to C-terminal, a variable domain that associates with the variable domain of (i) to form a NKp30-binding domain wherein the variable domain comprises an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 45-122 or 249, and a human CH1 or CL constant domain.
In one embodiment, the NKp30 ABD-cytokine unit comprises: (i) a first polypeptide chain comprising, from N- to C-terminal, an NKp30-binding domain or portion thereof comprising a variable domain comprising an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 123-212 or 250, a human CH1 or CL constant domain, optionally a domain linker, and a wild-type or variant IL- 2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b polypeptide comprising an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 213-248 or to a fragment thereof of at least 40, 50, 60, 80 or 100 contiguous amino acids thereof; and
(ii) a second polypeptide chain comprising, from N- to C-terminal, a variable domain that associates with the variable domain of (i) to form a NKp30-binding domain wherein the variable domain comprises an amino acid sequence at least 80%, 90%, 95%, 98% or 99% identical to an amino acid sequence of any of SEQ ID NOS: 45-122 or 249, and a human CH1 or CL constant domain.
In another embodiment where the NKp30 ABD-cytokine unit is placed on one polypeptide chain of a protein, the NKp30 ABD-cytokine unit can have the structure:
- L1 — Va-2 - L2 - Vb-2 - L3- Cyt wherein :
Va-2 and Vb-2 are each a VH domain or a VL domain, wherein one of Va-2 and Vb-2 is a VH and the other is a VL and wherein Va-2 and Vb-2 form an ABD that binds NKp30;
L1 , L2 and L3 are each an amino acid domain linker, wherein L1 , L2 and L3 can be different or the same, wherein L1 is a linker connecting the NKp30 ABD-cytokine unit to the rest of the multispecific protein (e.g., a protein comprising a VH and a VL and that associate to form an ABD that binds a cancer antigen); and Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b polypeptide. L1, L2 and L3 can each be independently specified as having a length of 15, 10 or 5 residues or less.
In one aspect of any embodiment herein, the cytokine or cytokine receptor ABD (either as a free cytokine or as incorporated into a multispecific protein) binds its receptor, as determined by SPR, with a binding affinity (KD) of 1 mM or lower, 200 nM or lower, 100 nM or lower, 50 nM or lower, or 25 nM or lower. In one embodiment, the cytokine or cytokine receptor ABD binds its receptor, as determined by SPR, with a binding affinity (KD) that is 1 nM or higher than 1 nM, optionally that is higher than 10 nM optionally that is higher than 15 nM. In one embodiment, the cytokine or cytokine receptor ABD binds its receptor, as determined by SPR, with a binding affinity (KD) between about 1 nm and about 200 nm, optionally between about 1 nm and about 100 nm, optionally between about 10 nM and about 1 pM, optionally between about 10 nM and about 200 pM, optionally between about 10 nM and about 100 nM, optionally between about 15 nM and about 1 pM, or optionally between about 15 nM and about 200 nM.
In one embodiment, the cytokine is a wild-type cytokine or a fragment or variant thereof that has at least 80% of the ability of a wild-type cytokine counterpart to induce signaling in NK cells, optionally wherein signaling is assessed by bringing the isolated cytokine moiety into contact with an NK cell and measuring STAT phosphorylation in the NK cells. In one embodiment, the cytokine is a wild-type cytokine or a fragment thereof that retains at least 70%, 80% or 90% of the affinity for its cytokine receptor present on NK cells, compared to the wild-type cytokine counterpart. In one embodiment, the cytokine is a variant cytokine, wherein the cytokine retains at least 70%, 80% or 90% of the affinity for its cytokine receptor present on NK cells, compared to the wild-type cytokine counterpart. In one embodiment, the cytokine does not comprise mutations that substantially reduce the affinity of the cytokine for the cytokine receptor present on NK cells. In one embodiment, the multispecific protein (or the cytokine when included in the multispecific protein) exhibits an EC50 for cytokine pathway signaling in NK cells that is lower than that observed with its wild-type cytokine counterpart alone. In one embodiment, the multispecific protein (or the cytokine when included in the multispecific protein) exhibits an EC50 for cytokine pathway signaling in NK cells that is lower than that observed with the cytokine alone or in a protein of comparable structure but lacking a NKp30 ABD and/or a CD16 ABD. Optionally the EC50 for cytokine pathway signaling in NK cells is at least 10-fold or 100-fold lower, optionally wherein cytokine pathway signaling is assessed by bringing the respective cytokine or multispecific protein into contact with an NK cell and measuring STAT phosphorylation in the NK cells.
In one embodiment, the multispecific protein is configured such that an Fc domain (or CD16-binding domain), the NKp30-binding domain and the cytokine receptor-binding domain are each capable of binding to their respective NKp30, CD16A or cytokine receptor binding partner when such binding partners are present together at the surface of a cell (e.g. an NK cell). In some embodiments, the multispecific protein can be characterized by monovalent binding to NKp30 (e.g. the multispecific protein comprises only one NKp30 ABD), monovalent (or optionally bivalent) binding to antigen of interest, monovalent binding to CD16A (e.g. the multispecific protein comprises only one Fc domain dimer), and monovalent binding to cytokine receptor (e.g., the multispecific protein comprises only one cytokine receptor ABD).
In one embodiment, the multispecific protein is configured e.g., through placement or configuration of the domain within a multispecific protein, optionally through use of one or more using domain linkers having a maximal potential length of 18 Angstroms (5 amino acid residues), 36 Angstroms (10 residues) or 54 Angstroms (15 residues) when in a stretched configuration such that the NKp30-binding domain and the cytokine receptor-binding domain, and the CD16-binding domain when present and capable of binding CD16, can assume a membrane planar binding conformation such that each of NKp30, CD16A and cytokine receptor are bound at the surface of an NK cell.
The multispecific protein can thus be configured such that the cytokine receptor binding domain is placed, topologically within the multimeric protein, terminally (e.g. C- terminal) to both the NKp30-binding domain and the CD16A-binding domain within the multispecific protein. For example the cytokine receptor ABD can be placed C-terminally on a polypeptide chain of the multispecific protein such that the positioning allows the cytokine receptor ABD to be topologically C-terminal within the multimeric protein). The NKp30 ABD and the CD16A ABD (e.g. a dimeric Fc domain) can be positioned adjacent to one another topologically in the protein, optionally connected to one another via a short domain linker.The NKp30 ABD, CD16A ABD and cytokine (or parts thereof) can thus be connected or positioned in series in the protein (or on polypeptide chain(s) thereof. Advantageously, the protein comprises a dimeric Fc domain (the Fc domain for example specified as consisting of two Fc monomers placed on separate polypeptide chains). Thus, in a multispecific protein the NKp30 ABD, the dimeric Fc domain and the cytokine are advantageously positioned adjacent to one another topologically (within the topology of the multispecific protein). In one embodiment, an NKp30-binding domain (or a part thereof, e.g. a VH or VL) and an Fc domain monomer (or CD16A-binding domain) are placed adjacent to one another on a same polypeptide chain(s), e.g. the adjacent NKp30-binding domain and the CD16A-binding domain can be separated by a domain linker but without any intervening protein domain (e.g. without an intervening domain that binds an antigen, and the cytokine moiety can be placed C-terminally thereto. Figures 2A to 20 show exemplary domain configurations.
For example, in preferred multispecific proteins having particularly advantageous NK cell potentiation activity, the multispecific protein comprises a Fc domain dimer comprised of a first and second Fc domain monomer positioned on different polypeptide chains (that dimerize via CH3-CH3 association). The first Fc domain monomer can be fused at its C- terminus to an anti-NKp30 ABD (or portion thereof), and the anti-NKp30 ABD (or portion thereof) is in turn fused at its C-terminus to a cytokine. The portion of an anti-NKp30 ABD can be for example a ((VH or VL)-CH1) unit or ((VH or VL)-CL) unit where the ABD is a Fab.
In any embodiment, the cytokine receptor-binding domain (cytokine receptor ABD), the NKp30-binding domain (NKp30 ABD) and the CD16-binding domain (CD16 ABD) can be specified as being placed within the one or more polypeptide chains that make up the multispecific protein so that the domains are oriented in a configuration in which they are adjacent to one another or in series, from N to C terminal, on the multimeric (e.g. heteromultimeric) protein. Domains can be optionally separated by a domain linker e.g. a linking peptide of 5-20 residues that does not itself bind to a predetermined antigen.
In any embodiment, the multispecific protein can be specified as being configured e.g., through placement or configuration of the domain within a multispecific protein, such that the NKp30 ABD and the cytokine receptor ABD (e.g. the cytokine moiety) have the ability to assume a position where they are on the same side or face of the Fc domain dimer within the multispecific protein molecule, so as to enhance the ability to bind NKp30, CD16A and cytokine receptor in a membrane planar binding conformation. This configuration can be readily implemented in any in any of the heterodimeric, heterotri meric or heterotetrameric proteins of the disclosure, for example by positioning the NKp30 ABD (or a part thereof, if the ABD is formed from association of two polypeptide chains) and the cytokine receptor ABD (or a part thereof, if the ABD is formed from association of two polypeptide chains) on the same polypeptide chain together with one of the Fc domain monomers.
The multispecific protein can have the formula (Xi)-l_i-(X2)-l-2-(X3), where one of Xi and X2 is an NKp30 ABD (e.g. a Fab, an scFv, a VHH) or a part thereof (e.g. the part may be an a VH or VL, a VH-CH1, VH-CL, VL-CL, VL-CH1) and the other is a Fc dimer or part thereof (e.g. an Fc monomer), and X3 is a cytokine, wherein and l_2 are each an optional domain linker. The cytokine is positioned at the C-terminus of the polypeptide chain on which it (or a part thereof) is placed. connects Xi and X2 via a covalent bond (e.g. peptide bond). I_2 connects X2 and X3 via a covalent bond (e.g. peptide bond). Xi to X3 can optionally be specified as being arranged from the topological N- to C-terminus of the protein. The multispecific protein can further comprise an ABD that binds an antigen of interest, e.g. connected at the N-terminus oif Xi or positioned N-terminally within the topology of the protein.
As demonstrated herein, it will be appreciated that the different elements Xi, X2 and X3 (and further the ABD that binds an antigen of interest) can each be readily distributed onto two or more different polypeptide chains within the protein. In one embodiment, the multispecific protein can optionally be characterized as comprising a first polypeptide chain comprising the formula (Xi)-I_i-(X2)-I_2-(X3), where one of Xi and X2 is an NKp30 ABD or part thereof and the other is a Fc monomer, and X3 is a cytokine or a part thereof, wherein and l_2 are each an optional domain linker. The cytokine can thus be positioned at the C-terminus of the polypeptide chain on which it (or a part thereof) is placed. The multispecific protein can further comprise an ABD that binds an antigen of interest, wherein the ABD (or a part thereof) is placed on the first polypeptide chain or on a separate polypeptide chain that associates (e.g. dimerizes) with first polypeptide chain (or with any other chain of the protein). The protein can then comprise one, two or more additional polypeptide chains that provide the complementary domains for the NKp30 ABD (when the NKp30 ABD is a part of an ABD,) the Fc monomer (so as to form an Fc dimer), the cytokine (where the cytokine is a part of a cytokine) and/or the ABD that binds an antigen of interest. Such additional polypeptide chains can thus associate (e.g. dimerize) with first or other polypeptide chain of the protein via non-covalent interactions and optionally further covalent interactions.
In another embodiment, the multispecific protein can optionally be characterized as comprising (i) a first polypeptide chain comprising the formula (Xi)-l_i-(X2a), where X1 is a first Fc monomer and za is a first part of a NKp30 ABD, and (ii) a second polypeptide chain comprising the formula (X2b)-l-2-(X3), wherein X2b is a second part of a NKp30 ABD (e.g. a VH or VL, a VH-CH1, VH-CL, VL-CL, VL-CH1) that associates with X2a to form an NKp30 ABD, and X3 is a cytokine or a part thereof. and l_2 are each a domain linker. The multispecific protein can further comprise an ABD that binds an antigen of interest, wherein the ABD (or a part thereof) is placed on the first polypeptide chain (e.g. N-terminal to Xi) or on a separate polypeptide chain that associates (e.g. dimerizes) with first polypeptide chain (or with any other chain of the protein). The multispecific protein can further com prise an ABD that binds CD16A, optionally the ABD is a dimeric Fc domain; the ABD that binds CD16A (or a part thereof) ca be placed on the first polypeptide chain (e.g. N-terminal to Xi), and when the ABD is a dimeric Fc domain one of the Fc monomers can be placed on the first polypeptide chain and the second Fc monomer can be placed on a separate polypeptide chain that associates (e.g. dimerizes) with first polypeptide chain.
In any embodiment herein, the multispecific protein can be characterized by having only one cytokine receptor binding domain.
In any embodiment herein, the multispecific protein can be characterized by having only one NKp30 binding domain.
Certain exemplary heteromultimeric proteins can comprise the following general domain organization of structure 1a or 1b, where the CD16 ADC (e.g. Fc domain) and NKp30 ABD are immediately adjacent to other another within the protein, and NKp30 ABD is immediately adjacent to the cytokine receptor ABD (embodied as a cytokine (Cyt)), and wherein the NKp30 ABD is interposed between the CD16 ABD and the Cyt:
(CD16 ABD) (NKp30 ABD) (Cyt) (Structure 1a) or
(Fc domain dimer) (NKp30 ABD) (Cyt) (Structure 1b) wherein the NKp30 ABD and the CD16ABD (e.g. Fc domain dimer) are connected by a domain linker and the NKp30 ABD and Cyt are connected by a domain linker.
The NKp30 ABD can conveniently be a Fab, a single domain antibody or an scFv. The CD16 ABD can be an Fc domain, a Fc domain dimer, an Fc domain of human lgG1 subtype. The Cyt can be for example an IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b) polypeptide, optionally wherein the polypeptide is a variant cytokine that differs by at least one residue from the wild-type human cytokine counterpart.
The protein of structure 1a can comprise an ABD that binds an antigen of interest on a target cell (Antigen ABD) placed terminal to (e.g. N-terminal to) the CD16 ABD (e.g. Fc domain dimer), as in a heteromultimeric protein having the structure 1c or 1d below: (Antigen ABD)n (CD16 ABD) (NKp30 ABD) (Cyt) (Structure 1c) or
(Antigen ABD)n (Fc domain dimer) (NKp30 ABD) (Cyt) (Structure 1d) wherein “n” is 1 or 2, and the Antigen ABD and the CD16 ABD (e.g. Fc domain dimer) are connected by a linker, optionally wherein the linker is an immunoglobulin hinge polypeptide, wherein the CD16 ABD and the NKp30 ABD are connected by a linker and the NKp30 ABD and Cyt are connected by a linker. Where “n” is 2, Structure 1d can also be represented as Structure 1 e:
(Antigen ABD)
(Fc domain dimer) (NKp30 ABD) (Cyt) (Structure 1e)
(Antigen ABD)
In any embodiment, the multispecific protein can be characterized as binding monovalently to each of the NKp30 polypeptide and the cytokine receptor, and being capable of directing an NKp30-expressing NK cell to lyse a target cell expressing the antigen of interest. Advantageously, in on embodiment, the presence of NK cells and target cells, the multi-specific protein can bind (i) to antigen of interest on target cells, (ii) to NKp30 on NK cells, (iii) to CD16A on NK cells and (iv) to the cytokine receptor on NK cells (e.g. CD122, IL-21 R, IL-7Ra, IL-27Ra, IL-12R, IL-18R, IFNAR), and when bound to such proteins on the target cell and NK cells, can induce signaling in and/or activation of the NK cells through NKp30 (the protein acts as an NKp30 agonist) and the cytokine receptor (the protein acts as a cytokine receptor agonist), thereby promoting activation of NK cells and/or lysis of target cells, notably via the activating signal transmitted by NKp30.
In one embodiment, in the presence of NK cells and target cells, the multi-specific protein can induce the cytotoxicity of, cytokine receptor pathway signaling in (as assessed by STAT signaling) and/or activation of the NK cells, wherein such cytotoxicity, activation and/or signaling is greater (e.g. at least 100-fold or 1000-fold lower ECso value) than that observed when the multi-specific protein is contacted with NK cells in the absence of target cells.
Optionally, the multi-specific protein can bind NKp30 and CD122 on NK cells (e.g. the protein comprises an IL2 or IL15 moiety, optionally an modified or variant IL2 or IL15 with decreased binding to CD25), and, when bound to both NKp30 and CD122, can induce signaling in the NK cells through both NKp30 and CD122. Optionally, the multi-specific protein can bind NKp30, CD16A and CD122 on NK cells, and, when bound to NKp30, CD16 and CD122, can induce signaling in the NK cells through NKp30, CD16A and CD122. Cytokine receptor signaling can be assessed by measuring STAT5, optionally wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
Optionally, the multi-specific protein can bind NKp30 and IL-21 R on NK cells (e.g. the protein comprises an IL21 moiety), and, when bound to both NKp30 and IL-21R, can induce signaling in the NK cells through both NKp30 and IL-21 R. Optionally, the multi-specific protein can bind NKp30, CD16A and IL-21 R on NK cells, and, when bound to NKp30, CD16A and IL- 21 R, can induce signaling in the NK cells through NKp30, CD16A and IL-21 R. Optionally, cytokine signaling is assessed by measuring STAT3, wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
Optionally, the multi-specific protein can bind NKp30 and IL-18R on NK cells (e.g. the protein comprises an IL18 moiety), and, when bound to both NKp30 and IL-18R (IL-18Ra and/or IL-18R ), can induce signaling in the NK cells through both NKp30 and IL-18R. Optionally, the multi-specific protein can bind NKp30, CD16A and IL-18R on NK cells, and, when bound to NKp30, CD16A and IL-18R, can induce signaling in the NK cells through NKp30, CD16A and IL-18R. Optionally, cytokine signaling signaling is assessed by measuring STAT3, wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
Optionally, the multi-specific protein can bind NKp30 and IL-7R (e.g. IL-7Ra (CD127) and/or CD132) on NK cells (e.g. the protein comprises an IL-7 moiety), and, when bound to both NKp30 and IL-7R, can induce signaling in the NK cells through both NKp30 and IL-7Ra. Optionally, the multi-specific protein can bind NKp30, CD16A and IL-7R on NK cells, and, when bound to NKp30, CD16A and IL-7R, can induce signaling in the NK cells through NKp30, CD16A and IL-7R. Optionally, cytokine signaling signaling is assessed by measuring STAT5, wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
Optionally, the multi-specific protein can bind NKp30 and IL-27R (e.g. IL-27Ra and/or GP130) on NK cells (e.g. the protein comprises an IL-27 moiety), and, when bound to both NKp30 and IL-27R, can induce signaling in the NK cells through both NKp30 and IL-27R. Optionally, the multi-specific protein can bind NKp30, CD16A and IL-27R on NK cells, and, when bound to NKp30, CD16A and IL-27R, can induce signaling in the NK cells through NKp30, CD16A and IL-27R. Signalling via NKp30 and/or CD16A can be assessed by a marker of NK cell activation (e.g. a marker used in the Examples, CD69 expression, etc.). Optionally, cytokine signaling signaling is assessed by measuring STAT 1 , wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
Optionally, the multi-specific protein can bind NKp30 and IL-12R (e.g., II_-12Bb1 and/or II_-12Bb2) on NK cells (e.g. the protein comprises an IL-27 moiety), and, when bound to both NKp30 and IL-12R, can induce signaling in the NK cells through both NKp30 and IL-12R. Optionally, the multi-specific protein can bind NKp30, CD16A and IL-12R on NK cells, and, when bound to NKp30, CD16A and IL-12R, can induce signaling in the NK cells through NKp30, CD16A and IL-12R. Optionally, cytokine signaling signaling is assessed by measuring STAT4, wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
Optionally, the multi-specific protein can bind NKp30 and IFNAR on NK cells, and, when bound to both NKp30 and IFNAR (IFNAR1 and/or IFNAR2), can induce signaling in the NK cells through both NKp30 and IFNAR. For example, the multi-specific protein can comprise an IFN-a or IFN-b moiety) Optionally, the multi-specific protein can bind NKp30, CD16A and IFNAR on NK cells, and, when bound to both NKp30, CD16A and IFNAR, can induce signaling in the NK cells through NKp30, CD16A and IFNAR. Optionally, cytokine signaling signaling is assessed by measuring STAT (e.g., STAT1, STAT2 or lFN regulatory factor (I RF)-9), wherein the observed signaling is greater than that observed with a comparator protein in which the NKp30 binding domain is replaced with a control ABD (e.g. that does not bind to any protein present in the assay system).
Signalling via NKp30 and/or CD16A can be assessed by a marker of NK cell activation (e.g. a marker used in the Examples, CD69 expression, etc.).
In some embodiment, the multispecific protein comprises at least a portion of a human Fc domain, e.g. a portion sufficient such that the Fc domain is bound by a human FcRn polypeptide, optionally wherein said FcRn binding affinity as assessed by SPR is within 1-log of that of a conventional human lgG1 antibody.
The multispecific proteins advantageously are able to potently mobilize both CD16+ and CD16- NK cells (all NK cells are NKp30+).
In one aspect, the multispecific protein comprises two or more polypeptide chains, i.e. it comprises a multi-chain protein (also referred as a multimeric protein). For example, the multispecific protein or multi-chain protein can be a hetero-dimer, hetero-trimer or hetero- tetramer or may comprise more than four polypeptide chains.
Any antigen binding domain (e.g. the ABD that binds the antigen of interest (e.g. tumor antigen), NKp30, or cytokine receptor) can be contained entirely on a single polypeptide chain, for example as an scFv or single antigen binding domain such as a sdAb or nanobody, a VNAR or VHH domain or a DARPin® protein module). Alternatively, an antigen binding domain can be made of two or more protein domains placed on separate polypeptide chains, such that the antigen binding domain binds its target when two or more complementary protein domains (e.g. as VH/VL pairs) are associated in the multimeric protein.
The multispecific protein can bind to the antigen of interest (e.g. cancer antigen) in monovalent or multivalent manner. Where the multispecific protein binds the antigen of interest monovalently, binds NKp30 monovalently and binds the cytokine receptor monovalently, the multispecific protein can be indicated as having a 1 :1:1 configuration. Where the multispecific protein binds the antigen of interest bivalently, binds NKp30 monovalently and binds the cytokine receptor monovalently, the multispecific protein can be indicated as having a 2:1:1 configuration. Representative examples of different 1 :1:1 and 2:1:1 configurations are shown in Figure 2.
In any aspect, the multispecific protein can be characterized as having a structure in which the freedom of motion (intrachain domain motion) or flexibility of one or more antigen binding domains (ABDs) is increased, e.g. compared to the ABDs of a conventional human IgG antibody. In one embodiment, provided is a multispecific protein comprising a structure that permits the antigen binding site of the first antigen binding domain and the antigen binding site of the second antigen binding domain to be separated by a distance that results in enhanced function, e.g., the ability of the multispecific protein to induce NKp30 signaling and lysis of target cells, e.g., optionally a distance of less than 80 angstrom (A). Multispecific proteins wherein the ABDs possess greater flexibility and/or are separated by an optimized distance may enhance the formation of a lytic NKp30-target synapse, thereby potentiating NKp30-mediated signaling. Such flexibility and/or domain of motion can be readily achieved through the use of linkers (e.g. flexible amino acid based linkers) that separate the NKp30 binding domain from the Fc domain (e.g. the Fc domain dimer, or more generally the rest of the multispecific protein).
In any aspect, the multispecific protein can be characterized as having increased freedom of motion of the antigen binding domains (e.g. compared to the ABDs of a conventional human IgG antibody, e.g., a human lgG1 antibody). One example of such a protein is a multimeric Fc domain-containing protein (e.g. a heterodimer or heterotrimer) in which an antigen binding domain (e.g., the ABD that binds NKp30) is linked or fused to an Fc domain via a flexible linker. The linker can provide flexibility or freedom of motion of one or more ABDs by conferring the ability to bend thereby potentially decreasing the angle between the ABD and the Fc domain (or between the two ABDs) at the linker. Optionally, both antigen binding domains (and optionally more if additional ABDs are present in the multispecific protein) are linked or fused to the Fc domain via a linker, typically a flexible peptide linker. Optionally, the protein with increased freedom of motion permits the protein to adopt a conformation in which the distance between the NKp30 binding site and the antigen of interest binding site is less that than observed in proteins in which both binding domains were Fabs, or less than in full length antibodies.
An ABD can be connected to an Fc domain monomer (or CH2 or CH3 domain thereof) via a domain linker. The linker can be a polypeptide linker, for example peptide linkers comprising a length of at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, or more. In other embodiments, the linkers comprises a length of between 2-4 residues, between 2-5 residues, between 2-6 residues, between 2-8 residues, between 5-10 residues, between 2-15 residues, between 4-15 residues, between 3-15 residues, between 5- 15 residues, between 10-15 residues, between 4-20 residues, between 5-20 residues, between 2-20 residues, between 10-30 residues, or between 10-50 residues. Optionally a linker comprises an amino acid sequence derived from an antibody constant region, e.g., an amino acid sequence from a CH1 or CK domain (e..g an an N-terminal sequence from a CH1 or CK domain) or from a hinge. Optionally a linker comprises the amino acid sequence RTVA. Optionally a linker is a flexible linker predominantly or exclusively comprised of glycine and/or serine residues, e.g., comprising an amino acid sequence (GxS)n where G is 1, 2, 3 or 4 and n is an integer from 1-10, from 1-6 or from 1-4. Optionally the linker comprises 1-20 or 1-10 further amino acid residues.
In one embodiment, provided is a heterotrimer having a polypeptide chain 1 , 2 and 3:
Vb-i - (CH1 or CL)c - Hinge - CH2 - CH3 (chain 2)
I I
Va - (CH1 or CL)a — Hinge - CH2 - CH3- L1 -Va-2 - (CH1 or CL) (chain 1)
I
Vb-2 - (CH1 or CL)d- L2- Cyt (chain 3) wherein :
Va-i, Vb-i, Va-2 and V -2 are each a VH domain or a VL domain, wherein one of Va-i and V -i is a Vhnand the other is a VL, and wherein Va-i and VM form a first antigen binding domain (ABD) that binds an antigen of interest, wherein one of Va-2and V -2 is a VH and the other is a VL and wherein Va-2 and V -2 form a second ABD that binds NKp30;
CH1 is a human immunoglobulin CH1 domain and CL is a human light chain constant domain; one of (CH1 or CL)a and (CH1 or CL)c is a CH1 and the other is a CL such that a (CH1/CL) pair is formed; one of (CH1 or CL)b and (CH1 or CL)d is a CH1 and the other is a CL such that a (CH1/CL) pair is formed; Hinge is an immunoglobulin hinge region or portion thereof;
L1 and L2 are each an amino acid domain linker, wherein L1 and L2 can be different or the same;
CH2 and CH3 are human immunoglobulin CH2 and CH3 domains, respectively; and
Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21, IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide. In another embodiment that can be made using the same domains and domain linkers, provided is a heterotrimer having a polypeptide chain 1, 2 and 3:
Vb-i - (CH1 or CL)c - Hinge - CH2 - CH3 (chain 2)
I I
Va - (CH1 or CL)a — Hinge - CH2 - CH3- L1 -Va-2 - (CH1 or CL) - L2- Cyt (chain 1)
I
Vb - (CH1 or CL)d (chain 3)
In one embodiment, provided is a heterodimer having a polypeptide chain 1 and 2:
Vai - (CH1 or CK)a- (hinge or L2) - CH2 - CH3 - Va2 - Vb2- L1 -Cyt (chain 1)
I I
V i - (CH1 or CK) - (hinge or L3) - CH2 - CH3 (chain 2). wherein :
Va-i, Vb-i, Va and V are each a VH domain or a VL domain wherein one of Va-i and V -i is a Vhnand the other is a VL, and wherein Va-i and VM form a first antigen binding domain (ABD) that binds an antigen of interest), wherein one of Va-2 and Vb-2 is a VH and the other is a VL and wherein Va-2and Vb-2form a second ABD that binds NKp30;
CH1 is a human immunoglobulin CH1 domain and CL is a light chain constant domain; one of (CH1 or CL)a and (CH1 or CL)b is a CH1 and the other is a CL such that a (CH1/CL) pair is formed;
Hinge is an immunoglobulin hinge region or portion thereof;
L1, L2 and L3 are each an amino acid domain linker, wherein L1, L2 and L3 can be different or the same;
CH2 and CH3 are human immunoglobulin CH2 and CH3 domains, respectively; and
Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21, IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
The disclosure further provides further heterodimer, heterotrimer and heterotetramer multispecific molecules and domain arrangements as further described herein. In one aspect, a multispecific protein is a heteromultimer, heterodimer, heterotrimer, heterotetramer having a structure or domain arrangement as shown in any of Figures 2A to 20. In one aspect of any of the embodiments described herein, an ABD (e.g., the anti- NKp30 ABD, the ABD that binds the antigen of interest or tumor antigen) can be specified as comprising an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR-1 to CDR-3). In one aspect of any of the embodiments described herein, a VH can be specified as having the amino acid sequence of a human VH domain. In one aspect of any of the embodiments described herein, a VL can be specified as having the amino acid sequence of a human VL domain.
In one aspect of any of the embodiments, a VH region comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence encoded by a gene of a human V gene group selected from the group consisting of IGHV1-18, IGHV1-2, IGHV1-24, IGHV1-3, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV1-69-2, IGHV1-69D, IGHV1-8, IGHV2-26, IGHV2-5, IGHV2-70, IGHV2-70D, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21 , IGHV3-23, IGHV3-23D, IGHV3-30, IGHV3-30-3, IGHV3-30-5, IGHV3-33, IGHV3-43, IGHV3-43D, IGHV3-48, IGHV3-49, IGHV3- 53, IGHV3-62, IGHV3-64, IGHV3-64D, IGHV3-66, IGHV3-7, IGHV3-72, IGHV3-73, IGHV3- 74, IGHV3-9, IGHV3-NL1 , IGHV4-28, IGHV4-30-2, IGHV4-30-4, IGHV4-31, IGHV4-34, IGHV4-38-2, IGHV4-39, IGHV4-4, IGHV4-59, IGHV4-61, IGHV5-10-1 , IGHV5-51 , IGHV6-1, IGHV7-4-1 , IGHV1-38-4, IGHV1/OR15-1, IGHV1/OR15-5, IGHV1/OR15-9, IGHV1/OR21-1, IGHV2-70, IGHV2/OR16-5, IGHV3-16, IGHV3-20, IGHV3-25, IGHV3-35, IGHV3-38, IGHV3- 38-3, IGHV3/OR15-7, IGHV3/OR16-10, IGHV3/OR16-12, IGHV3/OR16-13, IGHV3/OR16-17, IGHV3/OR16-20, IGHV3/OR16-6, IGHV3/OR16-8, IGHV3/OR16-9, IGHV4-61 , IGHV4/OR15- 8, IGHV7-81, and IGHV8-51-1. Optionally, a VH region comprises a VH comprising an amino acid sequence (e.g. CDR(s) and/or a human framework region(s), for example according to Kabat numbering) from said gene. In one aspect of any of the embodiments, a VH region comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence of SEQ ID NOS: 45-122.
In one aspect of any of the embodiments, a VL region comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence encoded by a gene of a human V gene group selected from the group consisting of IGKV1-12, IGKV1-13, IGKV1-16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-39, IGKV1-5, IGKV1-6, IGKV1-8, IGKV1-9, IGKV1-NL1 , IGKV1 D-12, IGKV1 D-13, IGKV1 D-16, IGKV1 D-17, IGKV1 D-33, IGKV1 D-43, IGKV1 D-8, IGKV2-24, IGKV2-28, IGKV2-29, IGKV2- 30, IGKV2-40, IGKV2D-26, IGKV2D-28, IGKV2D-29, IGKV2D-30, IGKV2D-40, IGKV3-11, IGKV3-15, IGKV3-20, IGKV3D-11 , IGKV3D-15, IGKV3D-20, IGKV3D-7, IGKV4-1, IGKV5-2, IGKV6-21, IGKV6D-21 , IGKV1-37, IGKV1/OR2-0, IGKV1/OR2-108, IGKV1 D-37, IGKV1 D-42, IGKV2D-24, IGKV3-7, IGKV3/OR2-268, IGKV3D-20, IGKV6D-41 , IGLV1-36, IGLV1-40, IGLV1-44, IGLV1-47, IGLV1-51, IGLV10-54, IGLV2-11, IGLV2-14, IGLV2-18, IGLV2-23, IGLV2-8, IGLV3-1 , IGLV3-10, IGLV3-12, IGLV3-16, IGLV3-19, IGLV3-21 , IGLV3-22, IGLV3- 25, IGLV3-27, IGLV3-9, IGLV4-3, IGLV4-60, IGLV4-69, IGLV5-37, IGLV5-39, IGLV5-45, IGLV5-52, IGLV6-57, IGLV7-43, IGLV7-46, IGLV8-61, IGLV9-49, IGLV1-41, IGLV1-50, IGLV11-55, IGLV2-33, IGLV3-32, IGLV5-48 and IGLV8/OR8-1. Optionally, a VL region comprises a VL comprising an amino acid sequence (e.g. CDR(s) and/or a human framework region(s), for example according to Kabat numbering) from said gene. In one aspect of any of the embodiments, a VL region comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence of SEQ ID NOS: 123- 212.
In one aspect of any of the embodiments described herein, an ABD comprises an scFv or Fab, wherein the scFv comprises a VH comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, any of 45-122, and 249, a domain linker, and a VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, any of 123-212, and 250; and wherein the Fab comprises one VH comprising an amino acid sequence at least 90% identical to a selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, any of 45-122, and 249, one VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, any of 123-212, and 250, one human CH1 domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 26 and one human CL domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 29, wherein the VH is fused to one of the CH1 or CL domains, and the VL is fused to the other of the CH1 or CL domains.
In one aspect of any of the embodiments described herein, an IL2 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-2 polypeptide of any of SEQ ID NOS: 213-226, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof. Optionally the IL2 further comprises 2, 3, 4, 5 or more amino acid substitutions that reduce binding to CD25, e.g. substitutions at any of the residues disclosed herein.
In one aspect of any of the embodiments described herein, an IL15 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-15 polypeptide of any of SEQ ID NO: 227, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
In one aspect of any of the embodiments described herein, an IL12 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-12 polypeptide of any of SEQ ID NOS: 247 and/or 248, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
In one aspect of any of the embodiments described herein, an IL7 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-7 polypeptide of any of SEQ ID NO: 244, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
In one aspect of any of the embodiments described herein, an IL27 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-21 polypeptide of any of SEQ ID NOS: 245 and/or 246, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
In one aspect of any of the embodiments described herein, an IL21 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-27 polypeptide of any of SEQ ID NOS: 229 or 230, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
In one aspect of any of the embodiments described herein, an IL18 comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IL-18 polypeptide of any of SEQ ID NO: 231 , or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
In one aspect of any of the embodiments described herein, an IFN-a comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IFN-a polypeptide of any of SEQ ID NOS: 232-242, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
In one aspect of any of the embodiments described herein, an IFN-b comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the IFN-a polypeptide of any of SEQ ID NO: 243, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
In one aspect of any of the embodiments described herein, an Fc domain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the Fc polypeptide of any of SEQ ID NOS: 30-35, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
In one aspect of any of the embodiments described herein, a CH1, CH2 and CH3 domain respectively comprise an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the CH1, CH2 or CH3 polypeptide of SEQ ID NO: 26, 27 or 28, respectively, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof. In one aspect of any of the embodiments described herein, a CK or CL domain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the CK polypeptide of any of SEQ ID NO: 29, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
In one aspect of any of the embodiments described herein, a hinge domain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the CK polypeptide of any of SEQ ID NOS: 36-40.
In one aspect of any of the embodiments described herein, a multispecific protein comprises:
(a) an ABD that binds to the antigen of interest, wherein the ABD comprises an scFv or Fab, a. wherein the scFv comprises a VH comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and any of 45-122, a domain linker, and a VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25 and any of 123-212; and b. wherein the Fab comprises one VH comprising an amino acid sequence at least 90% identical to a selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and any of 45-122, one VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25 and any of 123-212, one human CH1 domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 26 and one human CL domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 29, wherein the VH is fused to one of the CH1 or CL domains, and the VL is fused to the other of the CH1 or CL domains,
(b) an ABD that binds to a human NKp30 polypeptide, wherein the ABD comprises an scFv or Fab, a. wherein the scFv comprises a VH comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 45-122 or 249, a domain linker, and a VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 123-212 or 250; and b. wherein the Fab comprises one VH comprising an amino acid sequence at least 90% identical to a selected from SEQ ID NOS: 45-122 or 249 and any of 236-313, one VL comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 123-212 or 250, one human CH1 domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 26 and one human CL domain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 29, wherein the VH is fused to one of the CH1 or CL domains, and the VL is fused to the other of the CH1 or CL domains,
(c) a Fc domain dimer comprising two Fc domain monomer polypeptides, wherein each Fc domain monomer polypeptide comprises an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 30-35; and
(d) a cytokine polypeptide comprising an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 213-248, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof, fused, via a domain linker, to the C-terminus of one of the polypeptide chains of the multispecific protein.
In one embodiment, a multispecific protein comprises: a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of a first chain of a heterotrimeric protein described herein, a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of a second chain of a heterotrimeric protein described herein and a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of a third chain of a heterotrimeric protein described herein. In one embodiment, a multispecific protein comprises: a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of a first chain of a heterodimeric protein described herein, and a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of a second chain of a heterodimeric protein described herein.
In one embodiment, a multispecific protein comprises: a polypeptide comprising an amino acid sequence of a first chain of a heterotrimeric protein described herein, a polypeptide comprising an amino acid sequence of a heterotrimeric protein described herein and a polypeptide comprising an amino acid sequence of a third chain of a heterotrimeric protein described herein. In one embodiment, a multispecific protein comprises: a polypeptide comprising an amino acid sequence of a first chain of a heterodimeric protein described herein, and a polypeptide comprising an amino acid sequence of a second chain of a heterodimeric protein described herein. In one embodiment, a multispecific protein comprises: a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 251 , a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 252 and a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 253.
In one embodiment, a multispecific protein comprises: a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 257, a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 258; and a polypeptide comprising an amino acid sequence having at least 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 259.
In one aspect of any of the embodiments described herein, provided is a recombinant nucleic acid encoding a first polypeptide chain, and/or a second polypeptide chain, and/or a third polypeptide chain and/or a fourth polypeptide. In one aspect of any of the embodiments described herein, the invention provides a recombinant host cell comprising a nucleic acid encoding a first polypeptide chain, and/or a second polypeptide chain and/or a third polypeptide chain, optionally wherein the host cell produces a multimeric or other protein according to the invention with a yield (final productivity or concentration before or after purification) of at least 1 , 2, 3 or 4 mg/L. Also provided is a kit or set of nucleic acids comprising a recombinant nucleic acid encoding a first polypeptide chain of the according to the invention, a recombinant nucleic acid encoding a second polypeptide chain according to the invention, and, optionally, a recombinant nucleic acid encoding a third polypeptide chain according to the invention. Also provided are methods of making dimeric, trimeric and tetrameric proteins according to the invention.
Any of the methods can further be characterized as comprising any step described in the application, including notably in the “Detailed Description of the Invention”). The invention further relates to methods of identifying, testing and/or making proteins described herein. The invention further relates to a multispecific protein obtainable by any of present methods. The disclosure further relates to pharmaceutical or diagnostic formulations containing at least one of the multispecific proteins disclosed herein. The disclosure further relates to methods of using the subject multispecific proteins in methods of treatment or diagnosis.
These and additional advantageous aspects and features of the invention may be further described elsewhere herein.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the topology of the multispecific NK cell engager (NKCE) protein that binds on one face to a tumor antigen on a tumor cell, and on another face to an NK cell via a triple receptor cis-presentation of II_2bg complex, NKp30 and CD16A. IL2v capture on NK cells may improve binding to CD122 and mimic CD25-mediated IL-2 presentation.
Figures 2A and 2B show an exemplary multispecific protein in T5 format that binds to NKp30, CD16A and cytokiner receptor (e.g. CD122) on an NK cell, and to tumor antigen (e.g. TA, Tag, CD20) on a tumor cell. Figures 2C-20 show other multimeric protein structures with or without CD16A binding.
Figure 3 shows activation of TReg cells by heterotrimer proteins that contained either a wild-type IL-2 or a variant IL2, and that lacked binding to NKp30, CD16A and antigen of interest. The protein containing the variant IL2 showed a strongly decreased ability to activate Treg cells compared to wild-type IL-2 and to the heterotrimer protein containing wild-type IL- 2.
Figure 4 shows ability to direct purified NK cells to lyse CD20-positive RAJ I tumor target cells by CD20 x NKp30 binding proteins. GA101-T5-Com_mAb8-IL2v proteins, coengaging CD16A and NKp30 on NK cells and GA101-T6-Com_mAb8-IL2v proteins, engaging only NKp30 were highly potent in mediating NK cell lysis of tumor target cells.
Figures 5, 6, 7 and 8 show % of pSTAT5 cells among NK cells, CD4 T cells CD8 T cells and Treg cells, respectively. GA101-T5-Com_mAb8-IL2v, GA101-T6-Com_mAb8-IL2v and GA101-T6-IC-IL2v displayed comparable activation of each cell type. However, GA101- T5-Com_mAb8-IL2v resulted in a strong increase in potency in the ability to cause an increase in percent of pSTAT5+ cells among the NK cells, compared to that observed in CD4 T cells CD8 T cells and Treg cells. The GA101-T5-Com_mAb8-IL2v protein therefore permitted a selective activation of NK cells over Treg cells, CD4 T cells and CD8 T cells.
Figure 9 shows percentage of IFN-g expressing NK cells within PBMCs in presence of RAJI targeted tumor cells, upon treatment with GA101-T5-Com_mAb8-IL2v, GA101-T6- Com_mAb8-IL2v, IC-T6-IC-IL2v that did not bind CD16A or NKp30 on NK cells or human IgG isotype antibody having the GA101 VH/VL domains (HuG1).
Figure 10 shows MIR1b MedFI on NK cells within PBMCs in presence of RAJI targeted tumor cells, induced by GA101-T5-Com_mAb8-IL2v, GA101-T6-Com_mAb8-IL2v and IC-T6- IC-IL2v that did not bind CD16A or NKp30 on NK cells.
Figures 11A and B show respectively induction of IFN-g production and MIR1b MedFI, by GA101-T5-Com_mAb8-IL2v and GA101-T6-Com_mAb8-IL2v in NK cells, without any effect on IFN-g production and MIR1b MedFI in CD4 T cells, CD8 T cells or in gd T cells within PBMCs in presence of RAJI targeted tumor cells. DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used in the specification, "a" or "an" may mean one or more. As used in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.
Where "comprising" is used, this can optionally be replaced by "consisting essentially of", or optionally by "consisting of".
As used herein, the term "antigen binding domain" or ”ABD” refers to a domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope. Thus, in one embodiment, said domain can comprise a hypervariable region, optionally a VH and/or VL domain of an antibody chain, optionally at least a VH domain. In another embodiment, the binding domain may comprise at least one complementarity determining region (CDR) of an antibody chain. In another embodiment, the binding domain may comprise a polypeptide domain from a non-immunoglobulin scaffold.
The term "antibody" herein is used in the broadest sense and specifically includes full- length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and derivatives, so long as they exhibit the desired biological activity. Various techniques relevant to the production of antibodies are provided in, e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988). An "antibody fragment" comprises a portion of a full- length antibody, e.g. antigen-binding or variable regions thereof. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab’)2, F(ab)3, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VH and CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10: 949-57); camel IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments, and one or more isolated CDRs or a functional paratope, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 23, 1126-1136; W02005040219, and published U.S. Patent Applications 20050238646 and 20020161201.
The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity-determining region" or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. 1991) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as “Kabat position”, "variable domain residue numbering as in Kabat" and "according to Kabat" herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
By "framework" or "FR" residues as used herein is meant the region of an antibody variable domain exclusive of those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into the contiguous regions separated by the CDRs (FR1 , FR2, FR3 and FR4).
By "constant region" as defined herein is meant an antibody-derived constant region that is encoded by one of the light or heavy chain immunoglobulin constant region genes.
By "constant light chain" or "light chain constant region" or “CL” as used herein is meant the region of an antibody encoded by the kappa (CK) or lambda (C l) light chains. The constant light chain typically comprises a single domain, and as defined herein refers to positions 10S- 214 of CK, or CA, wherein numbering is according to the EU index (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda).
By "constant heavy chain" or "heavy chain constant region" as used herein is meant the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes to define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For full length IgG antibodies, the constant heavy chain, as defined herein, refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 118-447, wherein numbering is according to the EU index.
By "Fab" or "Fab region" as used herein is meant a unit that comprises the VH, CH1 , VL, and CL immunoglobulin domains. The term Fab includes a unit that comprises a VH-CH 1 moiety that associates with a VL-CL moiety, as well as crossover Fab structures in which there is crossing over or interchange between light- and heavy-chain domains. For example a Fab may have a VH-CL unit that associates with a VL-CH1 unit. Fab may refer to this region in isolation, or this region in the context of a protein, multispecific protein or ABD, or any other embodiments as outlined herein.
By "single-chain Fv" or "scFv" as used herein are meant antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
By "Fv" or "Fv fragment" or "Fv region" as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody.
By "Fc" or "Fc region", as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N- terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cy2 (CH2) and Cy3 (CH3) and optionally the hinge between Cy1 and Cy2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226, P230 or A231 to its carboxyl-terminus, wherein the numbering is according to the EU index. Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, as described below. By "Fc polypeptide" or “Fc-derived polypeptide” as used herein is meant a polypeptide that comprises all or part of an Fc region. Fc polypeptides herein include but are not limited to antibodies, Fc fusions and Fc fragments. Also, Fc regions according to the invention include variants containing at least one modification that alters (enhances or diminishes) an Fc associated effector function. Also, Fc regions according to the invention include chimeric Fc regions comprising different portions or domains of different Fc regions, e.g., derived from antibodies of different isotype or species.
By "variable region" as used herein is meant the region of an antibody that comprises one or more Ig domains substantially encoded by any of the VL (including VK (VK) and VA) and/or VH genes that make up the light chain (including k and l) and heavy chain immunoglobulin genetic loci respectively. A light or heavy chain variable region (VL or VH) consists of a "framework" or "FR" region interrupted by three hypervariable regions referred to as "complementarity determining regions" or "CDRs". The extent of the framework region and CDRs have been precisely defined, for example as in Kabat (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., U.S. Department of Health and Human Services, (1983)), and as in Chothia. The framework regions of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs, which are primarily responsible for binding to an antigen.
The term “specifically binds to” means that an antibody or polypeptide can bind preferably in a competitive binding assay to the binding partner, e.g. NKp30, as assessed using either recombinant forms of the proteins, epitopes therein, or native proteins present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.
When an antibody or polypeptide (e.g. a multispecific protein) is said to “compete with” a particular multispecific protein or a particular monoclonal antibody), it means that the antibody or polypeptide competes with the particular multispecific protein or monoclonal antibody in a binding assay using either recombinant target (e.g. NKp30) molecules or surface expressed target (e.g. NKp30) molecules. For example, if a test multispecific protein or antibody reduces the binding of multispecific protein GA101-T5-Com_mAb8-IL2v to a NKp30 polypeptide or NKp30-expressing cell in a binding assay, the test multispecific protein or antibody is said to “compete” respectively with GA101-T5-Com_mAb8-IL2v.
The term “affinity”, as used herein, means the strength of the binding of an antibody or protein to an epitope. The affinity of an antibody is given by the dissociation constant KD, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody- antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant KA is defined by 1/KD. Preferred methods for determining the affinity of proteins can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of proteins is the use of surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analytical device).
Within the context of this invention a “determinant” designates a site of interaction or binding on a polypeptide.
The term “epitope” refers to an antigenic determinant, and is the area or region on an antigen to which an antibody or protein binds. A protein epitope may comprise amino acid residues directly involved in the binding as well as amino acid residues which are effectively blocked by the specific antigen binding antibody or peptide, /.e., amino acid residues within the "footprint" of the antibody. It is the simplest form or smallest structural area on a complex antigen molecule that can combine with e.g., an antibody or a receptor. Epitopes can be linear or conformational/structural. The term “linear epitope” is defined as an epitope composed of amino acid residues that are contiguous on the linear sequence of amino acids (primary structure). The term “conformational or structural epitope” is defined as an epitope composed of amino acid residues that are not all contiguous and thus represent separated parts of the linear sequence of amino acids that are brought into proximity to one another by folding of the molecule (secondary, tertiary and/or quaternary structures). A conformational epitope is dependent on the 3-dimensional structure. The term ‘conformational’ is therefore often used interchangeably with ‘structural’. Epitopes may be identified by different methods known in the art including but not limited to alanine scanning, phage display, X-ray crystallography, array- based oligo-peptide scanning or pepscan analysis, site-directed mutagenesis, high throughput mutagenesis mapping, H/D-Ex Mass Spectroscopy, homology modeling, docking, hydrogen- deuterium exchange, among others. (See e.g., Tong et al., Methods and Protocols for prediction of immunogenic epitopes”, Briefings in Bioinformatics 8(2):96-108; Gershoni, Jonathan M; Roitburd-Berman, Anna; Siman-Tov, Dror D; Tarnovitski Freund, Natalia; Weiss, Yael (2007). "Epitope Mapping". BioDrugs 21 (3): 145-56; and Flanagan, Nina (May 15, 2011); "Mapping Epitopes with H/D-Ex Mass Spec: ExSAR Expands Repertoire of Technology Platform Beyond Protein Characterization", Genetic Engineering & Biotechnology News 31 (10).
“Valent” or “valency” denotes the presence of a determined number of antigen-binding moieties in the antigen-binding protein. A natural IgG has two antigen-binding moieties and is bivalent. A molecule having one binding moiety for a particular antigen is monovalent for that antigen.
By "amino acid modification" herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. An example of amino acid modification herein is a substitution. By "amino acid modification" herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a given position in a protein sequence with another amino acid. For example, the substitution Y50W refers to a variant of a parent polypeptide, in which the tyrosine at position 50 is replaced with tryptophan. Amino acid substitutions are indicated by listing the residue present in wild-type protein / position of residue / residue present in mutant protein. A "variant" of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or “parent” polypeptide. The polypeptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence.
"Conservative” amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Families of amino acid residues having similar side chains are known in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
The term “identity” or “identical”, when used in a relationship between the sequences of two or more polypeptides, refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e. , "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).
Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm may also be used to determine identity. An “isolated” molecule is a molecule that is the predominant species in the composition wherein it is found with respect to the class of molecules to which it belongs (i.e., it makes up at least about 50% of the type of molecule in the composition and typically will make up at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the species of molecule, e.g., peptide, in the composition). Commonly, a composition of a polypeptide will exhibit 98%, 98%, or 99% homogeneity for polypeptides in the context of all present peptide species in the composition or at least with respect to substantially active peptide species in the context of proposed use.
In the context herein, “treatment” or “treating” refers to preventing, alleviating, managing, curing or reducing one or more symptoms or clinically relevant manifestations of a disease or disorder, unless contradicted by context. For example, “treatment” of a patient in whom no symptoms or clinically relevant manifestations of a disease or disorder have been identified is preventive or prophylactic therapy, whereas “treatment” of a patient in whom symptoms or clinically relevant manifestations of a disease or disorder have been identified generally does not constitute preventive or prophylactic therapy.
As used herein, the phrase “NK cells” refers to a sub-population of lymphocytes that is involved in non-conventional immunity. NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD56 and/or NKp46 for human NK cells, the absence of the alpha/beta or gamma/delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express "self" MHC/HLA antigens by the activation of specific cytolytic machinery, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art. Any subpopulation of NK cells will also be encompassed by the term NK cells. Within the context herein “active” NK cells designate biologically active NK cells, including NK cells having the capacity of lysing target cells or enhancing the immune function of other cells. NK cells can be obtained by various techniques known in the art, such as isolation from blood samples, cytapheresis, tissue or cell collections, etc. Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). Humana Press pp. 219-238 (2000).
As used herein, an agent that has “agonist” activity at NKp30 is an agent that can cause or increase "NKp30 signaling". “NKp30 signaling” refers to an ability of an NKp30 polypeptide to activate or transduce an intracellular signaling pathway. Changes in NKp30 signaling activity can be measured, for example, by assays designed to measure changes in NKp30 signaling pathways, e.g. by monitoring phosphorylation of signal transduction components, assays to measure the association of certain signal transduction components with other proteins or intracellular structures, or in the biochemical activity of components such as kinases, or assays designed to measure expression of reporter genes under control of NKp30-sensitive promoters and enhancers, or indirectly by a downstream effect mediated by the NKp30 polypeptide (e.g. activation of specific cytolytic machinery in NK cells). Reporter genes can be naturally occurring genes (e.g. monitoring cytokine production) or they can be genes artificially introduced into a cell. Other genes can be placed under the control of such regulatory elements and thus serve to report the level of NKp30 signaling.
“NKp30” refers to a protein or polypeptide encoded by the Ncr3 (natural cytotoxicity triggering receptor 3) gene or by a cDNA prepared from such a gene. Any naturally occurring isoform, allele, ortholog or variant is encompassed by the term NKp30 polypeptide (e.g., an NKp30 polypeptide 90%, 95%, 98% or 99% identical to SEQ ID NO 1 , or a contiguous sequence of at least 20, 30, 50, 100 or 200 amino acid residues thereof). The 201 amino acid residue sequence of human NKp30 (including the 18 amino acid signal sequence) is shown below:
MAWMLLLILI MVHPGSCALW VSQPPEIRTL EGSSAFLPCS FNASQGRLAI GSVTWFRDEV VPGKEVRNGT PEFRGRLAPL ASSRFLHDHQ AELHIRDVRG HDASIYVCRV EVLGLGVGTG NGTRLVVEKE HPQLGAGTVL LLRAGFYAVS FLSVAVGSTV YYQGKCLTWK GPRRQLPAVV PAPLPPPCGS SAHLLPPVPG G (SEQ I D NO: 1).
SEQ ID NO: 1 corresponds to NCBI accession number 014931, the disclosure of which is incorporated herein by reference.
Producing polypeptides
The proteins described herein can be conveniently configured and produced using well known immunoglobulin-derived domains, notably heavy and light chain variable domains, hinge regions, CH1 , CL, CH2 and CH3 constant domains, and wild-type or variant cytokine polypeptides. Domains placed on a common polypeptide chain can be fused to one another either directly or connected via linkers, depending on the particular domains concerned. The immunoglobulin-derived domains will preferably be humanized or of human origin, thereby providing decreased risk of immunogenicity when administered to humans. As shown herein, advantageous protein formats are described that use minimal non-immunoglobulin linking amino acid sequences (e.g. not more than 4 or 5 domain linkers, in some cases as few as 1 or 2 domain linkers, and use of domains linkers of short length), thereby further reducing risk of immunogenicity.
Immunoglobulin variable domains are commonly derived from antibodies (immunoglobulin chains), for example in the form of associated VL and VH domains found on two polypeptide chains, or a single chain antigen binding domain such as an scFv, a VH domain, a VL domain, a dAb, a V-NAR domain or a VHH domain. In certain advantageous proteins formats disclosed herein that directly enable the use of a wide range of variable regions from Fab or scFv without substantial further requirements for pairing and/or folding, the an antigen binding domain (e.g., ABDi and ABD2) can also be readily derived from antibodies as a Fab or scFv.
The term “antigen-binding protein” can be used to refer to an immunoglobulin derivative with antigen binding properties. The binding protein comprises an immunologically functional immunoglobulin portion capable of binding to a target antigen. The immunologically functional immunoglobulin portion may comprise immunoglobulins, or portions thereof, fusion peptides derived from immunoglobulin portions or conjugates combining immunoglobulin portions that form an antigen binding site. Each antigen binding moiety comprises at least the necessarily one, two or three CDRs of the immunoglobulin heavy and/or light chains from which the antigen binding moiety was derived. In some aspects, an antigen-binding protein can consist of a single polypeptide chain (a monomer). In other embodiments the antigen binding protein comprises at least two polypeptide chains. Such an antigen-binding protein is a multimer, e.g., dimer, trimer or tetramer. Examples of antigen binding proteins includes antibody fragments, antibody derivatives or antibody-like binding proteins that retain specificity and affinity for their antigen.
Typically, antibodies are initially obtained by immunization of a non-human animal, e.g., a mouse, rat, guinea pig or rabbit, with an immunogen comprising a polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment, for which it is desired to obtain antibodies (e.g. a human polypeptide). The step of immunizing a non-human mammal with an antigen may be carried out in any manner well known in the art for stimulating the production of antibodies in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is herein incorporated by reference). Human antibodies may also be produced by using, for immunization, transgenic animals that have been engineered to express a human antibody repertoire (Jakobovitz et al. Nature 362 (1993) 255), or by selection of antibody repertoires using phage display methods. For example, a XenoMouse (Abgenix, Fremont, CA) can be used for immunization. A XenoMouse is a murine host that has had its immunoglobulin genes replaced by functional human immunoglobulin genes. Thus, antibodies produced by this mouse or in hybridomas made from the B cells of this mouse, are already humanized. The XenoMouse is described in United States Patent No. 6,162,963, which is herein incorporated in its entirety by reference. Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in (Ward et al. Nature , 341 (1989) p. 544, the entire disclosure of which is herein incorporated by reference). Phage display technology (McCafferty et al (1990) Nature 348:552-553) can be used to produce antibodies from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. See, e.g., Griffith et al (1993) EMBO J. 12:725- 734; US 5,565,332; US 5,573,905; US 5,567,610; and US 5,229,275). When combinatorial libraries comprise variable (V) domain gene repertoires of human origin, selection from combinatorial libraries will yield human antibodies.
In any embodiment, an antigen binding domain can be obtained from a humanized antibody in which residues from a complementary-determining region (CDR) of a human antibody are replaced by residues from a CDR of the original antibody (the parent or donor antibody, e.g. a murine or rat antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. The CDRs of the parent antibody, some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted in whole or in part into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs. The creation of such antibodies is described in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al. , 1988, Science 239:1534-1536. An antigen binding domain can thus have non-human hypervariable regions or CDRs and human frameworks region sequences (optionally with back mutations).
Additionally, a wide range of antibodies are available in the scientific and patent literature, including DNA and/or amino acid sequences, or from commercial suppliers. Antibodies will typically be directed to a pre-determined antigen. Examples of antibodies include antibodies that recognize an antigen expressed by a target cell that is to be eliminated, for example a proliferating cell or a cell contributing to a disease pathology. Examples include antibodies that recognize tumor antigens, microbial (e.g. bacterial or parasite) antigens or viral antigens.
Alternatively, antigen binding domains used in the proteins described herein can be readily derived from any of a variety of non-immunoglobulin scaffolds, for example affibodies based on the Z-domain of staphylococcal protein A, engineered Kunitz domains, monobodies or adnectins based on the 10th extracellular domain of human fibronectin III, anticalins derived from lipocalins, DARPins® (designed ankyrin repeat domains, multimerized LDLR-A module, avimers or cysteine-rich knottin peptides. See, e.g., Gebauer and Skerra (2009) Current Opinion in Chemical Biology 13:245-255, the disclosure of which is incorporated herein by reference.
As further exemplified herein, an antigen binding domain can conveniently comprise a VH and a VL (a VH/VL pair). In some embodiments, the VH/VL pair can be integrated in a Fab structure further comprising a CH1 and CL domain (a CH1/CL pair). A VH/VL pair refers to one VH and one VL domain that associate with one another to form an antigen binding domain. A CH1/CL pair refers to one CH1 and one CL domain bound to one another by covalent or non-covalent interactions, preferably non-covalent interactions, thus forming a heterodimer (e.g., within a protein such as a heterotrimer, heterotetramer, heteropentamer that can comprise one or more further polypeptide chains). The constant chain domains forming the pair can be present on the same or on different polypeptide chain, in any suitable combination.
Exemplary CDRs or VH and VL domains that bind NKp30 can be derived from the anti- NKp30 antibodies provided herein, or can be selected from known anti-NKp30 antibodies, for example the CDRs, VH and VL domains of PCT publication no. WO2019/226617, the disclosure of which is incorporated herein by reference. Variable regions can be used directly, or can be modified by selecting hypervariable or CDR regions from the NKp30 antibodies and placing them into the desired VL or VH framework, for example human frameworks. Antigen binding domains that bind NKp30 can also be derived de novo using methods for generating antibodies. Antibodies can be tested for binding to NKp30 polypeptides. In one aspect of any embodiment herein, a polypeptide (e.g. multispecific protein) that binds to NKp30 will be capable of binding NKp30 expressed on the surface of a cell, e.g. native NKp30 expressed by a NK cell. The antibody can be a function-conservative variant of mAb8. “Function- conservative variants” are those in which a given amino acid residue in a protein (e.g. an antibody or antibody fragment) has been changed without altering the overall conformation and function of the protein, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A “function-conservative variant” also includes a polypeptide which has at least 60% amino acid identity with the antibody capable of specifically binding to a NKp30 polypeptide as defined hereinabove as determined by BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, still preferably at least 90%, and even more preferably at least 95%, and which has the same or substantially similar properties or functions as the antibodies capable of specifically binding to a NKp30 polypeptide as defined hereinabove.
Exemplary anti-NKp30 VH and VL domains can comprise all the amino acid sequence of SEQ ID NO: 249 and SEQ ID NO: 250 respectively. In one example anti-NKp30 VH domain comprises the CDR1, CDR2 and CDR3 of the VH domain of SEQ ID NO: 249, optionally wherein CDR numbering is according to Kabat, Chothia or IMGT. In one example anti-NKp30 VL domain comprises the CDR1, CDR2 and CDR3 of the VL domain of SEQ ID NO: 250, optionally wherein CDR numbering is according to Kabat. Optionally, any of the CDR1 , 2 or 3 can be specified as having an amino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR or set of CDRs.
Antigen binding domains (ABDs) that bind an antigen of interest can be selected based on the desired predetermined antigen of interest (e.g. an antigen other than NKp30), and may include for example cancer antigens such as antigens present on tumor cells and/or on immune cells capable of mediating a pro-tumoral effect, e.g. a monocyte or a macrophage, optionally a suppressor T cell, regulatory T cell, or myeloid-derived suppressor cell (for the treatment of cancer); bacterial or viral antigens (for the treatment of infectious disease); or antigens present on pro-inflammatory immune cells, e.g. T cells, neutrophils, macrophages, etc. (for the treatment of inflammatory and/or autoimmune disorder).
As used herein, the term "bacterial antigen" includes, but is not limited to, intact, attenuated or killed bacteria, any structural or functional bacterial protein or carbohydrate, or any peptide portion of a bacterial protein of sufficient length (typically about 8 amino acids or longer) to be antigenic. Examples include gram-positive bacterial antigens and gram-negative bacterial antigens. In some embodiments the bacterial antigen is derived from a bacterium selected from the group consisting of Helicobacter species, in particular Helicobacter pyloris; Borrelia species, in particular Borrelia burgdorferi ; Legionella species, in particular Legionella pneumophilia ; Mycobacteria s species, in particular M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae ; Staphylococcus species, in particular Staphylococcus aureus; Neisseria species, in particular N. gonorrhoeae, N. meningitidis; Listeria species, in particular Listeria monocytogenes; Streptococcus species, in particular S. pyogenes, S. agalactiae; S. faecalis; S. bovis, S. pneumoniae; anaerobic Streptococcus species; pathogenic Campylobacter species; Enterococcus species; Haemophilus species, in particular Haemophilus influenzae; Bacillus species, in particular Bacillus anthracis; Corynebacterium species, in particular Corynebacterium diphtheriae; Erysipelothrix species, in particular Erysipelothrix rhusiopathiae; Clostridium species, in particular C. perfringens, C. tetani; Enterobacter species, in particular Enterobacter aerogenes, Klebsiella species, in particular Klebsiella 1S pneumoniae, Pasteurella species, in particular Pasteurella multocida, Bacteroides species; Fusobacterium species, in particular Fusobacterium nucleatum; Streptobacillus species, in particular Streptobacillus moniliformis; Treponema species, in particular Treponema pertenue; Leptospira; pathogenic Escherichia species; and Actinomyces species, in particular Actinomyces Israeli.
As used herein, the term "viral antigen" includes, but is not limited to, intact, attenuated or killed whole virus, any structural or functional viral protein, or any peptide portion of a viral protein of sufficient length (typically about 8 amino acids or longer) to be antigenic. Sources of a viral antigen include, but are not limited to viruses from the families: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV- 11 l/LAV, or HIV-Ill; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., Ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses); Bornaviridae ; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), Hepatitis C; Norwalk and related viruses, and astroviruses). Alternatively, a viral antigen may be produced recombinantly.
As used herein, the terms "cancer antigen" and "tumor antigen" are used interchangeably and refer to antigens (other than the cytokine receptor expressed on NK cells, NKp30, and CD16) that are differentially expressed by cancer cells or are expressed by non- tumoral cells (e.g. immune cells) having a pro-tumoral effect (e.g. an immunosuppressive effect), and can thereby be exploited in order to target cancer cells. Cancer antigens can be antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, or expressed at lower levels or less frequently, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Still other cancer antigens can be expressed on immune cells capable of contributing to or mediating a pro-tumoral effect, e.g. cell that contributes to immune evasion, a monocyte or a macrophage, optionally a suppressor T cell, regulatory T cell, or myeloid-derived suppressor cell.
The cancer antigens are usually normal cell surface antigens which are either over expressed or expressed at abnormal times, or are expressed by a targeted population of cells. Ideally the target antigen is expressed only on proliferative cells (e.g., tumor cells) or pro- tumoral cells (e.g. immune cells having an immunosuppressive effect), however this is rarely observed in practice. As a result, target antigens are in many cases selected on the basis of differential expression between proliferative/disease tissue and healthy tissue. Example of cancer antigens include: Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), Crypto, CD4, CD19, CD20, CD30, CD38, CD47, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), a Siglec family member, for example CD22 (Siglec2) or CD33 (Siglec3), CD79, CD123, CD138, CD171 , PSCA, L1-CAM, PSMA (prostate specific membrane antigen), BCMA, CD52, CD56, CD80, CD70, E-selectin, EphB2, Melanotransferrin, Mud 6 and TMEFF2. Examples of cancer antigens also include Immunoglobulin superfamily (IgSF) such as cytokine receptors, Killer-lg Like Receptor, CD28 family proteins, for example, Killer-lg Like Receptor 3DL2 (KIR3DL2), B7-H3, B7-H4, B7-H6, PD-L1. Examples also include MAGE, MART-1/Melan-A, gp100, major histocompatibility complex class l-related chain A and B polypeptides (MICA and MICB), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A/GA733, protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), nectins (e.g. nectin-4), major histocompatibility complex class l-related chain A and B polypeptides (MICA and MICB), proteins of the UL16-binding protein (ULBP) family, proteins of the retinoic acid early transcript-1 (RAET1) family, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate specific antigen (PSA), T-cell receptor/C D3-zeta chain, MAGE-family of tumor antigens, GAGE-family of tumor antigens, anti-Mullerian hormone Type II receptor, delta-like ligand 4 (DLL4), DR5, ROR1 (also known as Receptor Tyrosine Kinase-Like Orphan Receptor 1 or NTRKR1 (EC 2.7.10.1), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC family, VEGF, VEGF receptors, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor, members of the human EGF-like receptor family, e.g., HER-2/neu, HER-3, HER-4 or a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, Muc-1, CA125, integrin receptors, anb3 integrins, a5b1 integrins, allbB3-integrins, PDGF beta receptor, SVE- cadherin, hCG, CSF1R (tumor-associated monocytes and macrophages), a-fetoprotein, E- cadherin, a-catenin, b-catenin and g-catenin, p120ctn, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2, although this is not intended to be exhaustive.
Optionally, a multispecific protein can be specified as excluding or not requiring a stromal modifying moiety, e.g., a moiety capable of altering or degrading a component of, the stroma such as an ECM component, e.g., a glycosaminoglycan, e.g., hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparin sulfate, heparin, entactin, tenascin, aggrecan and keratin sulfate; or an extracellular protein, e.g., collagen, laminin, elastin, fibrinogen, fibronectin, and vitronectin. For example, the stromal modifying moiety can be a hyaluronan degrading enzyme, an agent that inhibits hyaluronan synthesis, or an antibody molecule against hyaluronic acid. Optionally, a multispecific protein can be specified as excluding a mesothelin targeting moiety or mesothelin-binding ABD. Optionally, a multispecific protein can be specified as excluding a PD-L1 targeting moiety, a HER3 targeting moiety, an IGFIR targeting moiety or a hyaluronidase 1 targeting moiety, or a combination a stroma targeting moiety or ABD and a cancer-antigen targeting moiety. Optionally a cancer antigen or antigen of interest can be specified as being other than a PD- L1, a HER3, an IGFIR or hyaluronidase 1.
By way of examples, when the ABD that binds an antigen of interest binds to a HER2 polypeptide, exemplary VH and VL pairs can be selected from antibodies trastuzumab, pertuzumab or margetuximab:
Trastuzumab heavy chain variable region
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS (SEQ ID NO: 2).
Trastuzumab light chain variable region
DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIK (SEQ ID NO: 3).
Margetuximab VH:
QVQLQQSGPE LVKPGASLKL SCTASGFNIK DTYIHWVKQR PEQGLEWIGRIYPTNGYTRY DPKFQDKATI TADTSSNTAY LQVSRLTSED TAVYYCSRWG GDGFYAMDYW GQGASVTVSS (SEQ ID NO: 4).
Margetuximab VL:
DIVMTQSHKF MSTSVGDRVS ITCKASQDVN TAVAWYQQKP GHSPKLLIYS ASFRYTGVPD RFTGSRSGTD FTFTISSVQA EDLAVYYCQQ HYTTPPTFGG GTKVEIK (SEQ ID NO: 5). In another example, when the ABD that binds an antigen of interest binds to a CD19 polypeptide, exemplary VH and VL pairs can be selected from the VH and VL pair from blinatumomab.
Blinatumomab VH:
QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDT NYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQG TTVTVSS (SEQ ID NO: 6).
Blinatumomab VL:
DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGI PPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO: 7).
In another example, when the ABD that binds an antigen of interest binds to a CD20 polypeptide, exemplary VH and VL pairs can be selected from VH and VL pair from rituximab and obinutuzumab :
Rituximab VH:
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTS YNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTV SA (SEQ ID NO: 8).
Rituximab VL:
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFS GSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 9). Obinutuzumab VH:
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTD YNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS (SEQ ID NO: 10).
Obinutuzumab VL:
DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSG VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK (SEQ ID NO: 11).
In another example, when the ABD that binds an antigen of interest binds to a EGFR polypeptide, exemplary VH and VL pairs can be selected from the EGFR-binding VH and VL pair from cetuximab, panitumumab, nimotuzumab, depatuxizumab and necitumumab: Cetuximab VH:
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDY NTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 12).
Cetuximab VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSG SGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 13). Panitumumab VH:
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTN YNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS (SEQ ID NO: 14).
Panitumumab VL:
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF SGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK (SEQ ID NO: 15). Nimotuzumab VH:
QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNF NEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVT VSS (SEQ ID NO: 16).
Nimotuzumab VL:
DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFS GVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQI (SEQ ID NO: 17). Necitumumab VH:
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDY NPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 18).
Necitumumab VL:
EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARF SGSGSGTDFTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAEIK (SEQ ID NO: 19). Depatuxizumab VH:
QVQLQESGPGLVKPSQTLSLTCTVSGYSISSDFAWNWIRQPPGKGLEWMGYISYSGNTRY QPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAGRGFPYWGQGTLVTVSS (SEQ ID NO: 20).
Depatuxizumab VL:
DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGWLQQKPGKSFKGLIYHGTNLDDGVPSR FSGSGSGTDYTLTISSLQPEDFATYYCVQYAQFPWTFGGGTKLEIK (SEQ ID NO: 21).
In another example, when the ABD that binds an antigen of interest binds to a BCMA polypeptide, exemplary VH and VL pairs can be selected from the BCMA-binding VH and VL pair from belantamab, teclistamab, elranatamab or pavurutamab:
Belantamab VH:
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSD TYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVT VSS (SEQ ID NO: 22).
Belantamab VL:
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIK (SEQ ID NO: 23). Pavurutamab VH:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNHIIHWVRQAPGQCLEWMGYINPYPGYHAY NEKFQGRATMTSDTSTSTVYMELSSLRSEDTAVYYCARDGYYRDTDVLDYWGQGTLVTVS S (SEQ ID NO: 24).
Pavurutamab VL:
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLHTGVPSRF SGSGSGTDFTFTISSLEPEDIATYYCQQGNTLPWTFGCGTKVEIK (SEQ ID NO: 25).
In another example, when the ABD that binds an antigen of interest binds to a PD-L1 polypeptide, exemplary VH and VL pairs can be selected from the PD-L1 -binding VH and VL pair from antibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1 B12, 7H1, 11 E6, 12B7, and 13G4 shown in US Patent no. 7,943,743, the disclosure of which is incorporated herein by reference, or of any of the antibodies MPDL3280A (atezolizumab, Tecentriq™, see, e.g., US patent no. 8,217,149, anti-PD-L1 from Roche/Genentech), MDX-1105 (anti-PD-L1 from Bristol-Myers Squibb), MSB0010718C (avelumab; anti-PD-L1 from Pfizer) and MEDI4736 (durvalumab; anti-PD-L1 from AstraZeneca).
In another example, when the ABD that binds an antigen of interest binds to a B7-H3 polypeptide, exemplary VH and VL pairs can be selected from the B7-H3 -binding VH and VL pairs of enoblituzumab, of TRL4542 shown in PCT publication no. W02018/129090, of 8H9 shown in PCT publication no. WO2018/209346, or of any of the antibodies of PCT publication nos. WO2016/106004, W02017/180813, W02019/024911, WO2019/225787,
W02020/063673, W02020/094120, W02020/102779, W02020/140094 and WO2020/151384. Examples of single domain B7H3 ABDs that can be used include Affibody™ formats described in PCT publication W02020/041626 and single domain antibodies (sdAb) of PCT publication nos. W02020/076970 and WO2021/247794. The disclosures of VH, VL and CDRs sequences of the above are incorporated herein by reference.
In another example, when the ABD that binds an antigen of interest binds to a B7-H6 polypeptide, exemplary VH and VL pairs can be selected from the B7-H6-binding VH and VL pairs shown in US Patent nos. US 11,034,766; US 8,822,652; US 9,676,855; US 11,034,766; US 11 ,034,767 or in PCT publication nos. WO2013/037727 or WO2021/064137.
In another example, when the ABD that binds an antigen of interest binds to a B7-H4 polypeptide, exemplary VH and VL pairs can be selected from the B7-H4-binding VH and VL of alsevalimab or the VH and VL pairs shown in US Patent nos. US 10,626,176; US 9,676,854; US 9,574,000; US 10,150,813; US 10,814,011 or in PCT publication nos. W02009/073533, WO2019/165077, WO2019/169212, WO2019/147670, WO2021/155307, W02022/039490, WO2019/154315 or WO2021/185934.
In one embodiment, the ABD that binds an antigen of interest binds to a cancer antigen, a viral antigen, a microbial antigen, or an antigen present on an infected cell (e.g. virally infected) or on a pro-inflammatory immune cell. In one embodiment, said antigen is a polypeptide selectively expressed or overexpressed on a tumor cell, and infected cell or a pro- inflammatory cell. In one embodiment, said antigen is a polypeptide that when inhibited, decreases the proliferation and/or survival of a tumor cell, an infected cell or a pro- inflammatory cell.
The ABDs which are incorporated into the polypeptides can be tested for any desired activity prior to inclusion in a multispecific NKp30-binding protein, for example the ABD can be tested in a suitable format (e.g. as conventional IgG antibody, fab, Fab’2 or scFv) for binding to (e.g. binding affinity) for its binding partner.
An ABD derived from an antibody will generally comprise at minimum a hypervariable region sufficient to confer binding activity. It will be appreciated that an ABD may comprise other amino acids or functional domains as may be desired, including but not limited to linker elements (e.g. linker peptides, CH1 , CK or CA domains, hinges, or fragments thereof). In one example an ABD comprises an scFv, a VH domain and a VL domain, or a single domain antibody (nanobody or dAb) such as a V-NAR domain or a VHH domain. ABDs can be made of a VH and a VL domain that associate with one another to form the ABD.
In one embodiment, one or both of the VH and VL pairs that form an ABD for NKp30 and antigen of interest are within a tandem variable region (a VH and VL domain separated by a flexible polypeptide linker), such as an scFv.
In one embodiment, one or both ABDs for NKp30 and antigen of interest can have a conventional or non-conventional Fab structure. A Fab structure can be characterized as a VH or VL variable domain linked to a CH1 domain and a complementary variable domain (VL or VH, respectively) linked to a complementary CK (or CA) constant domain, wherein the CH1 and CK (or CA) constant domains associate (dimerize). For example a Fab can be formed from a VH-CH1 unit (VH fused to a CH1) on a first polypeptide chain that dimerizes with a VL-CK unit (VL fused to a CK) on a second chain. Alternatively, a Fab can be formed from a VH-CK unit (VH fused to a CK) on a first polypeptide chain that dimerizes with a VL-CH1 unit (VL fused to a CH1) on a second chain.
In some embodiments, one of the ABDs for NKp30 and antigen of interest comprises a Fab structure, in which a variable domain is linked to a CH1 domain and a complementary variable domain is linked to a complementary CK (or CA) constant domain, wherein the CH1 and CK (or CA) constant domains associate to form a heterodimeric protein, and the other ABD comprise or consists of an scFv or a single binding domain (e.g. VhH domain, DARPin). The scFv or a single binding domain can optionally be fused to a CK or CA domain or hinge domain.
The CH1 and/or CK domains can then be linked to a CH2 domain, optionally in each case via a hinge region (or a suitable domain linker). The CH2 domain(s) is/are then linked to a CH3 domain. The CH2-CH3 domains can thus optionally be embodied as a full-length Fc domain (optionally a full-length Fc domain, except that the CH3 domain that lacks the C- terminal lysine).
The CD16 ABD, when present, can readily be embodied as a Fc domain dimer that is capable of binding to human CD16A and optionally other Fey receptors, e.g., CD16B, CD32A, CD32B and/or CD64). In one embodiment, an Fc moiety may be obtained by production of the polypeptide in a host cell or by a process that yields N297-linked glycosylation, e.g. a mammalian cell. In one embodiment, an Fc moiety comprises a human gamma isotype constant region comprising one or more amino acid modifications, e.g. in the CH2 domain, that increases binding to CD16 or CD16A.
The cytokine receptor antigen binding domain can readily be embodied as a cytokine, (e.g. a type 1 cytokine such as an IL-2, IL-15, IL-21 , IL-7, IL-27 or IL-12 cytokine, an IL-18 cytokine or a type 1 interferon such as IFN-a or IFN-b). Exemplary cytokine receptor ABDs and modified cytokines are further described herein.
Once appropriate antigen binding domains having desired specificity and/or activity are identified, nucleic acids encoding each of the or ABD can be separately placed, in suitable arrangements, in an appropriate expression vector or set of vectors, together with DNA encoding any elements such as CH1, CK, CH2 and CH3 domains or portions thereof, mutant IL2 polypeptides and any other optional elements (e.g. DNA encoding a hinge-derived or linker elements) for transfection into an appropriate host. ABDs will be arranged in an expression vector, or in separate vectors as a function of which type of polypeptide is to be produced, so as to produce the Fc-polypeptides having the desired domains operably linked to one another. The host is then used for the recombinant production of the multispecific polypeptide.
For example, a polypeptide fusion product can be produced from a vector in which one ABD or a part thereof (e.g. a VH, VL or a VH/VL pair) is operably linked (e.g. directly, or via a CH1 , CK or CA constant region and/or hinge region) to the N-terminus of a CH2 domain, and the CH2 domain is operably linked at its C-terminus to the N-terminus a CH3 domain. Another ABD or part thereof can be on a second polypeptide chain that forms a dimer, e.g. heterodimer, with the polypeptide comprising the first ABD. The multispecific polypeptide can then be produced in an appropriate host cell or by any suitable synthetic process. A host cell chosen for expression of the multispecific polypeptide is an important contributor to the final composition, including, without limitation, the variation in composition of the oligosaccharide moieties decorating the protein in the immunoglobulin CH2 domain. Thus, one aspect of the invention involves the selection of appropriate host cells for use and/or development of a production cell expressing the desired therapeutic protein such that the multispecific polypeptide retains FcRn and CD16 binding. The host cell may be of mammalian origin or may be selected from COS-1 , COS-7, HEK293, BHK21, CHO, BSC-1 , Hep G2, 653, SP2/0, 293, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. The host cell may be any suitable species or organism capable of producing N-linked glycosylated polypeptides, e.g. a mammalian host cell capable of producing human or rodent IgG type N-linked glycosylation.
Protein formats
Multimeric, multispecific proteins such as heterodimers, heterotrimers and hetero- tetramers can be produced according to a variety of formats. Different domains onto different polypeptide chain that associate to form a multimeric protein. Accordingly, a wide range of protein formats can be constructed around Fc domain dimers that are capable of binding to human FcRn polypeptide (neonatal Fc receptor), with or without additionally binding to CD16 or CD16A and optionally other Fey receptors, e.g., CD16B, CD32A, CD32B and/or CD64), depending on whether or not the CD16 binding ABD is desired to be present. As shown herein, greatest potentiation of NK cell cytotoxicity can be obtained through use of Fc moieties that have substantial binding to the activating human CD16 receptor (CD16A) binding; such CD16 binding can be obtained through the use of suitable CH2 and/or CH3 domains, as further described herein. In one embodiment, an Fc moiety is derived from a human lgG1 isotype constant region. Use of modified CH3 domains also contributes to the possibility of use a wide range of heteromultimeric protein structures. Accordingly, a protein comprises a first and a second polypeptide chain each comprising a variable domain fused to a human Fc domain monomer (i.e. a CH2-CH3 unit), optionally a Fc domain monomercomprising a CH3 domain capable of undergoing preferential CH3-CH3 hetero-dimerization, wherein the first and second chain associate via CH3-CH3 dimerization and the protein consequently comprises a Fc domain dimer. The variable domains of each chain can be part of the same or different antigen binding domains.
Multispecific proteins can thus be conveniently constructed using VH and VL pairs arranged as scFv or Fab structures, together with CH1 domains, CL domain, Fc domains and cytokines, and domain linkers. Preferably, the proteins will use minimal non-natural sequences, e.g. minimal use of non-lg linkers, optionally no more than 5, 4, 3, 2 or 1 domain linker(s) that is not an antibody-derived sequence, optionally wherein domain linker(s) are no more than 15, 10 or 5 amino acid residues in length. In one embodiment, the CD16 ABD is a Fc domain dimer.
Fc domain) and NKp30 ABD are immediately adjacent to other another within the protein, and NKp30 ABD is immediately adjacent to the cytokine receptor ABD (embodied as a cytokine (Cyt)), and wherein the NKp30 ABD is interposed between the CD16 ABD and the Cyt:
(CD16 ABD) (NKp30 ABD) (Cyt) (Structure 1a) or
(Fc domain dimer) (NKp30 ABD) (Cyt) (Structure 1b) wherein the NKp30 ABD and the CD16ABD (e.g. Fc domain dimer) are connected by a domain linker and the NKp30 ABD and Cyt are connected by a domain linker.
In some embodiment, the multispecific proteins (e.g. dimers, trimers, tetramers) may comprise a domain arrangement of any of the following in which domains can be placed on any of the 2, 3 or 4 polypeptide chains, wherein the NKp30 ABD is interposed between the Fc domain and the cytokine receptor ABD (e.g. the protein has a terminal or distal cytokine receptor ABD at the C-terminal end and a terminal or distal antigen of interest (Antigen) ABD at the topological N-terminal end), wherein the NKp30 ABD is connected to one of the polypeptide chains of the Fc domain dimer via a hinge polypeptide or a flexible linker, and wherein the ABD that binds the cytokine receptor is connected to NKp30 ABD (e.g. to one of the polypeptide chains thereof when the NKp30 ABD is contained on two chains) via a flexible linker (e.g. a linker comprising G and S residues), wherein “n” is 1 or 2:
(Anti-Antigen ABD)n - (Fc domain dimer) - (NKp30 ABD) - (cytokine receptor ABD).
In other configurations, the protein comprises an ABD that binds an antigen of interest on a target cell (Antigen ABD) and the ABD that binds NKp30 both placed topologically N- terminal to the Fc domain dimer, as in a heteromultimeric protein having the structure below:
(NKp30 ABD) (Fc domain dimer) (Cyt) wherein the Antigen ABD and the Fc domain dimer are connected by a linker or immunoglobulin hinge polypeptide, wherein the NKp30 ABD and the Fc domain dimer are connected by a linker or immunoglobulin hinge polypeptide, and wherein the Fc domain dimer and Cyt are connected by a linker.
The cytokine receptor ABD can be an IL2, IL15, IL18, IL21 or IFN-a polypeptide. The Fc domain can be specified to be an Fc domain dimer (e.g. that binds human FcRn and/or Fey receptors). In one embodiment, one or both of the antigen of interest (e.g. cancer antigen) ABD and NKp30 ABD is formed from two variable regions present within tandem variable regions, wherein the variable regions that associate to form a particular ABD can be on the same polypeptide chain or on different polypeptide chains. In another embodiment, one or both of the antigen of interest ABD and NKp30 ABD comprises a tandem variable region and the other comprises a Fab structure. In another embodiment, both of the antigen of interest and NKp30 ABD comprises a Fab structure. In another embodiment one of the antigen of interest and NKp30 ABD comprises a Fab structure and the other comprises an scFv structure.
Heterodimers and heterotrimers
The present disclosure provides advantageous approaches of making multimeric multispecific proteins which bind to the antigen of interest (monovalently or bivalently) and monovalently to each of NKp30, CD16A and cytokine receptor. The approaches readily allow domain configurations where the NKp30 ABD is positioned between the Fc domain and a cytokine polypeptide. These configurations can be achieved through the assembly of different polypeptide chains described herein that each comprise at least one heavy or light chain variable domain fused to a human CH1 or CK constant domain (a V-(CH1/CK) unit), wherein the protein chains undergo CH1-CK dimerization and are bound to one another by non- covalent bonds and optionally further disulfide bonds formed between respective CH1 and CK domains.
Exemplary heterodimeric or heterotri meric polypeptides have an NKp30 binding ABD, an antigen of interest binding ABD, a cytokine receptor-binding ABD (e.g. IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b polypeptide) and a Fc domain dimer can optionally be produced as one or more chains that each associate with a central chain, e.g. by CH1-CK heterodimerization and/or by CH3-CH3 dimerization. Different variants can be produced, as illustrated in the Examples herein.
In one embodiment, an isolated or purified heterodimeric or heterotri meric protein comprises at least two or three polypeptide chains, each comprising a V-(CH1/CK) unit, whereby the chains are bound to one another by non-covalent interactions and optionally further bound via disulfide bonds between CH1 and CK domains, and still further optionally, whereby the chains are bound by non-covalent interactions between the respective variable regions, CH1 and CK domains, and CH3 domains of the Fc portion.
In one example, the protein comprises a first and a second polypeptide chain each comprising a variable domain fused to a CH1 or CK domain (a V-(CH1/CK) unit), in turn fused at its C-terminus to a human Fc domain monomer comprising a CH2 domain and a CH3 domain capable of undergoing CH3-CH3 dimerization, wherein the first and second chain associate via CH1-CK and CH3-CH3 dimerization such that the protein comprises a Fcdomain dimer. The variable domains of each chain can be part of the same or different antigen binding domains.
The variable and constant regions can be selected and configured such that each chain will preferentially associate with its desired complementary partner chain. The resulting multimeric protein can be produced reliably and with high productivity using conventional production methods using recombinant host cells. The choice of which VH or VL to associate with a CH1 and CK in a unit is based on affinity between the units to be paired so as to drive the formation of the desired multimer. The resulting multimer will be bound by non-covalent interactions between complementary VH and VL domains, by non-covalent interactions between complementary CH1 and CK domains, and optionally by further disulfide bonding between complementary CH 1 and CK domains (and optionally further disulfide bonds between complementary hinge domains). VH-VL associations are stronger than VH-VH or VL-VL, consequently, as shown herein, one can place a VH or a VL next to either a CH1 or a CK, and the resulting V-C unit will partner preferably with its V-C counterpart. For example VH-CK will pair with VL-CH1 preferentially over VH-CH1. Additionally, by including an Fc domain, preferred chain pairing is further improved, as the two Fc monomer-containing chains are bound by non- covalent interactions between CH3 domains of the Fc domain monomers. The different V-C combinations, optionally further combined with Fc pairing thereby provides tools to make heteromultimeric proteins comprising a cytokine (e.g. IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL- 18, IFN-a or IFN-b polypeptide), represented as “Cyt” in the domain arrangements.
In one example, the multispecific protein is a heterodimer comprising a first and a second polypeptide chain each comprising a variable domain fused to a CH1 or CK domain (a V-(CH1/CK) unit), in turn fused at its C-terminus to a human Fc domain monomer, wherein the V-(CH1/CK) unit of the first chain has undergone CH1-CK dimerization with the V-(CH1/CK) unit of the second chain thereby forming a first antigen binding domain (ABDi) and a Fc domain dimer, wherein one of the polypeptide chains further comprises an antigen binding domain that forms a second antigen binding domain (ABD2), and wherein the Fc domain dimer binds to a human CD16 polypeptide, wherein one of ABDi and ABD2 binds NKp30 and the other binds the antigen of interest (e.g. tumor antigen).
In one example, the protein has a domain arrangement:
Vai - (CH1 or CK)a- (hinge or linker) - Fc domain - Va2 - Vb2- linker -Cyt (chain 1)
I
Vbi - (CH1 or CK)b- (hinge or linker) - Fc domain (chain 2) wherein Va-i , VM, Va-2 and Vb-2 are each a VH domain or a VL domain, and wherein one of Va-i and \ -i is a VH and the other is a VL such that Va-i and Vb-1 form a first antigen binding domain (ABD), wherein one of Va-2and Vb-2 is a VH and the other is a VL such that Va-2and Vb- 2 form a second antigen binding domain, wherein one of the ABD binds NKp30 and the other binds an antigen of interest.
In other examples, the protein is a heterotrimer and comprises three polypeptide chains, each comprising a variable domain fused to a CH1 or CK domain (a V-(CH1/CK) unit), wherein a first (central) chain comprises two V-(CH1/CK) units and a human Fc domain interposed between the units, the second chain comprises one V-(CH1/CK) unit and a human Fc domain monomer, and the third chain comprises one V-(CH1/CK) unit and a cytokine polypeptide (Cyt), wherein one of the V-(CH1/CK) units of the central chain has undergone CH1-CK dimerization with the V-(CH1/CK) unit of the second chain thereby forming a first antigen binding domain (ABDi) and a Fc domain dimer, and wherein the other of the V- (CH1/CK) units of the central chain has undergone CH1-CK dimerization with the V-(CH1/CK) unit of the third chain thereby forming a second antigen binding domain (ABD2), and wherein the Fc domain binds to a human CD16 polypeptide. In one embodiment, the Fc domain comprises N-linked glycosylation at residue N297 (Kabat EU numbering).
In one example, the protein has a domain arrangement:
VK - CK - Fc domain (second polypeptide)
I
VH - CH1 - Fc domain - VH - CK (first polypeptide)
I
VK - CH1 - Cyt (third polypeptide).
In another example, the protein has a domain arrangement:
VK - CK - Fc domain (second polypeptide)
I
VH - CH1 - Fc domain - VH - CH1 (first polypeptide)
I
VK - CK- Cyt (third polypeptide).
In one specific example of the above heterotrimeric proteins, the Fab structure interposed between the Fc domain and the cytokine is the NKp30 binding ABD (i.e. the NKp30 binding ABD is interposed between the Fc domain and the C-terminal cytokine). The Fc domain in the first polypeptide is connected (e.g. fused) at its C terminus to the N-terminus of the VH domain via a linker. The constant domain (the CH1 or CK domain in the respective domain arrangements) in the third polypeptide is connected (e.g. fused) at its C terminus to the N-terminus of the cytokine polypeptide via a linker. Each constant domain (the CH1 or CK domain in the respective domain arrangements) that is N-terminal to the Fc domain is fused at the C terminus of the constant domain to the N-terminus of the Fc domain via a hinge region.
Optionally, any of the multispecific proteins of the invention may include CH1, CL or CH3 domains which comprise amino acid modifications (e.g. substitutions) to promote heterodimerization. For example, heterodimerization modifications often involve steric repulsion, charge steering interaction, or interchain disulfide bond formation, wherein the CH3 domain interface of the antibody Fc region is mutated to create altered charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc chains supports favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation.
In one example, the first (central) polypeptide chain will provide one variable domain that will, together with a complementary variable domain on a second polypeptide chain, form a first antigen binding domain (e.g. the ABD that binds the antigen of interest), and an Fc domain. The first (central) polypeptide chain will also provide a second variable domain (e.g., placed on the opposite end of the interposed Fc domain from the first variable domain, at the C-terminus of the Fc domain) that will be paired with a complementary variable domain to form a second antigen binding domain (e.g. the ABD that binds NKp30); the variable domain that is complementary to the second variable domain can be placed on the central polypeptide (e.g. adjacent to the second variable domain in a tandem variable domain construct such as an scFv), or can be placed on a separate polypeptide chain, notably a third polypeptide chain. The second (and third, if present) polypeptide chains will associate with the central polypeptide chain by CH1-CK heterodimerization, forming non-covalent interactions and optionally further interchain disulfide bonds between complementary CH1 and CK domains (and optionally interchain disulfide bonds between hinge regions), with a primary multimeric polypeptide being formed so long as CH/CK and VH/VK domains are chosen to give rise to a preferred dimerization configuration that results preferentially in the desired VH-VL pairings. Remaining unwanted pairings can remain minimal during production and/or are removed during purification steps. In a trimer, or when polypeptides are constructed for preparation of a trimer, there will generally be one polypeptide chain that comprises a non-naturally occurring VH-CK or VK-CH1 domain arrangement. A cytokine (e.g., IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b) can then be placed at the C-terminus of one of polypeptide chains. The cytokine can be fused via a domain linker, and while not shown in certain domain arrangements herein, any domain arrangement can be specified as comprising a domain linker separating two domains. For example, in these structures, the cytokine can be placed at the C-terminus of the first (central) polypeptide chain or at the C-terminus of the third polypeptide chain (when such third chain is present).
Examples of the domain arrangements (N- to C-terminus, left to right) of central polypeptide chains for use in heterodimeric proteins in which the NKp30 ABD is interposed between the Fc domain and cytokine (Cyt) and wherein the cytokine moiety is placed on a different polypeptide chain (e.g. the second polypeptide chain) include any of the following, wherein each V is a variable domain:
Va-i - (CH1 or CK) - Fc domain - Va-2 - Vb-2 (first/central chain) or
Va-i - Vb-i - Fc domain - Va-2 - (CH1 or CK) (first/central chain)
Further examples of the domain arrangements of central polypeptide chains for use in heterodimeric proteins wherein the cytokine moiety is placed on the central chain include:
Va-i - (CH1 or CK) - FC domain - Va-2 - Vb-2 - Cyt (first/central chain) or
Va-i - Vb-i - Fc domain - Va-2 - (CH1 or CK) - Cyt (first/central chain)
In the above examples, when V domains are arranged immediately adjacent to one another in tandem on a chain, one of the V is a light chain and the other V is heavy chain variable domain, and the two V domains are separated by a flexible polypeptide linker and together form a scFv.
Further examples of central polypeptide chains include:
Va-i - (CH1 or CK)a — Fc domain - V2- Cyt or
V2 - Fc domain - Va-i - (CH1 or CK) or
V2 - Fc domain - Va-i - (CH 1 or CK) - Cyt wherein V2 is a single domain ABD (e.g. dAb, VHH, DARPin).
The Fc domain of the central chain may be a full Fc domain or a portion thereof sufficient to confer the desired functionality (e.g. binding to FcRn, binding to CD16, CH3-CH3 dimerization) when it forms a dimeric Fc with the Fc domain of a second polypeptide chain.
A second polypeptide chain can then be configured which will comprise an immunoglobulin variable domain and a CH1 or CK constant region, e.g., a (CH1 or CK)b unit, selected so as to permit CH1-CK heterodimerization with the central polypeptide chain; the immunoglobulin variable domain will be selected so as to complement the variable domain of the central chain that is adjacent to the CH1 or CK domain, whereby the complementary variable domains form an antigen binding domain for a first antigen of interest.
For example, a second polypeptide chain for use in a protein in which the NKp30 ABD is interposed between Fc and cytokine can comprise a domain arrangement:
Vb-i - (CH1 or CK)b- Fc domain such that the (CH1 or CK)b dimerizes with the (CH1 or CK)3 on the central chain, and the Vb-i forms an antigen binding domain together with Va-i of the central chain. If the Va-i of the central chain is a light chain variable domain, then Vw will be a heavy chain variable domain; and if Va-i of the central chain is a heavy chain variable domain, then Vw will be a light chain variable domain. Where the first chain does not comprise a Cyt, the second chain can comprise a Cyt fused to its C-terminus (e.g., via a domain linker).
In heterodimers, the antigen binding domain for the second antigen of interest can then be formed from Va-2 and Vb-2 which are configured as tandem variable domains on the central chain forming an ABD (e.g. forming an scFv unit).
The resulting heterodimer can, for example, have the following configuration (see further examples of such proteins shown as formats T13 and T13A shown in Figure 2:
Va-i - (CH1 or CK)3- FC domain - Va-2 - Vb-2 - Cyt (first/central polypeptide chain)
I
Vb-i - (CH1 or CK)b- Fc domain (second polypeptide chain) wherein one of Va-i of the first polypeptide chain and VM of the second polypeptide chain is a light chain variable domain and the other is a heavy chain variable domain, and wherein one of Va-2 and Vb-2 is a light chain variable domain and the other is a heavy chain variable domain. Va-2 and Vb-2 can be specified as being separated by a polypeptide linker (Va-2and Vb-2 form an scFv). Va-2and Vb-2 forms the ABD that binds NKp30 and Va-i and VM forms the ABD that binds the antigen of interest (e.g. cancer antigen).
Examples of domain arrangements of heteromultimeric proteins include the following, optionally wherein one or both of the hinge domains are replaced by a flexible linker polypeptide, wherein the NKp30 ABD is an scFv or a single domain ABD (e.g. dAb, VHH, DARPin) and the Fc domain is fused to the NKp30 ABD a linker polypeptide, and wherein the NKp30 ABD is fused to the cytokine polypeptide by a domain linker (e.g. a flexible polypeptide linker):
(VK-CK - hinge) - Fc domain - (NKp30 ABD) - Cyt (VH-CH1- hinge) - Fc domain (VH-CH1- hinge) - Fc domain - (NKp30 ABD) - Cyt (VK-CK- hinge) - Fc domain or
(VK-CH1- hinge) - Fc domain - (NKp30 ABD) - Cyt
(VH-CK- hinge) - Fc domain
Heterotrimeric proteins in which the NKp30 ABD is interposed between the Fc domain and the cytokine polypeptide can for example be formed by using a central (first) polypeptide chain comprising a first variable domain (V) fused to a first CH1 or CK constant region, a second variable domain (V) fused to a second CH1 or CK constant region, and an Fc domain or portion thereof interposed between the first and second variable domains (i.e. the Fc domain is interposed between the first and second (V-(CH1/CK) units. For example, a central polypeptide chain for use in a heterotrimeric protein according to the invention can have the domain arrangements (N- to C- termini) as follows:
Va-i - (CH1 or CK)3- FC domain - Va.2 - (CH1 or CK
The first polypeptide chain can optionally further have a Cyt is placed at its C-terminus.
A second polypeptide chain can then comprise a domain arrangement (N- to C- termini from left to right):
Vb-i - (CH1 or CK)c- FC domain such that the (CH1 or CK)C dimerizes with the (CH1 or CK)a on the central chain, and the Va-i and Vn form an antigen binding domain that binds the antigen of interest.
A third polypeptide chain can then comprise the following domain arrangement (N- to C- termini from left to right):
Vb-2 - (CH1 or CK)d - Cyt. such that the (CH1 or CK)d dimerizes with the (CH1 or CK)b unit on the central chain, and the Va-2and Vb-2 form the NKp30 binding domain.
Optionally, where the Cyt is placed at the C-terminus of the first polypeptide chain, the third polypeptide chain can then comprise the following domain arrangement (N- to C- termini from left to right):
Vb-2 - (CH1 or CK)d.
An example of a domain configuration of a resulting heterotrimer where the Cyt is placed on the third polypeptide chain (also shown as formats T5, T6 in Figure 2) is shown below:
Vb-i - (CH1 or CK)C - Fc domain (second polypeptide) Va-i - (CH1 or CK)3- FC domain - Va.2 - (CH1 or CK)b (first polypeptide)
I
Vb-2 - (CH1 or CK)d- Cyt (third polypeptide).
A domain configuration of a resulting heterotrimer where the Cyt is placed on the first polypeptide chain is shown below:
Vb-i - (CH1 or CK)C - Fc domain (second polypeptide)
I
Va-i - (CH1 or CK)3- FC domain - Va.2 - (CH1 or CK)b- Cyt (first polypeptide)
I
Vb-2 - (CH1 or CK)d (third polypeptide).
Thus, in a trimeric polypeptide in which the NKp30 ABD is interposed between the Fc domain and the cytokine polypeptide, the first polypeptide can have two variable domains that each form an antigen binding domain with a variable domain on a separate polypeptide chain (i.e. the variable domain of the second and third chains), the second polypeptide chain has one variable domain, and the third polypeptide has one variable domain, and one of the polypeptide chains comprises a cytokine polypeptide fused to its C-terminus.
A trimeric polypeptide can optionally comprise three polypeptide chains characterized as follows:
(a) a first polypeptide chain comprising from N-terminus to C-terminus: a first variable domain (V) fused to a first CH1 or CK constant region, a hinge domain or portion thereof, an Fc domain or portion thereof, and a second variable domain (V) fused to a second CH1 or CK constant region;
(b) a second polypeptide chain comprising from N-terminus to C-terminus: a variable domain fused to a CH1 or CK constant region selected to be complementary to the first CH1 or CK constant region of the first polypeptide chain such that the first and second polypeptides form a CH1-CK heterodimer, a hinge domain or portion thereof, and an Fc domain; and
(c) a third polypeptide chain comprising from N-terminus to C-terminus: a variable domain fused to a CH1 or CK constant region, and a cytokine polypeptide (e.g. fused to the constant region via a flexible polypeptide linker), wherein the variable domain and the constant region are selected to be complementary to the second variable domain and second CH1 or CK constant region of the first polypeptide chain such that the first and third polypeptides form a CH1-CK heterodimer bound by non-covalent interactions and optionally further disulfide bond(s) formed between the CH 1 or CK constant region of the third polypeptide and the second CH1 or CK constant region of the first polypeptide, but not between the CH1 or CK constant region of the third polypeptide and the first CH1 or CK constant region of the first polypeptide wherein the first, second and third polypeptides form a heterotrimer, and wherein the first variable domain of the first polypeptide chain and the variable domain of the second polypeptide chain form an antigen binding domain specific for an antigen of interest (e.g. on a target cell, a cancer antigen), and the second variable domain of the first polypeptide chain and the variable domain on the third polypeptide chain form an antigen binding domain that binds NKp30.
Examples of potential domain arrangements for such trimeric bispecific polypeptides include but are not limited to those shown in Table 1 below:
Table 1
Figure imgf000060_0001
In another aspect, a trimeric polypeptide can optionally be characterized as comprising three polypeptide chains:
(a) a first polypeptide chain comprising from N-terminus to C-terminus: a first variable domain (V) fused to a first CH1 or CK constant region, a hinge domain or portion thereof, an Fc domain or portion thereof, and a second variable domain (V) fused to a second CH1 or CK constant region, and a cytokine polypeptide (e.g. fused to the second CH1 or CK constant region via a flexible polypeptide linker);
(b) a second polypeptide chain comprising from N-terminus to C-terminus: a variable domain fused to a CH1 or CK constant region selected to be complementary to the first CH1 or CK constant region of the first polypeptide chain such that the first and second polypeptides form a C -CK heterodimer, a hinge domain or portion thereof, and an Fc domain; and (c) a third polypeptide chain comprising from N-terminus to C-terminus: a variable domain fused to a CH1 or CK constant region, wherein the variable domain and the constant region are selected to be complementary to the second variable domain and second CH1 or CK constant region of the first polypeptide chain such that the first and third polypeptides form a CH1-CK heterodimer bound by non-covalent interactions and optionally further disulfide bond(s) formed between the CH 1 or CK constant region of the third polypeptide and the second CH1 or CK constant region of the first polypeptide, but not between the CH1 or CK constant region of the third polypeptide and the first CH1 or CK constant region of the first polypeptide wherein the first, second and third polypeptides form a CH1-CK heterotrimer, and wherein the first variable domain of the first polypeptide chain and the variable domain of the second polypeptide chain form an antigen binding domain specific for an antigen of interest (e.g. on a target cell, a cancer antigen), and the second variable domain of the first polypeptide chain and the variable domain on the third polypeptide chain form an antigen binding domain that binds NKp30. Examples of potential domain arrangements for such trimeric bispecific polypeptides include but are not limited to those shown in Table 2 below:
Table 2
Figure imgf000062_0001
Using similar architecture, yet further multispecific proteins can be produced that binds the antigen of interest bivalently, binds NKp30 monovalently and binds the cytokine receptor monovalently, i.e., the multispecific protein has a 2:1:1 configuration. Examples are shown in
Figure 2.
In one example of a multispecific protein having two ABDs that each binds an antigen of interest, a heterodimer protein comprises the domain arrangement:
ABD2 - (CH1 or Ci )b - Fc domain (second polypeptide)
I
ABD3 - (CH1 or CK)3- FC domain - ABDI - Cyt (first polypeptide)
In these structures, the Fc domains of the first and second chains associate via CH3-CH3 dimerization, and (CH1 or Ci )b on the second chain and the (CH1 or CK)3 on the first chain undergo CH1- CK dimerization, wherein ABDi that binds NKp30 and ABD2 and ABD3 are each self-contained antigen binding domains that can bind an antigen of interest (e.g. a cancer antigen) without association with a complementary domain on a different polypeptide chain, wherein each (CH1 or CK)b and (CH1 or CK)3 is fused to the Fc domain via an immunoglobulin hinge amino acid sequence, and wherein Cyt is a cytokine polypeptide (e.g. fused to the ABDi via a flexible polypeptide linker). Another representation of the heterodimeric protein is:
Va2 - Vb2 - (CH1 or CK)b - Fc domain (second polypeptide)
I
Va3 - Vb3 - (CH1 or CK)3- FC domain - Vai - Vbi - Cyt (first polypeptide) where each V domain pair Vai - Vbi , Va2 - Vb2 and Va3 - Vb3 comprises a VH and VL fused via a domain linker such the pair forms an scFv antigen binding domain. In an example of a multispecific protein having two ABDs that each binds an antigen of interest, a heterotrimer protein comprises the domain arrangement:
ABD2 - (CH1 or CK)C - Fc domain (third polypeptide)
I
ABD3 - (CH1 or CK)3- FC domain - Vai - (CH1 or CK)b (first polypeptide)
I
Vbi - (CH1 or CK)CI- Cyt (second polypeptide) or
ABD2 - (CH1 or CK)C - Fc domain (third polypeptide)
I
ABD3 - (CH1 or CK)3- FC domain - Vai - (CH1 or Ci )b- Cyt (first polypeptide)
I
Vbi - (CH1 or CK)d (second polypeptide)
In these structures, the Fc domains of the first and third chains associate via CH3-CH3 dimerization, (CH1 orCi )c On the third chain and the (CH1 orCi )a On the central chain undergo CH1- CK dimerization, and the (CH1 or Ci )b on the first chain and the (CH1 or Ci )d on the second chain undergo CH1- CK dimerization. The Vai and \ i form a first antigen binding domain that bind NKp30. ABD2 and ABD3 are each self-contained antigen binding domains that can bind to an antigen of interest (e.g. a cancer antigen), e.g. without requiring association with a complementary variable domain on a different polypeptide chain. ABD2 and ABDs can for example each comprise a single domain ABD or a VH and a VK pair (in any desired order), placed on a single chain and separated by a flexible peptide linker (e.g. as an scFv), such that a heterotrimer protein can comprise:
VK - VH - (CH1 or CK)C - Fc domain (third polypeptide)
I
VK - VH - (CH1 or CK)3- Fc domain - Vai - (CH1 or CK)b (first polypeptide)
I
Vbi - (CH1 or CK)CI- Cyt (second polypeptide)
VH - VK - (CH1 or CK)C - Fc domain (third polypeptide)
I
VH - VK - (CH1 or CK)3- FC domain - Va1 - (CH1 or CK) (first polypeptide)
Vbi - (CH1 or CK)CI- Cyt (second polypeptide)
Examples of possible configurations of a resulting heterotrimer are structures having domain arrangement, from N- to C-terminus: VK - VH - CK - Fc domain (third polypeptide)
I
VK - VH - CH1 - Fc domain - VHi -CK (first polypeptide)
I
VKi -CH1 - Cyt (second polypeptide) or
VH - VK - CK - Fc domain (third polypeptide)
I
VH - VK - CH1 - Fc domain - VHi -CK (first polypeptide)
I
VKi -CH1 - Cyt (second polypeptide) or
VK - VH - CK - Fc domain (third polypeptide)
I
VK - VH - CH1 - Fc domain - VKi -CK (first polypeptide)
I
VHi -CH1 - Cyt (second polypeptide) or
VH - VK - CK - Fc domain (third polypeptide)
I
VH - VK - CH1 - Fc domain - VKi -CK (first polypeptide)
I
VHi -CH1 - Cyt (second polypeptide) or
VH - VK - CK - Fc domain (third polypeptide)
I
VH - VK - CH1 - Fc domain - VKi -CH1 (first polypeptide)
I
VHi -CK - Cyt (second polypeptide) or
VK - VH - CK - Fc domain (third polypeptide)
I
VK - VH - CH1 - Fc domain - VKi -CH1 (first polypeptide)
I
VHi -CK - Cyt (second polypeptide) or
VH - VK - CH1 - Fc domain (third polypeptide)
I
VH - VK - CK- FC domain - VHi -CK (first polypeptide)
I
VKi -CH1 - Cyt (second polypeptide) or
VK- VH - CH1 - Fc domain (third polypeptide)
I
VK - VH - CK- FC domain - VHi -CK (first polypeptide)
I
VKi -CH1 - Cyt (second polypeptide) or
VK- VH - CH1 - Fc domain (third polypeptide)
I
VK - VH - CK - FC domain - VKi -CK (first polypeptide)
I
VHi -CH1 - Cyt (second polypeptide) or
VH - VK- CH1 - Fc domain (third polypeptide)
I
VH - VK - CK- FC domain - VKi -CK (first polypeptide)
I
VHi -CH1 - Cyt (second polypeptide) or
VH - VK- CH1 - Fc domain (third polypeptide)
I
VH - VK - CK- FC domain - VKi -CH1 (first polypeptide)
I
VHi -CK - Cyt (second polypeptide). or
VK- VH - CH1 - Fc domain (third polypeptide)
I
VK - VH - CK- FC domain - VKi -CH1 (first polypeptide)
I
VHi -CK - Cyt (second polypeptide).
In another example of a multispecific protein having two ABDs that each binds an antigen of interest, a heterotetramer protein can be constructed in which the NKp30 ABD is interposed between the Fc domain and the cytokine, for example molecules having the following domain arrangement: (fourth chain) domain (second chain) domain - Vai - Vbi - Cyt (first chain)
Figure imgf000065_0001
(Vb-2- (CH1 or CK) (third chain) wherein the first chain and the second chain associate by CH3-CH3 dimerization and the first chain and the third chain associate by the CH1 or CK dimerization, wherein the domains of the first chain and the third chain are selected to be complementary to permit the first and third chains to associate by CH1-CK dimerization, wherein the domains of the second chain and the fourth chain are selected to be complementary to permit the second and fourth chains to associate by CH1-CK dimerization, and wherein each V domain pair Vai and Vbi , Va2 and Vb2 and Va3 and Vb3 comprises a VH and a VL such the pair forms ABD, wherein Va-i and Vn are separated by a domain linker to form an scFv that binds NKp30, and wherein the Vai and Vbi pair and the Va2 and Vb2 pair each forms and ABD that binds an antigen of interest.
Examples of such configurations include shown in the Table 3 below:
Table 3
Figure imgf000066_0001
The domain arrangements can also be respectively represented as follows, in which each L is a domain linker:
(Vb-3- (CH1 or CK) (Chain 4) l
(Va-3 — (CH1 or CK) - (hinge or L) - CH2 - CH3 (Chain 2) l
(Va-2 — (CH1 or CK) - (hinge or L) - CH2 - CH3 -L- Va1 - V i - L - Cyt (Chain 1) I
(Vb-2- (CH1 or CK) (Chain 3)
In other configurations, multimeric proteins with the Fc domain interposed between NKp30 ABD and the cytokine receptor ABD can be constructed. Such proteins may be composed of a central (first) polypeptide chain comprising one or two immunoglobulin variable domains, connected or fused, optionally via a CH1 or CL constant region or via a linker, to the N-terminus of an Fc domain, wherein the Fc domain is connected at its C-terminus to a cytokine polypeptide (e.g. to the C-terminus of the cytokine polypeptide. When only one variable domain is present on the central chain, an additional polypeptide chain (e.g. a light chain comprising a V and C domain, optionally a VK and a CK domain) will provide the complementary variable region to form and ABD with the variable domain of the central chain. When two variable domains are present on the central chain they can be arranged as an scFv to form an ABD. A further one or two additional chains (i.e. the third and fourth chain) can then provide at least one further ABD.
Thus, in addition to the first/central chain, a further one, two or three polypeptide chains will provide the complementary Fc domain and variable domains depending on whether the ABDs are configured as scFv or Fab. In this configuration, the Fc domain is interposed between the NKp30 binding ABD and the cytokine polypeptide in the multispecific protein. In this way, heterodimeric, heterotrimeric or heterotetrameric multispecific proteins can be constructed.
Examples of the domain arrangements (N- to C-termini, left to right) of central polypeptide chains include any of the following, wherein each V is a variable domain:
(Va-i - (CH1 or CL) - (hinge or linker) - CH2 - CH3 - linker - Cyt (first/central chain) or
(Va-2 - Vb-2 ) - (hinge or linker) - CH2 - CH3 - linker - Cyt (first/central chain).
In these domain arrangements of the first/central chain, Va-i is a light chain or heavy chain variable domain which will form an ABD together with a variable region Vw on a further polypeptide chain (e.g. the further chain comprising the variable region VM fused to a constant region); Va-2 and Vb-2 together form an scFv (one of Va-2 and Vb-2 is a light chain variable domain and the other is a heavy chain variable domain and they are separated by a flexible polypeptide linker; in any embodiment, a V - V can accordingly be specified as comprising a linker placed between the two V regions); and the CH3 domain is connected or fused to the cytokine via a domain linker (e.g. a flexible chemical or polypeptide linker). A second polypeptide chain can then be configured to comprise one or two immunoglobulin variable domains, optionally a constant region, and an Fc domain suitable to undergo CH3-CH3 dimerization with the first/central polypeptide chain. When the second polypeptide chain has one immunoglobulin variable domain it can conveniently be fused to a CH1 or CL domain which is in turn fused to the CH2 domain via a hinge region. When the second polypeptide chain has two immunoglobulin variable domains, the two variable domains can together form an scFv and be fused to the CH2 domain via a polypeptide linker.
A second polypeptide chain for use in a protein in which the Fc domain is interposed between the NKp30 ABD and IL2v can comprise a domain arrangement:
(Va-4 - Vb-4 ) - (hinge or linker) - CH2 - CH3 (second chain) or
(Va-3 - (CH1 or CK) - (hinge or linker) - CH2 - CH3 (second chain).
In these domain arrangements of the second chain, Va-3 is a light chain or heavy chain variable domain which will form an ABD together with a variable region Vb-3 on a further polypeptide chain (e.g. a chain comprising the variable region Vb-3 fused to a light chain constant region); Va-4 and Vb-4 together form an scFv (one of Va-4 and Vb-4 is a light chain variable domain and the other is a heavy chain variable domain and they are separated by a flexible polypeptide linker).
When dimeric proteins are desired, the following heterodimer can be configured:
(Va-2 - Vb-2 ) - (hinge or linker) - CH2 - CH3 - linker - Cyt (first/central chain)
(Va-4 - Vb-4 ) - (hinge or linker) - CH2 - CH3 (second chain).
An exemplary heterodimeric protein can have a polypeptide chain 1 and 2:
Va-i - L1 - Vb-i - (hinge or L3) - CH2 - CH3 - L5 - Cyt (chain 1)
Va-2 - L2 - Vb-2 - (hinge or L4) - CH2 - CH3 (chain 2) or
Va-i - L1 - Vb-i - CL - (hinge or L3) - CH2 - CH3 - L5 - Cyt (chain 1)
Va-2 - L2 - Vb-2 - CH1 - (hinge or L4) - CH2 - CH3 (chain 2) or
Va-i - L1 - Vb-i - CH1 - (hinge or L3) - CH2 - CH3 - L5 - Cyt (chain 1)
Va-2 - L2 - Vb-2 - CL - (hinge or L4) - CH2 - CH3 (chain 2) wherein: Va-i, Vb-i, Va-2 and Vb-2 are each a VH domain or a VL domain, wherein one of Va-i and Vb-i is a VH and the other is a VL and wherein Va-i and Vw form a first antigen binding domain (ABD), wherein one of Va-2 and Vb-2 is a VH and the other is a VL and wherein Va-2 and Vb-2 form a second ABD, wherein one of the ABD binds NKp30 and the other binds antigen of interest;
CH1 is a heavy chain constant domain 1 and CL is a light chain constant domain;
Hinge is an immunoglobulin hinge region or portion thereof;
L1, L2, L3, L4 and L5 are each an amino acid domain linker, wherein L1, L2, L3, L4 and L5 can be different or the same;
CH2 and CH3 are human immunoglobulin CH2 and CH3 domains, respectively; and
Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21 , IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
For heterotrimeric multispecific proteins, one of the first and second chains will be selected to comprise two variable domains (e.g., as an scFv), and a third polypeptide chain can then be provided, comprising the following domain arrangement:
(V- (CH1 or Ci<) (third chain).
In the domain arrangement of the third chain, V, which can be also be designated VM or Vb-3, is a light chain or heavy chain variable domain which will form an ABD together with the variable region Va-i or Va-3 on the first or second polypeptide chain. The CH1 or CK domain in the third chain will be selected so as to undergo CH1-CK dimerization with the respective first or second polypeptide chain with which the third chain is selected to associate (one of the associating chains has CH1 and the other has CK).
The resulting heterotrimer proteins can then be constructed in which the Fc domain is interposed between the NKp30 ABD and the cytokine, for example molecules having the following domain arrangement:
(Va-2 - linker - Vb-2 ) - (hinge or linker) - CH2 - CH3 - linker - Cyt (second chain)
I
(Va-i - (CH1 or CK) - (hinge or linker) - CH2 - CH3 (first/central chain)
I
(Vb-i - (CH1 or CK) (third chain) wherein the first/central chain and the second chain associate by CH3-CH3 dimerization and the first/central chain and the third chain associate by the CH1 or CK dimerization, wherein the domains of the first/central chain and the third chain are selected to be complementary to permit the first and third chains to associate by CH1-CK dimerization, and wherein Va-i, VM, Va-2 and Vb-2 are each a VH domain or a VL domain, and wherein one of Va-i and Vw is a VH and the other is a VL such that Va-i and VM form a first antigen binding domain (ABD), wherein one of Va-2 and Vb-2 is a VH and the other is a VL such that Va-2 and Vb-2 form a second antigen binding domain (e.g. an scFv wherein Va-2 and Vb-2 are separated by a linker), wherein one of the ABD binds NKp30 and the other binds an antigen of interest. In one specific embodiment, Va-2 and Vb-2 form an ABD that binds NKp30 and Va-i and Vb-i form an ABD that binds the antigen of interest.
Another exemplary structure in which the Fc domain is interposed between the NKp30 ABD and the cytokine has following domain arrangements: (Va-2 - linker- Vb-2) - (hinge or linker) - CH2 - CH3 (second chain)
I
(Va-i - (CH1 or CK) - (hinge or linker) - CH2 - CH3 - linker -Cyt (first/central chain)
I
(Vb-i - (CH1 or CK) (third chain) In one specific embodiment, Va-i and Vw form an ABD that binds NKp30 and Va-2and
Vb-2form an ABD that binds the antigen of interest.
Examples of amino acid sequences of the polypeptide chains of such a protein are provided in Table 4 below.
Table 4
Figure imgf000070_0001
Figure imgf000071_0001
In heterotetrameric multispecific proteins, both of the first and second chains can be selected to comprise one variable domain fused to a CH1 or CK constant domain, and a third polypeptide chain as shown above can be provided, as well as a fourth polypeptide chain comprising the following domain arrangement:
(V- (CH1 or CK) (fourth chain).
In the domain arrangement of the fourth chain, V, which can be also be designated \ - 1 or Vb-3, is a light chain or heavy chain variable domain which will form an ABD together with the variable region Va-i or Va-3 on the first or second polypeptide chain. If the V of the third chain is designated VM and the third chain associates with the first chain such that Vw and Va-i form an ABD, then the V of the fourth chain will be Vb-3 and the fourth chain will associate with the second chain such that Vb-3 associates with Va-3 to form an ABD. The CH1 or CK domain in the fourth chain will be selected so as to undergo CH1-CK dimerization with the respective first or second polypeptide chain with which the fourth chain is selected to associate (one of the associating chains has CH1 and the other has CK).
The resulting heterotetramer proteins can then be constructed in which the Fc domain is interposed between the NKp30 ABD and the cytokine, for example molecules having the following domain arrangements:
(Vb-3- (CH1 or CK) (fourth chain)
I
(Va-3- (CH1 or CK) - (hinge or linker) - CH2 - CH3 (second chain)
I
(Va-i - (CH1 or CK) - (hinge or linker) - CH2 - CH3 - linker - Cyt (first/central chain)
I
(Vb-i - (CH1 or CK) (third chain) wherein the first/central chain and the second chain associate by CH3-CH3 dimerization and the first/central chain and the third chain associate by the CH1 or CK dimerization, wherein the domains of the first/central chain and the third chain are selected to be complementary to permit the first and third chains to associate by CH1-CK dimerization, wherein the domains of the second chain and the fourth chain are selected to be complementary to permit the second and fourth chains to associate by CH1-CK dimerization, and wherein Va-i, VM, Va-3 and Vb-3 are each a VH domain or a VL domain, and wherein one of Va-i and Vn is a VH and the other is a VL such that Va-i and Vn form a first antigen binding domain (ABD), wherein one of Va-3 and Vb-3 is a VH and the other is a VL such that Va-3 and Vb-3 form a second antigen binding domain, wherein one of the ABD binds NKp30 and the other binds an antigen of interest. In one specific embodiment, Va-3 and Vb-3 an ABD that binds NKp30 and Va-i and VM form an ABD that binds the antigen of interest. In another specific embodiment, Va-3 and Vb-3 an ABD that binds the antigen of interest and Va-i and Vn form an ABD that binds NKp30.
In this way, a range of different domain arrangements can be constructed from the variable domains, constant domains and IL2v polypeptides. Example of domain arrangements of resulting heterodimer, heterotrimer and heterotetramer proteins are shown in Table 5 below (domain linkers not shown).
Table 5
Figure imgf000072_0001
Figure imgf000073_0001
As illustrated in Table 5, in some embodiments, multispecific heterotetramer proteins can be generated on the natural immunoglobulin architecture containing two pairs of heavy chain and light chain combination with each pair having distinct binding specificity, with the cytokine polypeptide bound to the C-terminus of the Fc domain of one of the two heavy chains (one of the first and second chains), e.g. via a domain linker. Other multispecific heterotrimer and heterotetramer proteins can be constructed in which VH and VK domains are substituted by one another and/or in which CH1 and CK are domains are substituted by one another compared to the natural immunoglobulin architecture. If desired, CH1 and/or CL domains can be engineered to promote or enhance the desired heterodimerization through steric repulsion, or charge steering interaction. The correct dimerization of the light chains (third and where present fourth chains) can be enhanced through the introduction of amino acid substitutions that give rise to attractive/repulsive charge pairs into the CH1 and CK domains. Homodimerization of the two heavy chains is mediated by the CH3 interaction. To promote heterodimeric formation, amino acid substitutions can be introduced to the two respective CH3 regions.
In one embodiment, a multispecific protein can be generated by post-production assembly from structures based on half-antibodies, wherein one of the heavy chains bears at its C-terminus a cytokine fused via a domain linker (e.g., a ( - linker - Cyt) moiety fused to the C-terminus of the Fc domain monomer), thereby solving the issues of heavy and light chain mispairing. Such multispecific protein will advantageously contain modification to favor heterodimerization of the half-antibodies, for example comprising a F405L mutation in one Fc monomer and a K409R mutation in the other Fc monomer, see, e.g., Labrijn et al., (2013) PNAS 110 (13) 5145-515. Each half-antibody-type structure is individually produced in separate cell line and purified. The purified antibodies are then subjected to mild reduction to obtain half-antibodies, which were then assembled into multispecific proteins and purified from the mixture using conventional purifications methods.
Yet further multispecific proteins can be produced using similar architecture that binds the antigen of interest bivalently, binds NKp30 monovalently and binds the cytokine receptor monovalently, i.e., the multispecific protein has a 2:1:1 configuration. Examples are shown in
Figure 2.
One example is heterotetramer protein made from three different polypeptides which has two Fab structures and one scFv, where the two Fabs bind to the antigen of interest. The protein can be constructed in which the Fc domain is interposed between the NKp30 ABD and the cytokine, having the following domain arrangements:
(Vb-2 — (CH1 or CK) (2nd chain 3)
I
(Va-2- (CH1 or CK) - (hinge or linker) - CH2 - CH3 (chain 2)
I
(Va-2 — (CH 1 or CK) - (Va-i - Vb-i)- (hinge or linker) - CH2 - CH3 - linker - Cyt (chain 1)
I
(Vb-2 - (CH 1 or CK) (1 st chain 3) wherein the chain 1 and the chain 2 associate by CH3-CH3 dimerization, the chain 1 and the chain 3 (1st of two identical chain 3 polypeptides) associate by CH1 or CK dimerization, and the chain 2 and the chain 3 (2nd of two identical chain 3 polypeptides) associate by CH1 or CK dimerization, wherein the domains of the chains 1 , 2 and the chain 3 are selected to be complementary to permit the chains 1 and 2 to associate with chain 3 by CH1-CK dimerization, and wherein Va-i , VM, Va-2 and Vb-2 are each a VH domain or a VL domain, and wherein one of
Va-i and VM is a VH and the other is a VL such that Va-i and VM form a first antigen binding domain (ABD), wherein one of Va-2 and Vb-2 is a VH and the other is a VL such that Va-2 and Vb-
2 form a second antigen binding domain, wherein VM and V an ABD that binds NKp30 and
Va-2 and Vb-2 form the ABDs that binds the antigen of interest.
Another example is heteropentamer protein constructed from four different chains which has three Fab structures, two of which bind to the antigen of interest. The protein can be constructed in which the Fc domain is interposed between the NKp30 ABD and the cytokine, having the following domain arrangements:
(Vb-2 - (CH1 or CK) (1st chain 4)
I
(Va-2- (CH1 or CK) - (hinge or linker) - CH2 - CH3 (chain 2)
I
(Va-2 — (CH 1 or CK) - (Va-i - (CH1 or CK) - (hinge or linker) - CH2 - CH3 - linker- Cyt (chain 1)
I I
(Vb-2 - (CH1 or CK) (VM - (CH1 or CK)
(2nd chain 4) (chain 3) wherein the chain 1 and the chain 2 associate by CH3-CH3 dimerization, the chain 1 and the chain 3 associate by CH1 or CK dimerization, and the chain 1 and the chain 4 (2nd of two identical chain 4 polypeptides) associate by CH1 or CK dimerization, wherein the domains of the chain 1 and the chain 3 are selected to be complementary to permit the chains 1 and 3 to associate by CH1-CK dimerization, wherein the domains of the chains 1 and 2 and 4 are selected to be complementary to permit the chains 1 and 2 and chain 4 (each of the two identical chain 4 polypeptides) to associate by CH1-CK dimerization, and wherein Va-i , VM, Va-2 and Vb-2 are each a VH domain or a VL domain, and wherein one of Va-i and Vw is a VH and the other is a VL such that Va-i and VM form a first antigen binding domain (ABD), wherein one of Va-2 and Vb-2 is a VH and the other is a VL such that Va-2 and Vb-2 form a second antigen binding domain, wherein Va-i and Vn an ABD that binds NKp30 and Va-2 and Vb-2 form the ABDs that binds the antigen of interest.
In any embodiment, it may be specified that the protein has a Fc domain dimer comprised of a first and second Fc domain monomer placed on separate chains that dimerize via CH3-CH3 association, wherein one of the Fc domain monomers is connected to the both the anti-NKp30 ABD and the cytokine, and the other (second) Fc domain monomer has a free C-terminus (no anti-NKp30 ABD or cytokine fused to its C-terminus).
Optionally in any embodiment herein, fusions or linkages on the same polypeptide chain between different domains (e.g., between two V domains placed in tandem, between V domains and CH1 or CK domains, between CH1 or CK domains and Fc domains, between Fc domains monomers and V domains, between Fc domain monomers and cytokine) may occur via intervening amino acid sequences, for example via a hinge region or linker peptide. Generally, domain arrangements or structures herein are depicted without showing domain linkers, and it will be appreciated that the domain arrangements can be specified as having domain linkers between specified domain(s). For example, the cytokine can be specified as being fused to an adjacent domain via a domain linker, and a domain linker can be inserted in the relevant domain arrangement or structure. In another example, tandem variable domains (e.g. in an scFv) can be specified as being fused to one another via a domain linker, and a domain linker can be inserted between the two V regions in the relevant domain arrangement or structure. In another example, a CH1 or CL (or CK) constant region can be fused to an Fc domain or CH2 domain thereof via a domain linker or hinge domain or portion thereof, and accordingly a domain linker or hinge domain or portion thereof can be inserted between CH1 or CL domain and the Fc domain or CH2 domain in the relevant domain arrangement or structure. An example of the domain arrangement of a multispecific protein with linkers shown is shown in Figure 2A for the representative heterotrimer in format “T5”, shows domain linkers such as hinge and glycine-serine linkers, and interchain disulfide bridges.
In any embodiment herein, a polypeptide chain (e.g., chain 1, 2, 3 or 4) can be specified as having a free N and/or C terminus (no other protein domains at the terminus of the polypeptide chain). In any embodiment herein, the proteins domains described herein can optionally be specified as being indicated from N- to C- termini. Protein arrangements of the disclosure for purposes of illustration are shown from N-terminus (on the left) to C-terminus (on the right). Adjacent domains on a polypeptide chain can be referred to as being fused to one another (e.g. a domain can be said to be fused to the C-terminus of the domain on its left, and/or a domain can be said to be fused to the N-terminus of the domain on its right). The proteins domains described herein can be fused to one another directly (e.g. V domains fused directly to CH1 or CL domains) or via linkers or short intervening amino acid sequences that serve to connect the domains on a polypeptide chain (e.g. they may optionally be specified to lack other predetermined functionality, or to lack specific binding to a predetermined ligand). Two polypeptide chains will be bound to one another (indicated by “”), by non-covalent interactions, and optionally can further be attached via interchain disulfide bonds, formed between cysteine residues within complementary CH1 and CK domains.
Connections and linkers
Generally, there are a number of suitable linkers that can be used in the multispecific proteins, including traditional peptide bonds, generated by recombinant techniques. In some embodiments, the linker is a "domain linker”, used to link any two domains as outlined herein together. Adjacent protein domains can be specified as being connected or fused to one another by a domain linker. An exemplary domain linker is a (poly)peptide linker, optionally a flexible (poly)peptide linker. Peptide linkers or polypeptide linkers, used interchangeably herein, may have a subsequence derived from a particular domain such as a hinge, CH1 or CL domain, or may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity. In one embodiment, the linker is from about 1 to 50 amino acids in length, preferably about 2 to 30 amino acids in length. In one embodiment, linkers of 4 to 20 amino acids in length may be used, with from about 5 to about 15 amino acids finding use in some embodiments. While any suitable linker can be used, many embodiments, linkers (e.g. flexible linkers) can utilize a glycine-serine polypeptide or polymer, including for example comprising (GS)n, (GSG2S)n, (G4S)n, (GSs)n, (GS^n and (GsS)n, where n is an integer of at least one (optionally n is 1 , 2, 3 or 4), glycine-alanine polypeptide, alanine-serine polypeptide, and other flexible linkers. Linkers comprising glycine and serine residues generally provides protease resistance. One example of a (GS)i linker is a linker having the amino acid sequence STGS; such a linker can be useful to fuse a domain to the C-terminus of an Fc domain (or a CH3 domain thereof). In some embodiments peptide linkers comprising (G2S)n are used, wherein, for example, n = 1-20, e.g., (G2S), (G2S)2, (G2S)3, (G2S)4, (G2S)s, (G2S)6, (G2S)7 or(G2S)s, or (GsS)n, wherein, for example, n is an integer from 1-15. In one embodiment, a domain linker comprises a (G4S)n peptide, wherein, for example, n is an integer from 1-10, optionally 1-6, optionally 1-4. In some embodiments peptide linkers comprising (GS2)n (GSs)n or (GS4)n are used, wherein, for example, n = 1-20, e.g., (GS2), (GS2)2, (GS2)3, (GS3)I, (GS3)2, (GS3K (GS4)I, (GS4)2, (GS4)3, wherein, for example, n is an integer from 1-15. In one embodiment, a domain linker comprises a (GS4)n peptide, wherein, for example, n is an integer from 1-10, optionally 1-6, optionally 1-4. In one embodiment, a domain linker comprises a C-terminal GS dipeptide, e.g., a (GS4) comprising linker has the sequence (GS4)GS.
Any of the peptide or domain linkers may be specified to comprise a length of at least 2 residues, 3 residues, 4 residues, at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, or more. In other embodiments, the linkers comprise a length of between 2-4 residues, between 2-4 residues, between 2-6 residues, between 2-8 residues, between 2-10 residues, between 2-12 residues, between 2-14 residues, between 3-15 residues, between 4-15 residues, between 2-16 residues, between 2-18 residues, between 2- 20 residues, between 2-22 residues, between 2-24 residues, between 2-26 residues, between 2-28 residues, between 2-30 residues, between 2 and 50 residues, or between 10 and 50 residues.
Examples of polypeptide linkers may include sequence fragments from CH1 or CL domains; for example the first 4-12 or 5-12 amino acid residues of the CL/CH1 domains are particularly useful for use in linkages of scFv moieties. Linkers can be derived from immunoglobulin light chains, for example CK or CA. Linkers can be derived from immunoglobulin heavy chains of any isotype, including for example Cy1, Cy2, Cy3, Cy4 and Cp. Linker sequences may also be derived from other proteins such as Ig-like proteins (e.g. TCR, FcR, KIR), hinge region-derived sequences, and other natural sequences from other proteins. In certain domain arrangements, VH and VL domains are linked to another in tandem separated by a linker peptide (e.g. a scFv) and in turn be fused to the N- or C-terminus of an Fc domain (or CH2 domain thereof). Such tandem variable regions or scFv can be connected to the Fc domain via a hinge region or a portion thereof, an N-terminal fragment of a CH1 or CL domain, or a glycine- and serine-containing flexible polypeptide linker.
Fc domains can be connected to other domains via immunoglobulin-derived sequence or via non-immunoglobulin sequences, including any suitable linking amino acid sequence. Advantageously, immunoglobulin-derived sequences can be readily used between CH1 or CL domains and Fc domains, in particular, where a CH1 or CL domain is fused at its C-terminus to the N-terminus of an Fc domain (or CH2 domain). An immunoglobulin hinge region or portion of a hinge region can and generally will be present on a polypeptide chain between a CH1 domain and a CH2 domain. A hinge or portion thereof can also be placed on a polypeptide chain between a CL (e.g. CK) domain and the CH2 domain of an Fc domain when a CL is adjacent to an Fc domain on the polypeptide chain. However, it will be appreciated that a hinge region can optionally be replaced for example by a suitable linker peptide, e.g. a flexible polypeptide linker.
The NKp30 ABD and cytokine receptor ABD (e.g., a cytokine) are advantageously linked to the rest of the multispecific protein (e.g. or to a constant domain or Fc domain thereof) via a flexible linker (e.g. polypeptide linker) that leads to less structural rigidity or stiffness (e.g. between or amongst the ABD and Fc domain) compared to a conventional (e.g. wild-type full length human IgG) antibody. For example, the multispecific protein may have a structure or a flexible linker between the NKp30 ABD and constant domain or Fc domain that permits an increased range of domain motion compared to the two ABDs in a conventional (e.g. wild-type full length human IgG) antibody. In particular, the structure or a flexible linker can be configured to confer on the antigen binding sites greater intrachain domain movement compared to antigen binding sites in a conventional human lgG1 antibody. Rigidity or domain motion/interchain domain movement can be determined, e.g., by computer modeling, electron microscopy, spectroscopy such as Nuclear Magnetic Resonance (NMR), X-ray crystallography, or Sedimentation Velocity Analytical ultracentrifugation (AUC) to measure or compare the radius of gyration of proteins comprising the linker or hinge. A test protein or linker may have lower rigidity relative to a comparator protein if the test protein has a value obtained from one of the tests described in the previous sentence differs from the value of the comparator, e.g., an lgG1 antibody ora hinge, by at least 5%, 10%, 25%, 50%, 75%, or 100%. A cytokine can for example be fused to the C-terminus of a CH3 domain by a linker selected from GSSSS (SEQ ID NO: 41), GSSSSGSSSS (SEQ ID NO: 42), GSSSSGSSSSGS (SEQ ID NO: 43) or GSSSSGSSSSGSSSS (SEQ ID NO: 44).
In one embodiment, the multispecific protein may have a structure or a flexible linker between the NKp30 ABD and Fc domain that permits the NKp30 ABD and the ABD which binds an antigen of interest to have a spacing between said ABDs comprising less than about 80 angstroms, less than about 60 angstroms or ranges from about 40-60 angstroms.
At its C-terminus, an Fc domain (or a CH3 domain thereof) can be connected to the N- terminus of a NKp30 ABD or a cytokine polypeptide via a polypeptide linker, for example a glycine-serine-containing linker, optionally a linker having the amino acid sequence STGS.
In certain embodiments, a CH1 or CL domain of a Fab (e.g. of an NKp30 ABD) is fused at its C-terminus to the N-terminus of the cytokine via a flexible polypeptide linker, for example a glycine-serine-containing linker. Preferably, the linker will have a chain length of at least 4 amino acid residues, optionally the linker has a length of 5, 6, 7, 8, 9 or 10 amino acid residues. In certain embodiments, the NKp30 ABD is placed C-terminal to the Fc domain, and the NKp30 is positioned between an Fc domain and the cytokine polypeptide in the multispecific protein. The NKp30 ABD will be connected or fused at its N-terminus (at the N- terminus of a VH or a VL domain) to the C-terminus of the Fc domain via a linker (e.g. a glycine and serine containing linker, a linker having the sequence STGS, a flexible polypeptide linker) of sufficient length to enable the NKp30 binding ABD to fold and/or adopt an orientation in such a way as to permit binding to NKp30 at the surface of an NK cell, while at the same time possesses a sufficient distance and range of motion relative to the adjacent Fc domain (or more generally to rest of the multispecific protein) such that the Fc domain can also simultaneously be found by CD16 expressed at the surface of the same NK cell. Additionally, when the NKp30 ABD is placed between an Fc domain and an cytokine polypeptide in the multispecific protein, the C-terminus of a VH or VL of an scFv NKp30 ABD, or the CH1 or CL domain of a Fab NKp30 ABD will be connected or fused to the N-terminus of the cytokine polypeptide via a flexible linker (e.g. a flexible polypeptide linker) of sufficient length to enable the NKp30 binding ABD to fold and/or adopt an orientation in such a way as to permit binding to NKp30 at the surface of an NK cell, while at the same time providing a sufficient distance and range of motion relative to the adjacent cytokine polypeptide such that the cytokine polypeptide can also simultaneously be bound by its cytokine receptor expressed at the surface of the NK cell. Preferably, the linker will have a chain length of at least 4 amino acid residues, optionally the linker has a length of 5, 6, 7, 8, 9 or 10 amino acid residues.
In tandem variable regions (e.g. scFv), two V domains (e.g. a VH domain and VL domains are generally linked together by a linker of sufficient length to enable the ABD to fold in such a way as to permit binding to the antigen for which the ABD is intended to bind. Examples of linkers include linkers comprising glycine and serine residues, e.g., the amino acid sequence GEGTSTGSGGSGGSGGAD (SEQ ID NO : 260). In another specific embodiment, the VH domain and VL domains of an scFv are linked together by the amino acid sequence (G4S)3.
In one embodiment, a (poly)peptide linker used to link a VH or VL domain of an scFv to a CH2 domain of an Fc domain comprises a fragment of a CH 1 domain or CL domain and/or hinge region. For example, an N-terminal amino acid sequence of CH1 can be fused to a variable domain in order to mimic as closely as possible the natural structure of a wild-type antibody. In one embodiment, the linker comprises an amino acid sequence from a hinge domain or an N-terminal CH1 amino acid. In one embodiment, the linker peptide mimics the regular VK-CK elbow junction, e.g., the linker comprises or consists of the amino acid sequence RTVA. In one embodiment, the hinge region used to connect the C-terminal end of a CH1 or CK domain (e.g. of a Fab) with the N-terminal end of a CH2 domain will be a fragment of a hinge region (e.g. a truncated hinge region without cysteine residues) or may comprise one or more amino acid modifications which remove (e.g. substitute by another amino acid, or delete) a cysteine residue, optionally both cysteine residues in a hinge region. Removing cysteines can be useful to prevent undesired disulfide bond formation, e.g., the formation of disulfide bridges in a monomeric polypeptide.
A "hinge" or "hinge region" or "antibody hinge region" herein refers to the flexible polypeptide or linker between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237. Thus for an IgG the hinge generally includes positions 221 (D221 in lgG1) to 236 (G236 in lgG1), wherein the numbering is according to the EU index as in Kabat. References to specific amino acid residues within constant region domains found within the polypeptides shall be, unless otherwise indicated or as otherwise dictated by context, be defined according to Kabat, in the context of an IgG antibody.
In one embodiment, the hinge region (or fragment thereof) is derived form a hinge domain of a human lgG1 antibody. For example a hinge domain may comprise the amino acid sequence: THTCPPCPAPELL (SEQ ID NO: 36), or a fragment comprising the first 8 resides thereof, or an amino acid sequence at least 60%, 70%, 80% or 90% identical to any of the foregoing, optionally wherein one or both cysteines are deleted or substituted by a different amino acid residue, optionally a serine.
In one embodiment, the hinge region (or fragment thereof) is derived from a Cp2-C Cp3 hinge domain of a human IgM antibody. For example a hinge domain may comprise the amino acid sequence: NASSMCVPSPAPELL (SEQ ID NO: 37), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto, optionally wherein one or both cysteines are deleted or substituted by a different amino acid residue.
Polypeptide chains that dimerize and associate with one another via non-covalent bonds or interactions may or may not additionally be bound by an interchain disulfide bond formed between respective CH1 and CK domains, and/or between respective hinge domains on the chains. CH1 , CK and/or hinge domains (or other suitable linking amino acid sequences) can optionally be configured such that interchain disulfide bonds are formed between chains such that the desired pairing of chains is favored and undesired or incorrect disulfide bond formation is avoided. For example, when two polypeptide chains to be paired each possess a CH1 or CK adjacent to a hinge domain, the polypeptide chains can be configured such that the number of available cysteines for interchain disulfide bond formation between respective CH1/CK-hinge segments is reduced (or is entirely eliminated). For example, the amino acid sequences of respective CH1 , CK and/or hinge domains can be modified to remove cysteine residues in both the CH1/CK and the hinge domain of a polypeptide; thereby the CH1 and CK domains of the two chains that dimerize will associate via non-covalent interaction(s).
In another example, the CH1 or CK domain adjacent to (e.g., N-terminal to) a hinge domain comprises a cysteine capable of interchain disulfide bond formation, and the hinge domain which is placed at the C-terminus of the CH1 or CK comprises a deletion or substitution of one or both cysteines of the hinge (e.g. Cys 239 and Cys 242, as numbered for human lgG1 hinge according to Kabat). In one embodiment, the hinge region (or fragment thereof) comprise the amino acid sequence: THTSPPSPAPELL (SEQ ID NO: 38), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto.
In another example, the CH1 or CK domain adjacent (e.g., N-terminal to) a hinge domain comprises a deletion or substitution at a cysteine residue capable of interchain disulfide bond formation, and the hinge domain placed at the C-terminus of the CH1 or CK comprises one or both cysteines of the hinge (e.g. Cys 239 and Cys 242, as numbered for human lgG1 hinge according to Kabat). In one embodiment, the hinge region (or fragment thereof) comprises the amino acid sequence: THTCSSCPAPELL (SEQ ID NO: 39), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto.
In another example, a hinge region is derived from an IgM antibody. In such embodiments, the CH1/CK pairing mimics the Cp2 domain homodimerization in IgM antibodies. For example, the CH1 or CK domain adjacent (e.g., N-terminal to) a hinge domain comprises a deletion or substitution at a cysteine capable of interchain disulfide bond formation, and an IgM hinge domain which is placed at the C-terminus of the CH1 or CK comprises one or both cysteines of the hinge. In one embodiment, the hinge region (or fragment thereof) comprises the amino acid sequence: THTCSSCPAPELL (SEQ ID NO: 40), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto.
Alternatively to the polypeptide linkers, a variety of nonproteinaceous polymer or chemical linkers may find use in the multispecific proteins. For example nonproteinaceous polymers including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers. In some examples, an amino acid sequence in a polypeptide chain of a multispecific protein may be modified to introduce a reactive group, optionally a protected reactive group, and the so-modified protein or chain is then reacted with a linker or a polypeptide comprising a complementary reactive group. In some examples, an amino acid residue in a polypeptide chain of a multispecific protein can be bound to a linker comprising a reactive group (for further reaction with a second polypeptide functionalized with a linker with a complementary reactive group) or directly a second polypeptide via an enzyme catalyzed reaction. For example, a polypeptide comprising an acceptor glutamine or lysine can reacted with linker comprising a primary amine in the presence of a transglutamine enzyme (e.g. Bacterial Transglutaminase, BTG) such that the transglutaminase enzyme catalyzes the conjugation of the linker to an acceptor glutamine residue within the primary structure of the polypeptide, for example within an immunoglobulin constant domain or within a TGase recognition tag inserted or appended to (e.g., fused to) a constant region. A second polypeptide can also be functionalized with a linker in a similar manner, and when the conjugated linkers each bear complementary reactive groups (e.g. R on the linker of one polypeptide and R’ on the linker of the other polypeptide), the two functionalized polypeptides can be reacted such that they are bound via the linker comprising the residue of the reaction or R with R’. Examples of reactive group pairs R and R’ include a range of groups capable of biorthogonal reaction, for example 1,3-dipolar cycloaddition between azides and cyclooctynes (copper-free click chemistry), between nitrones and cyclooctynes, oxime/hydrazone formation from aldehydes and ketones and the tetrazine ligation (see also WO2013/092983). The resulting linker and functionalized antibody, or the Y element thereof, can thus comprise a RR’ group resulting from the reaction of R and R’, for example a triazole. Methods and linkers for use in BTG-mediated conjugation to antibodies is described in PCT publication no. WO2014/202773, the disclosure of which is incorporated by reference. “Transglutaminase”, used interchangeably with “TGase” or “TG”, refers to an enzyme capable of cross-linking proteins through an acyl-transfer reaction between the y-carboxamide group of peptide-bound glutamine and the e-amino group of a lysine or a structurally related primary amine such as amino pentyl group, e.g. a peptide-bound lysine, resulting in a £-(Y-glutamyl)lysine isopeptide bond. TGases include, inter alia, bacterial transglutaminase (BTG) such as the enzyme having EC reference EC 2.3.2.13 (protein-glutamine-Y-glutamyltransferase). The term “acceptor glutamine” residue, when referring to a glutamine residue of an antibody, means a glutamine residue that is recognized by a TGase and can be cross-linked by a TGase through a reaction between the glutamine and a lysine or a structurally related primary amine such as amino pentyl group. Preferably the acceptor glutamine residue is a surface-exposed glutamine residue. The term “TGase recognition tag” refers to a sequence of amino acids comprising an acceptor glutamine residue and that when incorporated into (e.g. appended to) a polypeptide sequence, under suitable conditions, is recognized by a TGase and leads to cross-linking by the TGase through a reaction between an amino acid side chain within the sequence of amino acids and a reaction partner. The recognition tag may be a peptide sequence that is not naturally present in the polypeptide comprising the enzyme recognition tag. Examples of TGase recognition tags include the amino acid sequences disclosed in WO2012/059882 and WO2014/072482, the disclosure of which sequences are incorporated herein by reference.
Constant regions
Constant region domains can be derived from any suitable human antibody, particularly human antibodies of gamma isotype, including, the constant heavy (CH1) and light (CL, CK or Cl) domains, hinge domains, CH2 and CH3 domains.
With respect to heavy chain constant domains, "CH1" generally refers to positions l ie- 220 according to the EU index as in Kabat. Depending on the context, a CH1 domain (e.g. as shown in the domain arrangements), can optionally comprise residues that extend into the hinge region such that the CH1 comprises at least part of a hinge region. For example, when positioned C-terminal on a polypeptide chain and/or or C-terminal to the Fc domain, and/or within a Fab structure that is or C-terminal to the Fc domain, the CH1 domain can optionally comprise at least part of a hinge region, for example CH1 domains can comprise at least an upper hinge region, for example an upper hinge region of a human lgG1 hinge, optionally further in which the terminal threonine of the upper hinge can be replaced by a serine. Such a CH2 domain can therefore comprise at its C-terminus the amino acid sequence: EPKSCDKTHS. (SEQ ID NO : 261).
Exemplary human CH1 domain amino acid sequences include: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (SEQ ID NO: 26) or
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHS (SEQ ID NO: 27). or
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT (SEQ ID NO: 28).
Exemplary human CK domain amino acid sequences include: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29).
In some exemplary configurations, the multispecific protein can be a heterodimer, a heterotrimer or a heterotetramer comprising one or two Fabs (e.g. one Fab binding NKp30 and the other binding the antigen of interest), in which variable regions, CH1 and/or CL domains are engineered by introducing amino acid substitutions in a knob-into-holes or electrostatic steering approach to promote the desired chain pairings of CH1 domains with CK domains. In some exemplary configurations, the multispecific protein can be a heterodimer, a heterotrimer or a heterotetramer comprising one or two Fabs (e.g. one Fab binding NKp30 and the other binding the antigen of interest), wherein a Fab has a VH/VL crossover (VH and VL replace one another) or a CH1/CL crossover (the CH1 and CL replace one another), and wherein the CH1 and/or CL domains comprise amino acid substitutions to promote correct chain association by knob-into-holes or electrostatic steering.
"CH2" generally refers to positions 237-340 according to the EU index as in Kabat, and "CH3" generally refers to positions 341-447 according to the EU index as in Kabat. CH2 and CH3 domains can be derived from any suitable antibody. Such CH2 and CH3 domains can be used as wild-type domains or may serve as the basis for a modified CH2 or CH3 domain. Optionally the CH2 and/or CH3 domain is of human origin or may comprise that of another species (e.g., rodent, rabbit, non-human primate) or may comprise a modified or chimeric CH2 and/or CH3 domain, e.g., one comprising portions or residues from different CH2 or CH3 domains, e.g., from different antibody isotypes or species antibodies.
In any of the domain arrangements, the Fc domain monomer may comprise a CH2- CH3 unit (a full length CH2 and CH3 domain or a fragment thereof). In heterodimers or heterotrimers comprising two chains with Fc domain monomers (i.e. the heterodimers or heterotrimers comprise a Fc domain dimer), the CH3 domain will be capable of CH3-CH3 dimerization (e.g. it will comprise a wild-type CH3 domain or a CH3 domain with modifications to promote a desired CH3-CH3 dimerization).
Exemplary human lgG1 CH2-CH3 (Fc) domain amino acid sequences include: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 30).
An Fc domain may optionally further comprise a C-terminal lysine (K). In some exemplary configurations, the multispecific protein can be a heterodimer, a heterotrimer or a heterotetramer, wherein the polypeptide chains are engineered for heterodimerization among each other so as to produce the desired protein. In embodiments where the desired chain pairings are not driven by CH1-CK dimerization or generally where enhancement of pairing is desired, the chains may comprise constant or Fc domains with amino acid modifications (e.g., substitutions) that favor the preferential chain pairing, e.g. favor a desired hetero-dimerization of the two different chains over the homo-dimerization of two identical chains.
In some embodiments, a “knob-into-holes” approach is used in which the domain interfaces (e.g. CH3 domain interface of the antibody Fc region) are mutated so that the antibodies preferentially heterodimerize. Mutations can be introduced to create altered charge polarity across the interface (e.g. Fc dimer interface) such that co-expression of electrostatically matched chains (e.g. Fc-containing chains) support favorable attractive interactions thereby promoting desired heterodimer (e.g. Fc heterodimer) formation, whereas unfavorable repulsive charge interactions suppress unwanted heterodimer (e.g., Fc homodimer) formation. See for example mutations and approaches reviewed in Brinkmann and Kontermann, 2017 MAbs, 9(2): 182-212, the disclosure of which is incorporated herein by reference. For example one heavy chain comprises a T366W substitution and the second heavy chain comprises a T366S, L368A and Y407V substitution, see, e.g. Ridgway et al (1996) Protein Eng., 9, pp. 617-621; Atwell (1997) J. Mol. Biol., 270, pp. 26-35; and W02009/089004, the disclosures of which are incorporated herein by reference. For example the “Hole” mutations on a first Fc monomer can comprise Y349C/T366S/L368A/Y407V and the complementary “Knob” mutations on the second Fc monomer can comprise S354C/T366W (Kabat EU numbering). In another approach, one heavy chain comprises a F405L substitution and the second heavy chain comprises a K409R substitution, see, e.g., Labrijn et al. (2013) Proc. Natl. Acad. Sci. U.S.A., 110, pp. 5145-5150. In another approach, one heavy chain comprises T350V, L351Y, F405A, and Y407V substitutions and the second heavy chain comprises T350V, T366S, K392L, and T394W substitutions, see, e.g. Von Kreudenstein et al., (2013) mAbs 5:646-654. In another approach, one heavy chain comprises both K409D and K392D substitutions and the second heavy chain comprises both D399K and E356K substitutions, see, e.g. Gunasekaran et al. , (2010) J. Biol. Chem. 285:19637-19646. In another approach, one heavy chain comprises D221E, P228E and L368E substitutions and the second heavy chain comprises D221 R, P228R, and K409R substitutions, see, e.g. Strop et al., (2012) J. Mol. Biol. 420: 204-219. In another approach, one heavy chain comprises S364H and F405A substitutions and the second heavy chain comprises Y349T and T394F substitutions, see, e.g. Moore et al., (2011) mAbs 3: 546-557. In another approach, one heavy chain comprises a H435R substitution and the second heavy chain optionally may or may not comprise a substitution, see, e.g. US Patent no. 8,586,713. When such hetero-multimeric antibodies have Fc regions derived from a human lgG2 or lgG4, the Fc regions of these antibodies can be engineered to contain amino acid modifications that permit CD16 binding. In some embodiments, the antibody may comprise mammalian antibody-type N-linked glycosylation at residue N297 (Kabat EU numbering).
In some embodiments, a multispecific protein comprises one or more amino acid modifications (e.g. substitutions) in a CH3 domain that affect binding to an affinity purification medium, e.g. Protein A. Introduction into one of the CH3 domains of mutations that diminish binding to Protein A can be used to distinsguish unwanted chain pairings from the desired protein. For example, mutations can be introduced at amino acids H435 and Y436 (Kabat EU numbering), for example H435R and Y436F.
In some embodiments, one or more pairs of disulfide bonds such as A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C are introduced into the Fc region to increase stability, for example further to a loss of stability caused by other Fc modifications. Additional example includes introducing K338I, A339K, and K340S mutations to enhance Fc stability and aggregation resistance (Gao et al, 2019 Mol Pharm. 2019; 16:3647).
In some embodiments,, one of the Fc monomers that make up the dimeric domain can comprise mutations in the CH3 domain (H435R and Y436F, according to EU numbering, disclosed in Jendeberg et al. (1997) Journal of Immunological Methods 201: 25-34) to reduce binding to Protein A; these mutations which are optional do not affect the activity of the protein but can potentially improve efficiency of purification by permitting the elimination of homodimers.
In some embodiments, where a multispecific protein is intended to have reduced binding to a human Fc gamma receptor. In some embodiments, where a multispecific protein is intended to have reduced binding to a human CD16A polypeptide (and optionally further reduced binding to CD32A, CD32B and/or CD64), the Fc domain is a human lgG4 Fc domain, optionally further wherein the Fc domain comprises a S228P mutation to stabilize the hinge disulfide. In one embodiment, the Fc domain has an amino acid sequence at least 90%, 95% or 99% identical to a human lgG4 Fc domain, optionally further comprising a Kabat S228P mutation.
In embodiments, where a multispecific protein is intended to have reduced binding to human CD16A polypeptide (and optionally further reduced binding to CD32A, CD32B and/or CD64), a CH2 and/or CH3 domain (or Fc domain comprising same) may comprise a modification to decrease or abolish binding to FcyRIIIA (CD16). For example, CH2 mutations in a Fc domain dimer proteins at reside N297 (Kabat numbering) can substantially eliminate CD16A binding. However the person of skill in the art will appreciate that other configurations can be implemented. For example, substitutions into human lgG1 or lgG2 residues at positions 233-236 and/or residues at positions 327, 330 and 331 were shown to greatly reduce binding to Fey receptors and thus ADCC and CDC. Furthermore, Idusogie et al. (2000) J. Immunol. 164(8):4178-84 demonstrated that alanine substitution at different positions, including K322, significantly reduced complement activation.
In one embodiment, the asparagine (N) at Kabat heavy chain residue 297 can be substituted by a residue other than an asparagine (e.g. a glutamine, a residue other than glutamine, for example a serine). In one embodiment, an Fc domain modified to reduce binding to CD16A comprises a substitution in the Fc domain at Kabat residues 234, 235 and 322. In one embodiment, the protein comprises a substitution in the Fc domain at Kabat residues 234, 235 and 331. In one embodiment, the protein comprises a substitution in the Fc domain at Kabat residues 234, 235, 237 and 331. In one embodiment, the protein comprises a substitution in the Fc domain at Kabat residues 234, 235, 237, 330 and 331. In one embodiment, the Fc domain is of human lgG1 subtype. Amino acid residues are indicated according to EU numbering according to Kabat.
In one embodiment, an Fc domain modified to reduce binding to CD16A comprises an amino acid modification (e.g. substitution) at one or more of Kabat residue(s) 233-236, optionally one or more of residues 233-237, or at one, two or three of residues 234, 235 and/or 237, and an amino acid modification (e.g. substitution) at Kabat residue(s) 330 and/or 331. One example of such an Fc domain comprises substitutions at Kabat residues L234, L235 and P331 (e.g., L234A/L235E/P331S or (L234F/L235E/P331S). Another example of such an Fc domain comprises substitutions at Kabat residues L234, L235, G237 and P331 (e.g., L234A/L235E/G237A/P331S). Another example of such an Fc domain comprises substitutions at Kabat residues L234, L235, G237, A330 and P331 (e.g.,
L234A/L235E/G237A/A330S/P331S). In one embodiment, an antibody comprises an human lgG1 Fc domain comprising L234A/L235E/N297X/P331S substitutions, L234F/L235E/N297X/P331S substitutions, L234A/L235E/G237A/N297X/P331S substitutions, or L234A/L235E/G237A/ N297X/A330S/P331S substitutions, wherein X can be any amino acid other than an asparagine. In one embodiment, X is a glutamine; in another embodiment, X is a residue other than a glutamine (e.g. a serine).
In one embodiment, an Fc domain that has low or reduced binding to CD16A comprises a human lgG4 Fc domain, wherein the Fc domain has the amino acid sequence below (human lgG4 with S228P substitution), or an amino acid sequence at least 90%, 95% or 99% identical thereto.
ASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA
VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPPCPAPEFL GGPSVFLFPP KPKDTLMISR TPEVTCW VD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRW SV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC
SVMHEALHNH YTQKSLSLSL (SEQ ID NO: 31). In one embodiment, an Fc domain modified to reduce binding to CD16A comprises the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235 and 331 (underlined):
Figure imgf000088_0001
In one embodiment, an Fc domain modified to reduce binding to CD16A comprises the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235 and 331 (underlined):
Figure imgf000088_0002
In one embodiment, an Fc domain modified to reduce binding to CD16A comprises the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235, 237, 330 and 331
(underlined):
Figure imgf000088_0003
V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G (SEQ ID NO: 34).
In one embodiment, an Fc domain modified to reduce binding to CD16A comprises the amino acid sequence below, or a sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235, 237 and 331 (underlined):
A S T K G P S V F P L A P S S K S T S G G T A A L G C L V K D Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T K V D K R V E P K S C D K T H T C P P C P A P E A E G A P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A S I E K T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G (SEQ ID NO: 35).
Any of the above Fc domain sequences can optionally further comprise a C-terminal lysine (K), i.e. as in the naturally occurring sequence.
In certain embodiments herein where binding to CD16 (CD16A) is desired, a CH2 and/or CH3 domain (or Fc domain comprising same) may be a wild-type domain or a domain having CD16 interface residues from the wild-type human lgG1 domain, or may comprise one or more amino acid modifications (e.g. amino acid substitutions) which increase binding to human CD16 and optionally another receptor such as FcRn. Optionally, the modifications will not substantially decrease or abolish the ability of the Fc-derived polypeptide to bind to neonatal Fc receptor (FcRn), e.g. human FcRn. Typical modifications include modified human lgG1 -derived constant regions comprising at least one amino acid modification (e.g. substitution, deletions, insertions), and/or altered types of glycosylation, e.g., hypofucosylation. Such modifications can affect interaction with Fc receptors: FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16). FcyRI (CD64), FcyRIIA (CD32A) and FcyRIII (CD 16) are activating (i.e., immune system enhancing) receptors while FcyRI IB (CD32B) is an inhibiting (i.e., immune system dampening) receptor. A modification may, for example, increase binding of the Fc domain to FcyRII la on effector (e.g. NK) cells and/or decrease binding to FcyRIIB. Examples of modifications are provided in PCT publication no. WO2014/044686, the disclosure of which is incorporated herein by reference. Specific mutations (in lgG1 Fc domains) which affect (enhance) FcyRIIIa or FcRn binding are also set forth below.
Figure imgf000090_0001
In some embodiments, the multispecific protein comprises a variant Fc region comprise at least one amino acid modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 and/or CH3 domain of the Fc region, wherein the modification enhances binding to a human CD16 polypeptide. In other embodiments, the multispecific protein comprises at least one amino acid modification (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 domain of the Fc region from amino acids 237-341 , or within the lower hinge-CH2 region that comprises residues 231-341. In some embodiments, the multispecific protein comprises at least two amino acid modifications (for example, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications), wherein at least one of such modifications is within the CH3 region and at least one such modifications is within the CH2 region. Encompassed also are amino acid modifications in the hinge region. In one embodiment, encompassed are amino acid modifications in the CH1 domain, optionally in the upper hinge region that comprises residues 216-230 (Kabat EU numbering). Any suitable functional combination of Fc modifications can be made, for example any combination of the different Fc modifications which are disclosed in any of United States Patents Nos. US, 7,632,497; 7,521,542; 7,425,619; 7,416,727; 7,371,826; 7,355,008; 7,335,742; 7,332,581; 7,183,387; 7,122,637; 6,821,505 and 6,737,056; and/or in PCT Publications Nos. WO2011/109400; WO 2008/105886; WO 2008/002933; WO 2007/021841; WO 2007/106707; WO 06/088494; WO 05/115452; WO 05/110474; WO 04/1032269; WO 00/42072; WO 06/088494; WO 07/024249; WO 05/047327; WO 04/099249 and WO 04/063351; and/or in Lazar et al. (2006) Proc. Nat. Acad. Sci. USA 103(11): 405-410; Presta, L.G. et al. (2002) Biochem. Soc. Trans. 30(4):487-490; Shields, R.L. et al. (2002) J. Biol. Chem. 26; 277(30):26733-26740 and Shields, R.L. et al. (2001) J. Biol. Chem. 276(9):6591-6604).
In some embodiments, the multispecific protein comprises an Fc domain comprising at least one amino acid modification (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region, such that the molecule has an enhanced binding affinity for human CD16 relative to the same molecule comprising a wild-type Fc region, optionally wherein the variant Fc region comprises a substitution at any one or more of positions 221 , 239, 243, 247, 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 308, 309, 310, 311 , 312, 316, 320, 322, 326, 329, 330, 332, 331 , 332, 333, 334, 335, 337, 338, 339, 340, 359, 360, 370, 373, 376, 378, 392, 396, 399, 402, 404, 416, 419, 421, 430, 434, 435, 437, 438 and/or 439 (Kabat EU numbering).
In one embodiment, the multispecific protein comprises an Fc domain comprising at least one amino acid modification (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region, such that the molecule has enhanced binding affinity for human CD16 relative to a molecule comprising a wild-type Fc region, optionally wherein the variant Fc region comprises a substitution at any one or more of positions 239, 298, 330, 332, 333 and/or 334 (e.g. S239D, S298A, A330L, I332E, E333A and/or K334A substitutions), optionally wherein the variant Fc region comprises a substitution at residues S239 and I332, e.g. a S239D and I332E substitution (Kabat EU numbering).
In some embodiments, the multispecific protein comprises an Fc domain comprising N-linked glycosylation at Kabat residue N297. In some embodiments, the multispecific protein comprises an Fc domain comprising altered glycosylation patterns that increase binding affinity for human CD16. Such carbohydrate modifications can be accomplished by, for example, by expressing a nucleic acid encoding the multispecific protein in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery are known in the art and can be used as host cells in which to express recombinant antibodies to thereby produce an antibody with altered glycosylation. See, for example, Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1 , as well as, European Patent No: EP 1176195; PCT Publications WO 06/133148; WO 03/035835; WO 99/54342, each of which is incorporated herein by reference in its entirety. In one aspect, the multispecific protein contains one or more hypofucosylated constant regions. Such multispecific protein may comprise an amino acid alteration or may not comprise an amino acid alteration and/or may be expressed or synthesized or treated under conditions that result in hypofucosylation. In one aspect, a multispecific protein composition comprises a multispecific protein described herein, wherein at least 20, 30, 40, 50, 60, 75, 85, 90, 95% or substantially all of the antibody species in the composition have a constant region comprising a core carbohydrate structure (e.g. complex, hybrid and high mannose structures) which lacks fucose. In one embodiment, provided is a multispecific protein composition which is free of N- linked glycans comprising a core carbohydrate structure having fucose. The core carbohydrate will preferably be a sugar chain at Asn297.
Optionally, a multispecific protein comprising a Fc domain dimer can be characterized by having a binding affinity to a human CD16A polypeptide that is within 1-log of that of a conventional human lgG1 antibody, e.g., as assessed by surface plasmon resonance.
In one embodiment, the multispecific protein comprising a Fc domain dimer in which an Fc domain is engineered to enhance Fc receptor binding can be characterized by having a binding affinity to a human CD16A polypeptide that is at least 1-log greater than that of a conventional or wild-type human lgG1 antibody, e.g., as assessed by surface plasmon resonance.
In one embodiment, a multispecific protein comprising a Fc domain dimer can be characterized by having a binding affinity to a human FcRn (neonatal Fc receptor) polypeptide that is within 1-log of that of a conventional human lgG1 antibody, e.g., as assessed by surface plasmon resonance.
Optionally a multispecific protein comprising a Fc domain dimer can be characterized by a Kd for binding (monovalent) to a human Fc receptor polypeptide (e.g., CD16A) of less than 105 M (10 pmolar), optionally less than 10-6 M (1 pmolar), as assessed by surface plasmon resonance (e.g. as in the Examples herein, SPR measurements performed on a Biacore T100 apparatus (Biacore GE Healthcare), with bispecific antibodies immobilized on a Sensor Chip CM5 and serial dilutions of soluble CD16 polypeptide injected over the immobilized bispecific antibodies.
Cytokine receptor ABD
The antigen binding domain that binds to a cytokine receptor on NK cells (cytokine receptor ABD) can advantageously comprise a suitable cytokine polypeptide or polypeptide fragment such that the cytokine receptor ABD binds the cytokine receptor on the surface of an NK cell. The cytokine can for example be a full-length wild-type IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b polypeptide, a fragment thereof sufficient to bind to the NK cell receptor for such cytokine, or a variant of any of the foregoing. The cytokine molecule can be a fragment comprising at least 20, 30, 40, 50, 60, 70, 80 or 100 contiguous amino acids of a human cytokine, wherein the cytokine retains the ability to bind its cytokine receptor present on the surface of an NK cell. In certain embodiments, the cytokine is a variant of a human cytokine comprising one or more amino acid modifications (e.g. amino acid substitutions) compared to the wild-type human cytokine, for example to decrease binding affinity to a receptor present on non-NK cells, for example Treg cells, CD4 T cells, CD8 T cells. The cytokine can for example be a type I cytokine and a member of the common cytokine receptor gamma-chain (cg-chain) cytokine family, that signals via a heteromultimeric or heterdimeric receptor complex comprised of a receptor subunit (e.g., I L-2R /l L-15Rb or IL-21 R) subunit that associates with the common gamma-chain (CD132).
In one embodiment, the multispecific proteins that binds to NKp30 and optionally further CD16A incorporates a cytokine (or fragment of variant thereof) which is modified to attenuate (reduce) binding affinity at the cytokine receptor expressed on NK cells, in comparison to the human wild-type cytokine counterpart. The modified cytokine (or fragment of variant thereof) retains partial activity and/or binding affinity at the cytokine receptor expressed on NK cells, in comparison to the human wild-type cytokine counterpart. In one embodiment, the cytokine retains at least 5%, 10%, 20% or 50% of the ability of a wild-type cytokine counterpart to induce signaling through its receptor on NK cells.
In one embodiment, the multispecific proteins that binds to NKp30 and optionally further CD16A permits the incorporation of a wild-type cytokine (or fragment of variant thereof) that retain substantially full activity and/or binding affinity at the cytokine receptor expressed on NK cells, in comparison to the human wild-type cytokine counterpart. In one embodiment, the cytokine is a wild-type cytokine or fragment thereof, or is a modified cytokine, wherein the cytokine is does not have a substantially reduced ability to induce signaling and/or does not have a substantially reduced binding affinity at its receptor on NK cells (e.g. CD122, IL-21 R, IL-7Ra, IL-27Ra, IL-12R, IL-18R). In one embodiment, the cytokine does not comprise a modification (e.g., substitution, deletion, etc.) that substantially reduces its ability to induce signaling through its receptor on NK cells (e.g. CD122, IL-21 R, IL-7Ra, IL-27Ra, IL-12R, IL- 18R). In one embodiment, the cytokine retains at least 80%, 90% of the ability of a wild-type cytokine counterpart to induce signaling through its receptor on NK cells (e.g. CD122, IL-21R, IL-7Ra, IL-27Ra, IL-12R, IL-18R). Optionally, signaling is assessed by bringing the cytokine (e.g. as a recombinant protein domain or within a multispecific protein of the disclosure) into contact with an NK cell and measuring signaling, e.g. measuring ST AT phosphorylation in the NK cells.
In some embodiments, when the exemplary anti-NKp30 VH/VL pairs disclosed herein having a KD for NKp30 in the range of about 15 nM, or functionally conservative variants thereof, are used in the multispecific proteins, the cytokine or cytokine receptor ABD (either as a free cytokine or as incorporated into a multispecific protein) can be specified as binding its receptor, as determined by SPR, with a binding affinity (KD) of 200 nM or less, 100 nM or less, 50 nM or less or 25 nM or less. In one embodiment, the cytokine or cytokine receptor ABD binds its receptor, as determined by SPR, with a binding affinity (KD) that is 1 nM or higher than 1 nM, optionally that is higher than 10 nM optionally that is higher than 15 nM. In one embodiment, the cytokine or cytokine receptor ABD binds its receptor, as determined by SPR, with a binding affinity (KD) between about 1 nm and about 200 nm, optionally) between about 1 nm and about 100 nm optionally between about 10 nM and about 200 nM, optionally between about 10 nM and about 100 nM optionally between about 15 nM and about 100 nM.
When the cytokine-binding ABD is a CD122-binding ABD, the ABD can be or comprise a suitable interleukin-2 (IL-2) polypeptide such that the CD122 ABD binds CD122. As exemplified herein, the ABD is advantageously a variant or modified IL-2 polypeptide that has reduced binding to CD25 (IL-2Ra) (e.g. reduced or abolished binding affinity, for example as determined by SPR) compared to a wild-type human interleukin-2. Such a variant or modified IL-2 polypeptide is also referred herein to as an “IL2v” ora “not-alpha IL-2”. The CD122-binding ABD can optionally be specified to have a binding affinity for human CD122 that is substantially equivalent to that of wild-type human IL-2, or that is reduced compared to wild-type human IL- 2. The CD122-binding ABD can optionally be specified to have an ability to induce CD122 signaling and/or binding affinity for CD122 that is substantially equivalent to that of wild-type human IL-2. In one embodiment, the CD122-binding ABD has a reduction in binding affinity for CD25 that is greater than the reduction in binding affinity for CD122, for example a reduction of at least 1-log, 2-log or 3-log in binding affinity for CD25 and a reduction in binding affinity for CD122 that is less than 1-log.
For example, a heteromultimeric multispecific protein can be specified as being capable of binding to NKp30 and CD122, and optionally further CD16A, on an NK cell, and which is capable of potentiating NK cell cytotoxicity toward a target cell expressing an antigen of interest, and comprising:
(a) an ABD that binds to an antigen of interest,
(b) an ABD that binds to a human NKp30 polypeptide,
(c) an Fc domain or portion thereof capable of binding FcRn, and optionally further to CD16A, and
(d) an ABD that binds a human CD122 polypeptide, wherein the ABD is placed at the C-terminus of the polypeptide chain which comprises such ABD, optionally wherein such ABD comprises an IL-2 polypeptide or portion thereof that binds CD122 and displays reduced binding affinity for human CD25 respectively, compared to a human wild-type IL-2 polypeptide. The ABD that binds CD122 can thus be placed for example C-terminal and/or adjacent to the Fc domain and/or ABD that binds NKp30 on a polypeptide chain. IL-2 is believed to bind II_-2Rb (CD122) in its form as a monomeric IL-2 receptor (IL- 2R), followed by recruitment of the IL-2Ry (CD132; also termed common g chain) subunit. In cells that do not express CD25 at their surface, binding (e.g. reduced binding) to CD122 can therefore optionally be specified as being binding in or to a CD122:CD132 complex. The CD122 (or CD122:CD132 complex) can optionally be specified as being present at the surface of an NK cell. In cells that express CD25 at their surface, IL-2 is believed to bind CD25 (IL- 2Ra) in its form as a monomeric IL-2 receptor, followed by association of the subunits IL-2Rb and IL-2RY. Binding (e.g. reduced binding, partially reduced binding) to CD25 can therefore optionally be specified as being binding in or to a CD25:CD122 complex or a CD25:CD122:CD132 complex.
In a multispecific protein herein, the multispecific protein can optionally be specified as being configured and/or in a conformation (or capable of adopting a conformation) in which the CD122 ABD (e.g. IL2v) is capable of binding to CD122 at the surface of a cell (e.g. an NK cell, a CD122+CD25- cell) when the multispecific protein is bound to NKp30 (and optionally further to CD16) at the surface of said cell. Optionally further, the multispecific protein:CD122 complex is capable of binding to CD132 at the surface of said cell.
The CD122 ABD or IL2v can be a modified IL-2 polypeptide, for example a monomeric IL-2 polypeptide modified by introducing one more amino acid substitutions, insertions or deletions that decrease binding to CD25, with or without a decrease in binding to CD122.
In some embodiments, where binding to CD25 is sought to be selectively decreased, a IL-2 polypeptide can be modified by binding or associating it with one or more other additional molecules such as polymers or (poly)peptides that result in a further decrease of or abolished binding to CD25. For example a wild-type or mutated IL-2 polypeptide can be modified or further modified by binding to it another moiety that shields, masks, binds or interacts with CD25-binding site of human IL-2, thereby decreasing binding to CD25. In some examples, molecules such as polymers (e.g. PEG polymers) are conjugated to an IL-2 polypeptide to shield or mask the epitope on IL-2 that is bound by CD25, for example by introduction (e.g. substitution) to install an amino acid containing a dedicated chemical hook at a specific site on the IL-2 polypeptide. In other examples, a wild-type or variant IL-2 polypeptide is bound to anti-IL-2 monoclonal antibody or antibody fragment that binds or interacts with CD25-binding site of human IL-2, thereby decreasing binding to CD25.
In any embodiment, an IL2 polypeptide can be a full-length IL-2 polypeptide or it can be an IL-2 polypeptide fragment, so long as the fragment or IL2v that comprises it retains the specified activity (e.g. retaining at least partial CD122 binding, compared to wild-type IL-2 polypeptide). As shown herein, an IL2v polypeptide can advantageously comprise an IL-2 polypeptide comprising one or more amino acid mutations designed to reduce its ability to bind to human CD25 (IL-2Ra), while retaining at least at least some, or optionally substantially full, ability to bind human CD122.
Various IL2v or not-alpha IL-2 moieties have been described which reduce the activation bias of IL-2 on CD25+ cells. Such IL2v reduce binding to IL-2Ra and maintain at least partial binding to IL-2Rb. Several IL2v polypeptides have been described, many having mutations in amino acid residue regions 35-72 and/or 79-92 of the IL-2 polypeptide. For example, decreased affinity to IL-2Ra may be obtained by substituting one or more of the following residues in the sequence of a wild-type IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69, and L72 (amino acid residue numbering is with reference to the mature IL-2 polypeptide shown in SEQ ID NO: 213).
The wild-type mature human IL-2 protein and a wild-type mature IL-2p protein fragment lacking the three first residues APT are shown below in SEQ ID NOS: 213 and 214, respectively:
Wild-type mature human IL-2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEE VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS11STLT
(SEQ ID NO: 213).
Wild-type mature IL-2p:
SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 214).
An exemplary IL2v (also referred to herein as IL2v in the Examples) can have the amino acid of wild-type IL-2 with the five amino acid substitutions T3A, F42A, Y45A, L72G and C125A, as shown below, optionally further with deletion of the three N-terminal residues APA:
APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEE
VLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
(SEQ ID NO: 215).
As few as one or two mutations can reduce binding to IL-2Ra and E-2Rb. For example, as exemplified in the multispecific proteins herein, the IL2v polypeptide having two amino acid substitutions R38A and F42K in the wild-type IL-2p amino acid sequence displayed suitable reduced binding to IL-2Ra, with retention of binding to II_-2Rb resulting in highly active multispecific proteins, referred to herein as IL2v2.
IL2v2 (R38A/F42K substitutions):
SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 216).
In one embodiment, an IL2v polypeptide has the wild-type IL-2p amino acid sequence with the three amino acid substitutions R38A, F42K and T41A, as shown below, referred to herein as IL2v3:
IL2v3 (R38A/T41A/F42K substitutions):
SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLAKKFYMPKKATELKHLQCLEEELKPLEEVLN LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 217).
Thus, in one embodiment, an IL2 variant comprises at least one or at least two amino acid modifications (e.g. substitution, insertion, deletion) compared to a human wild type IL-2 polypeptide. In one embodiment, an IL2v comprises a R38 substitution (e.g. R38A) and an F42 substitution (e.g., F42K), compared to a human wild type IL-2 polypeptide. In one embodiment, an IL2v comprises a R38 substitution (e.g. R38A), an F42 substitution (e.g., F42K) and a T41 substitution (e.g. T41A), compared to a human wild type IL-2 polypeptide. In one embodiment, an IL2v comprises a T3 substitution (e.g. T3A), an F42 substitution (e.g., F42A), a Y45 substitution (e.g. Y45A), a L72 substitution (e.g. L72G) and a C125 substitution (e.g. C125A), compared to a human wild type IL-2 polypeptide. Optionally the IL2v comprises an amino acid sequence identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to the polypeptide of SEQ ID NOS: 213-217. Optionally the IL2v comprises a fragment of a human IL-2 polypeptide, wherein the fragment has an amino sequence identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NOS: 213-217.
Any combination of the positions can be modified. In some embodiments, the IL-2 variant comprises two or more modification. In some embodiments, the IL-2 variant comprises three or more modification. In some embodiments, the IL-2 variant comprises four, five, or six or more modifications. IL2 variant polypeptides can for example comprise two, three, four, five, six or seven amino acid modifications (e.g. substitutions). For example, US Patent No. 5,229,109, the disclosure of which is incorporated herein by reference, provides a human IL2 polypeptide having a R38A and F42K substitution. US Patent No. 9,447,159, the disclosure of which is incorporated herein by reference, describes human IL2 polypeptides having substitutions T3A, F42A, Y45A, and L72G substitutions. US Patent No. 9,266,938, the disclosure of which is incorporated herein by reference, describes human IL2 polypeptides having substitutions at residue L72 (e.g. L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K), residue F42 (e.g. F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42K); and at residue Y45 (e.g., Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R and Y45K), including for example the triple mutation F42A / Y45A / L72G to reduce or abolish the affinity for IL-2Ra receptor. Yet further W02020/057646, the disclosure of which is incorporated herein by reference, relates to amino acid sequence of IL-2v polypeptides comprising amino acid substitutions in various combinations among amino acid residues K35, T37, R38, F42, Y45, E61 and E68. Yet further, WO2020252418, the disclosure of which is incorporated herein by reference, relates to amino acid sequence of IL-2v polypeptides having at least one amino acid residues position R38, T41 , F42, F44, E62, P65, E68, Y107, or S125 substituted with another amino acid, for example wherein the amino acid substitution is selected from the group consisting of: the substitution of L19D, L19H, L19N, L19P, L19Q, L19R, L19S, L19Y at position 19, the substitution of R38A, R38F, R38G at position 38, the substitution of T41A, T41G, and T41V at position 41 , the substitution of F42A at position 42, the substitution of F44G and F44V at position 44, the substitution of E62A, E62F, E62H, and E62L at position 62, the substitution of P65A, P65E, P65G, P65H, P65K, P65N, P65Q, P65R at position 65, the substitution of E68E, E68F, E68H, E68L, and E68P at position 68, the substitution of Y107G, Y107H, Y107L and Y107V at position 107, and the substitution of S125I at position 125, the substitution of Q126E at position 126. Numbering of positions is with respect to Wild-type mature human IL-2.
A modified IL-2 can optionally be specified as exhibiting a KD for binding to CD25 or to a CD25:CD122:CD132 complex that is decreased by at least 1-log, optionally at least 2-log, optionally at least 3-log, compared to a wild-type human IL-2 polypeptide (e.g. comprising the amino acid sequence of SEQ ID NO: 213). A modified IL-2 can optionally be specified as exhibiting less than 20%, 30%, 40% or 50% of binding affinity to CD25 or to a CD25:CD122:CD132 complex compared to a wild-type human IL-2 polypeptide. An IL2 can optionally be specified as exhibiting at least 50%, 70%, 80% or 90% of binding affinity to CD122 or to a CD122:CD132 complex compared to a wild-type human IL-2 polypeptide. In some embodiments, an IL2 exhibits at least 50%, 60%, 70% or 80% but less than 100% of binding affinity to CD122 or to a CD122:CD132 complex compared to a wild-type human IL-2 polypeptide. In some embodiments, an IL2v exhibits less than 50% of binding affinity to CD25 and at least 50%, 60%, 70% or 80% of binding affinity to CD122, compared to wild-type IL-2 polypeptide.
Differences in binding affinity of wild-type and disclosed mutant polypeptide for CD25 and CD122 and complexes thereof can be measured, e.g., in standard surface plasmon resonance (SPR) assays that measure affinity of protein-protein interactions familiar to those skilled in the art.
Exemplary IL2 variant polypeptides have one or more, two or more, or three or more CD25-affinity-reducing amino acid substitutions relative to the wild-type mature IL-2 polypeptide having an amino acid sequence of SEQ ID NO: 213. In one embodiment, the exemplary IL2v polypeptides comprise one or more, two or more, or three or more substituted residues selected from the following group: Q11, H16, L18, L19, D20, D84, S87, Q22, R38, T41, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91 , I92, T123, Q126, S127, 1129, and S130.
In one embodiment, the exemplary IL2 variant polypeptide has one, two, three, four, five or more of amino acid residues position R38, T41, F42, F44, E62, P65, E68, Y107, or S125 substituted with another amino acid.
In one embodiment, decreased affinity to CD25 or a protein complex comprising such (e.g., a CD25:CD122:CD132 complex) may be obtained by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69, and L72.
In one embodiment, a CD122 ABD or IL-2 polypeptide is an IL-2 mimetic polypeptide. Synthetic IL-2/IL-15 polypeptide mimics can be computationally designed to bind to CD122, but not to CD25, for example as described in Silva et al, (2019) Nature 565(7738): 186-191 and W02020/005819, the disclosures of which are incorporated herein by reference, also provides IL-2 and IL-15 mimetic polypeptides that bind CD122 but not CD25.
For example, an IL-2 mimetic polypeptide can be characterized as a non-naturally occurring polypeptide comprising domains Xi, X2, X3, and X4, wherein:
(a) Xi is a peptide comprising the amino acid sequence at least 85% identical to EHALYDAL (SEQ ID NO: 218);
(b) X2 is a helical -peptide of at least 8 amino acids in length;
(c) X3 is a peptide comprising the amino acid sequence at least 85% identical to YAFNFELI (SEQ ID NO: 219);
(d) X4 is a peptide comprising the amino acid sequence at least 85% identical to ITILQSWIF (SEQ ID NO: 220); wherein Xi, X2, X3, and X* may be in any order in the polypeptide, wherein amino acid linkers may be present between any of the domains, and wherein the polypeptide binds to CD122 (or to the CD122:CD132 heterodimer). Optionally, the polypeptides bind the CD122:CD132 heterodimer with a binding affinity of 200 nM or less, 100 nM or less, 50 nM or less or 25 nM or less.
In one aspect, the invention provides non-naturally occurring polypeptides comprising domains Xi, X2, X3, and X4, wherein:
(a) Xi is a peptide comprising the ammo acid sequence EHALYDAL (SEQ ID NO: 218);
(b) X2 is a helical-peptide of at least 8 amino acids in length;
(c) X3 is a peptide comprising the amino acid sequence YAFNFELI (SEQ ID NO: 219);
(d) X4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ ID NO: 220); wherein Xi, X2, X3, and X4 may be in any order in the polypeptide, wherein amino acid linkers may be present between any of the domains, and wherein the polypeptide binds to CD122 (or to the CD122:CD132 heterodimer). Optionally, the polypeptides bind the CD122:CD132 heterodimer with a binding affinity of 200 nM or less, 100 nM or less, 50 nM or less or 25 nM or less, optionally between about 1 nm and about 100 nm, optionally between about 10 nM and about 200 nM, optionally between about 10 nM and about 100 nM optionally between about 15 nM and about 100 nM.
In one example, Xi, X3, and X4 may be any suitable length, meaning each domain may contain any suitable number of additional amino acids other than the peptides of SEQ I D NOS: 218, 219 and 220, respectively. In one embodiment, Xi is a peptide comprising the amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along its length to the peptide
PKKKIQLHAEHALYDALMILNI (SEQ ID NO: 221); X3 is a peptide comprising the amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along its length the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO: 222); and X4 is a peptide comprising the amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical along its length to the amino acid sequence EDEQEEMANAIITILQSWIFS (SEQ ID NO: 223).
In one example, a computationally designed synthetic IL-2 polypeptide or mimetic (or CD122 ABD) comprises the amino acid sequence (neoleukin) shown below (with or without a (084)3 domain linker:
PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKA KRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 224) or
GSSSSGSSSSGSSSSPKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIA RLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWI FS (SEQ ID NO: 225).
In yet other examples, an IL-2 polypeptide is modified by connecting, fusing, binding or associating it with one or more other additional compounds, chemical compounds, polymer (e.g. PEG), or polypeptides or polypeptide chains that result in a decrease of binding to CD25. For example a wild-type IL-2 polypeptide or fragment thereof can be modified by binding to it a CD25 binding peptide or polypeptide, including but not limited to an anti-IL-2 monoclonal antibody or antibody fragment thereof that binds or interacts with CD25-binding site of human IL-2, thereby decreasing binding to CD25.
In one example, an IL-2 further comprises a receptor domain, e.g., a cytokine receptor domain. In one embodiment, the cytokine molecule comprises an IL-2 receptor, or a fragment thereof (e.g., an IL-2 binding domain of an IL-2 receptor alpha). In one example, a CD25- derived polypeptide is fused to an IL-2 polypeptide, as described in Lopes et al, J Immunother Cancer. 2020; 8(1), the disclosure of which is incorporated herein by reference. In one example, the IL-2 is a variant fusion protein comprising a circularly permuted (cp) IL-2 fused to a CD25 polypeptide (see e.g., PCT publication no. WO2020/249693, the disclosure of which is incorporated herein by reference). Where the CD122 ABD comprises a circularly permuted (cp) IL-2 fused to a CD25 polypeptide, the ABD can comprise a cplL-2:IL-2Ra polypeptide or protein of described in PCT publication no. WO2013/184942, the disclosure of which is incorporated herein by reference. For example, the permuted (cp) IL-2 variant fused to a CD25 polypeptide can have the amino acid sequence:
SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGSS STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK PLEEVLNLAQGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLY MLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLP GHCREPPPWENEATERIYHFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWT QPQLICTG (SEQ ID NO: 226), or an amino acid sequence having sequence identity that is about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher over a contiguous stretch of about 20 amino acids up to the full length of SEQ ID NO: 226.
In one example, IL-2 is associated with a specific anti-IL-2 monoclonal antibody (mAb), thus forming an I L-2/anti-l L-2 mAb complex (IL-2cx). Such complexes have been shown to overcome the CD25 binding (Boyman et al., Science 311, 1924-1927 (2006). An exemplary anti-IL2 antibody is antibody NARA1. PCT publication no. W02017/122130, the disclosure of which is incorporated herein by reference, describes fusion proteins in which flexible linkers are used to connect IL-2 to the variable region of the light or heavy chain of NARA1. Sahin et al. (Nature Communications volume 11, Article number: 6440 (2020)) describe an improved construct in which IL-2 is brought into contact with and bound to the complementarity determining region 1 of the light chain (L-CDR1) of NARA1 , which resulted in a protein complex in which the IL-2 is bound to its antigen-binding groove on the antibody fragment (or the polypeptide chain(s) of the fragment).
In other examples, an IL-2 polypeptide or fragment thereof can be modified by binding to it a moiety of interest (e.g. a compound, chemical compounds, polymer, linear or branched PEG polymer), covalently attached to a natural amino acid or to an unnatural amino acid installed at a selected position. Such a modified interleukin 2 (IL-2) polypeptide can comprise at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-2 polypeptide and CD25 but retains significant binding to the CD122:CD132 signaling complex, wherein the reduced binding to CD25 is compared to binding between a wild-type IL-2 polypeptide and CD25. An unnatural amino acid can be positioned at any one or more of residues K35, T37, R38, T41 , F42, K43, F44, Y45, E60, E61 , E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107 of IL-2. As disclosed in PCT publication nos. WO2019/028419 and WO2019/014267, the disclosures of which are incorporated herein by reference, the unnatural amino acid can be incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNA pair. The unnatural amino acid can for example comprise a lysine analogue, an aromatic side chain, an azido group, an alkyne group, or an aldehyde or ketone group. The modified IL-2 polypeptide can then be covalently attached to a water-soluble polymer, a lipid, a protein, or a peptide through the unnatural amino acid. Examples of suitable polymers include polyethylene glycol (PEG), polypropylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), polyplefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly (a- hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), or a combination thereof, or a polysaccharide such as dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).
In some examples, an exemplary IL2v/not-alpha IL-2 conjugate can comprise a full- length or fragment of an IL-2 polypeptide in which an amino acid residue in the IL-2 polypeptide (e.g. a residue at position selected from K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, and L72) is replaced by a natural or non-natural amino acid residue attached to a polymer via a chemical linker. The polymer can be a PEG polymer, e.g. a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa.
A modified IL2 polypeptide can comprising at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-2 polypeptide and CD25 but retains significant binding with CD122:CD132 signaling complex to form a CD122:CD132 complex, wherein the reduced binding to CD25 is compared to binding between a wild-type IL-2 polypeptide and CD25 An exemplary N2v/not-alpha IL-2 conjugate is THOR-707 (Synthorx inc).
For example, as described in PCT publication no. W02020/163532, the disclosure of which is incorporated herein by reference, an amino acid residue in the IL-2 conjugate is replaced by the structure of Formula (I):
Figure imgf000103_0001
Y is CH2 and Z is
Figure imgf000103_0002
or,
Y is CH2 and Z is and wherein, W is a PEG group having an average molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa; and X has the structure:
Figure imgf000104_0001
In one embodiment, an IL-2 comprises a releasable polymer (e.g. a releasable PEG polymer), e.g. the IL-2 is conjugated, linked or bound to a releasable polymer that results in a decrease in CD25 binding in vivo and/or in vitro. Example of such modified IL-2 include bempegaldesleukin or RSLAIL-2 (Nektar Therapeutics inc.), which exhibits about a 60-fold decrease in affinity to CD25 relative to IL-2, but only about a 5-fold decrease in affinity CD122 relative to IL-2. “Bempegaldesleukin” (CAS No.1939126-74-5) is an IL-2 in which human interleukin-2 (des-1-alanine, 125-serine), is N-substituted with an average of six [(2,7- bis{[methylpoly(oxyethylene)iokD]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl moieties at its amino residues. As disclosed in PCT publication no. W02020/095183, the disclosure of which is incorporated herein by reference, the releasable PEG comprised can be based upon a 2,7,9-substituted fluorene, with poly(ethylene glycol) chains extending from the 2- and 7- positions on the fluorene ring via amide linkages (fluorene-C(O)-NH-), and having releasable covalent attachment to IL-2 via attachment to a carbamate nitrogen atom attached via a methylene group (-CH2-) to the 9-position of the fluorene ring. The modified IL-2 can comprise compounds encompassed by the following formula:
Figure imgf000104_0002
or wherein each “n”, the number of CH2CH2O units) is an integer from about 3 to about 4000, or more preferably is an integer from about 200-300. “m”, referring to the number of polyethylene glycol moieties attached to IL-2 is an integer selected from the group consisting of 1, 2, 3, 7 and more than 7. In some embodiments, each “n” is approximately the same, i.e. , the weight average molecular weight of each polyethylene glycol “arm” covalently attached to the fluorenyl core is about the same. Optionally, the weight average molecular weight of each PEG arm is about 10,000 daltons, such that the weight average molecular weight of the overall branched polymer moiety is about 20,000 daltons.
In another embodiment where the cytokine-binding ABD is a CD122-binding ABD, the ABD can be or comprise a suitable interleukin-15 (IL-15) polypeptide such that the CD122 ABD binds CD122. In some embodiments, the cytokine molecule is an IL-15 molecule, e.g., a full length, a fragment or a variant (I L-15v) of IL-15, e.g., human IL-15. In some embodiments, the IL-15 molecule comprises a wild-type human IL-15 amino acid sequence, e.g., having the amino acid sequence of SEQ ID NO: 227. In some embodiments, the IL-15 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-15 amino acid sequence of SEQ ID NO: 227. In other embodiments, the IL-15 molecule is a variant of human IL-15, e.g., having one or more amino acid modifications. Optionally the IL-15 comprises a fragment of a human IL-15 polypeptide, wherein the fragment has an amino sequence is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the wild-type mature human IL-15 polypeptide of SEQ ID NO: 227.
Wild-type mature human IL-15:
NW VNVISDLKKI EDLIQSMHID ATLYTESDVH PSCKVTAMKC FLLELQVISL ESGDASIHDT VENLIILANN SLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS (SEQ ID NO: 227).
In some embodiments, an IL-15 variant comprises a modification (e.g. substitution) at position 45, 51 , 52, or 72 (with reference to the sequence of human IL-15, SEQ ID NO: 227), e.g., as described in US 2016/0184399. In some embodiments, the IL-15 variant comprises four, five, or six or more modifications. In some embodiments, the IL-15 variant comprises one or more modification at amino acid position 8, 10, 61, 64, 65, 72, 101, or 108 (with reference to the sequence of human IL-15, SEQ ID NO: 227). In some embodiments the IL-15 variant possesses increased affinity for CD122 as compared with wild-type IL-15. In some embodiments the IL-15 variant possesses decreased affinity for CD122 as compared with wild-type IL-15. In some embodiments, the mutation is chosen from D8N, K10Q, D61 N, D61 H, E64H, N65H, N72A, N72H, Q101N, Q108N, or Q108H (with reference to the sequence of human IL-15, SEQ ID NO: 227). Any combination of the positions can be mutated. In some embodiments, the IL-15 variant comprises two or more mutations. In some embodiments, the IL-15 variant comprises three or more mutations. In some embodiments, the IL-15 variant comprises four, five, or six or more mutations. In some embodiments the IL-15 variant comprises mutations at positions 61 and 64. In some embodiments the mutations at positions 61 and 64 are D61 N or D61 H and E64Q or E64H. In some embodiments the IL-15 variant comprises mutations at positions 61 and 108. In some embodiments the mutations at positions 61 and 108 are D61 N or D61 H and Q108N or Q108H.
The extracellular domain of IL-15Ra comprises a domain referred to as the sushi domain, which binds IL-15. The general sushi domain, also referred to as complement control protein (CCP) modules or short consensus repeats (SCR), is a protein domain found in several proteins, including multiple members of the complement system. The sushi domain adopts a beta-sandwich fold, which is bounded by the first and fourth cysteine of four highly conserved cysteine residues, comprising a sequence stretch of approximately 60 amino acids (Norman, Barlow, et al. J Mol Biol. 1991 ;219(4):717-25). The amino acid residues bounded by the first and fourth cysteines of the sushi domain IL-15Ra comprise a 62 amino acid polypeptide referred to as the minimal domain. Including additional amino acids of IL-15Ra at the N- and C-terminus of the minimal sushi domain, such as inclusion of N-terminal lie and Thr and C- terminal lie and Arg residues result in a 65 amino acid extended sushi domain.
The CD122 ABD can further comprise a receptor domain, e.g., a cytokine receptor domain. In one embodiment, the cytokine molecule comprises an IL-15 receptor, or a fragment thereof (e.g., an IL-15 binding domain of an IL-15 receptor alpha).
In some embodiments, the CD122 ABD binds an IL-15 receptor alpha (IL-15Ra) sushi domain, a first domain linker, and an IL-15 polypeptide, e.g. from N- to C-terminus, a IL-15Ra sushi domain fused to a domain linker, in turn fused to an IL-15 polypeptide. Optionally the IL- 15 polypeptide is a variant IL-15 polypeptide, e.g., comprising one or more amino acid substitutions. In other embodiments, the variant IL-15 domain comprises the amino acid sequence of SEQ ID NO: 227 and amino acid substitutions selected from the group consisting of N4D/N65D, D30N/N65D, and D30N/E64Q/N65D. A sushi domain as described herein may comprise one or more mutations relative to a wild-type sushi domain. In some embodiments, the IL-l5Ra sushi domain comprises the amino acid sequence:
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
(SEQ ID NO: 228).
An IL-15 polypeptide can be modified by connecting, fusing, binding or associating it with one or more other additional compounds using any of several known techniques, for example by conjugation or binding to chemical compounds, polymer (e.g. PEG), or polypeptides or polypeptide chains that result in a decrease of binding to IL-15Ra. In one example, a wild-type IL-15 polypeptide or fragment thereof can be modified by binding to it a IL-15Ra binding peptide or polypeptide, including but not limited to an anti-IL-15 monoclonal antibody or antibody fragment thereof that binds or interacts with IL-15Ra -binding site of human IL-15, thereby decreasing binding to IL-15Ra.
In another example, an IL-15 polypeptide or fragment thereof can be modified by binding to it a moiety of interest (e.g. a compound, chemical compounds, polymer, linear or branched PEG polymer), covalently attached to a novel amino acid installed at a selected position. Such a modified IL-15 polypeptide can comprise at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-15 polypeptide and IL-15Ra but retains significant binding with CD122:CD132 signaling complex to form an CD122:CD132 complex, wherein the reduced binding to IL-15Ra is compared to binding between a wild-type IL-15 polypeptide and IL-15Ra. The unnatural amino acid can be positioned at any one or more of residues N1, W2, V3, N4, 16, S7, D8, K10, K11, E13, D14, L15, Q17, S18, M19, H20, 121, D22, A23, T24, L25, Y26, E28, S29, D30, V31, H32, P33, S34, C35, K36, V37, T38, K41, L44, E46, Q48, V49, S51, L52, E53, S54, G55, D56, A57, S58, H60, D61 , T62, V63, E64, N65, I67, I68, L69, N71, N72, S73, L74, S75, S76, N77, G78, N79, V80, T81 , E82, S83, G84, C85, K86, E87, C88, E89, E90, L91 , E92, E93, K94, N95, 196, K97, E98, L100, Q101, S102, V104, H105, Q108, M109, F110, 1111, N112, T113, and S114 of IL- 15. As disclosed in WO2019165453, WO2019/028419 and WO2019/014267, the disclosures of which are incorporated herein by reference, the unnatural amino acid can be incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNA pair. The unnatural amino acid can for example comprise a lysine analogue, an aromatic side chain, an azido group, an alkyne group, or an aldehyde or ketone group. The modified IL-15 polypeptide can then be covalently attached to a water-soluble polymer, a lipid, a protein, or a peptide through the unnatural amino acid. Examples of suitable polymers include polyethylene glycol (PEG), polypropylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, polypxyethylated polyol), polyplefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly (a- hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), or a combination thereof, or a polysaccharide such as dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).
For example, as described in W02020/163532, the disclosure of which is incorporated herein by reference, an amino acid residue in the IL-15 conjugate is replaced by the structure of Formula (I):
Figure imgf000108_0001
Y is CH2 and Z is
Figure imgf000108_0003
or,
Y is CH2 and Z is
Figure imgf000108_0002
and wherein, W is a PEG group having an average molecular weight selected from
5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa; and
X has the structure: In one embodiment, an IL-15 comprises a releasable polymer (e.g. a releasable PEG polymer), e.g. the IL-15 is conjugated, linked or bound to a releasable polymer that results in a decrease in IL-15R binding in vivo and/or in vitro. Examples include compounds disclosed in PCT publication no. W02020/097556, the disclosure of which is incorporated herein by reference. For example, the modified IL-15 can comprise comprising compound having the structure:
Figure imgf000109_0001
wherein (n) is an integer from about 150 to about 3,000, (m) is an integer selected from 2, 3, 4, and 5, (n’) is 1, and ~NH~ represents an amino group of the IL-15 polypeptide.
In another embodiment where the cytokine-binding ABD binds an IL-21 receptor (IL- 21R)-binding ABD, the ABD can be or comprise a suitable interleukin-21 (IL-21) polypeptide such that the IL-21 R ABD binds IL-21 R on the surface of NK cells. IL-21 R is similar in structure to the IL-2 receptor and the IL-15 receptor, in that each of these cytokine receptors comprises a common gamma chain (yc). In some embodiments, the cytokine molecule is an IL-21 molecule, e.g., a full length, a fragment or a variant of IL-21, e.g., human IL-21. In embodiments, the IL-21 molecule is a wild-type, human IL-21 , e.g., having the amino acid sequence of SEQ ID NO: 229. In some embodiments, the IL-15 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-21 amino acid sequence of SEQ ID NO: 229. In other embodiments, the IL-21 molecule is a variant of human IL-21, e.g., having one or more amino acid modifications. Optionally the IL-21 comprises a fragment of a human IL-21 polypeptide, wherein the fragment has an amino sequence is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 229.
Wild-type mature human IL-21:
HKSSSQ GQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DS (SEQ ID NO: 229) . In some embodiments, the IL-21 variant can comprise an IL-21 polypeptide comprising one or more amino acid mutations designed to reduce its ability to bind to human IL-21 R, while retaining substantial ability to bind human IL-21 R. For example, the IL-21 can be characterized as binding to human IL-21 R with a KD that is greater than or is about 0.04 nM, as determined by SPR.
Examples of such IL-21 variants are provided in PCT publication no. WO2019028316, the disclosure of which is incorporated herein by reference. In exemplary aspects, the amino acid substitutions are located at two amino acid positions selected from the group consisting of 10, 14, 20, 75, 76, 77, 78 and 81 according to numbering of SEQ ID NO: 229 or at two amino acid positions selected from the group consisting of 5, 9, 15, 70, 71 , 72, 73, and 76, according to the amino acid position numbering of SEQ ID NO: 230. In exemplary aspects, the IL-21 variant comprises the amino acid sequence:
QGQDX HMXXM XXXXX XVDXL KNXVN DLVPE FLPAP EDVET NCEWS AFSCF QKAQL KSANT GNNEX XIXXX XXXLX XXXXX TNAGR RQKHR LTCPS CDSYE KKPPK EFLXX FXXLL XXMXX QHXSS RTHGS EDS (SEQ ID NO: 230), wherein X represents any amino acid, and wherein the IL-21 variant amino acid sequence differs from the amino acid sequence of human IL-21 (SEQ ID NO: 229) by at least 1 amino acid.
In exemplary aspects, the IL-21 variant comprises the sequence of SEQ ID NO: 230, wherein SEQ ID NO: 230 differs from SEQ ID NO: 229 by at least one amino acid at a position designated by X in SEQ ID NO: 230. In exemplary aspects, the IL-21 variant has at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NO: 229.
In exemplary embodiments, the IL-21 variant comprises an amino acid substitution relative to the wild-type IL-21 amino acid sequence within the N-terminal half of the amino acid sequence, e.g. at a position within positions 10-30 or 13-28 (both inclusive), according to the amino acid position numbering of SEQ ID NO: 229. In other exemplary embodiments, the IL- 21 variant comprises an amino acid substitution relative to the wild-type IL-21 amino acid sequence within the C-terminal half of the amino acid sequence, e.g., at a position within positions 105-138 or 114-128 (both inclusive), according to the amino acid position numbering of SEQ ID NO: 229. In other exemplary embodiments, the IL-21 variant comprises an amino acid substitution relative to the wild-type IL-21 amino acid sequence in the middle third of the amino acid sequence, e.g., at a position within positions 60-90 or 70-85 (both inclusive), according to the amino acid position numbering of SEQ ID NO: 229. Optionally, the IL-21 variant comprises only one amino acid substitution, relative to the wild-type IL-21 amino acid sequence. Optionally, the amino acid substitution is located at an amino acid position selected from the group consisting of: 10, 13, 14, 16, 17, 18, 19, 20, 21, 24, 28, 70, 71, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 114, 115, 117, 118, 121, 122, 124, 125, or 128, according to the amino acid position numbering of SEQ ID NO: 229.
In another embodiment where the cytokine-binding ABD binds an IL-18 receptor (IL- 18Ra)-binding ABD, the ABD can be or comprise a suitable interleukin-18 (IL-18) polypeptide such that the IL-18R ABD binds IL-18Ra on the surface of NK cells. In some embodiments, the cytokine molecule is an IL-18 molecule, e.g., a full length, a fragment or a variant of IL-18, e.g., human IL-18. In embodiments, the IL-18 molecule is a wild-type, human IL-18, e.g., having the amino acid sequence of SEQ ID NO: 422. In some embodiments, the IL-18 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-18 amino acid sequence of SEQ ID NO: 231. In other embodiments, the IL-18 molecule is a variant of human IL-18, e.g., having one or more amino acid modifications. Optionally the IL-18 comprises a fragment of a human IL-18 polypeptide, wherein the fragment has an amino sequence is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 231.
Wild-type mature human IL-18:
YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKC EKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKL ILKKEDELGDRSIMFTVQNED (SEQ ID NO: 231).
In one embodiment, an IL-18 is modified to decrease its binding affinity for IL-18BP while not substantially decreasing affinity for IL-18Ra. For example, an IL-18 may comprise of a modification such as amino acid substitutions at positions M51 , S55, R104 and/or N 110 that are not involved in IL-18Ra binding, optionally further in combination with a substitution at K53 and/or M60 (positions are with reference to the wild-type mature IL-18 amino acid sequence). In one embodiment, the IL-18 has a M51S, S55A, R104Q, R104K or R104S and/or N110A substitution. In one embodiment, the IL-18 comprises a K53S or K53A substitution. In one embodiment, the IL-18 comprises a M60S or M60K substitution.
In another embodiment, the cytokine-binding ABD binds a type I interferon receptor, for example interferon-a receptor (IFN-aR). The ABD can be or comprise a suitable type I interferon, for example an interferon-a (IFN-a) or interferon-b (IFN-b) polypeptide such that the IFN-a ABD binds IFN-aR on the surface of NK cells. The interferon-a receptor is also known as the interferon a/b receptor (IFNAR), a heterodimeric transmembrane receptor that is composed of the two subunits IFNAR1 and IFNAR2. For type I IFNs, the main STAT signaling complex is formed by IFN-stimulated gene factor 3 consisting of STAT 1 , STAT2, and IFN regulatory factor (IRF)-9. In some embodiments, the cytokine molecule is an IFN-a or IFN- b molecule, e.g., a full length, a fragment or a variant of IFN-a or IFN-b, e.g., human IFN-a or IFN-b, for example a human IFN-a1, IFN-a2, IFN-a4, IFN-a5, IFN-a6, IFN-a7, IFN-a8, IFN- a10, IFN-a12, IFN-a14, IFN-a16 or IFN-a17 polypeptide. In some embodiments, the IFN-a or IFN-b molecule is a wild-type, human IFN-a or IFN-b, e.g., having the amino acid sequence of any of SEQ ID NOS: 232-243. In other embodiments, the IFN-a or IFN-b molecule is a variant of human IFN-a or IFN-b, e.g., having one or more amino acid modifications. In some embodiments, the IFN-a or IFN-b molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IFN-a or IFN-b amino acid sequence of SEQ ID NOS: 232-243, respectively. In other embodiments, the IFN-a or IFN-b molecule is a variant of human IFN-a or IFN-b, e.g., having one or more amino acid modifications. Optionally the IFN-a or IFN-b comprises a fragment of a human IFN-a or IFN-b polypeptide, wherein the fragment has an amino sequence is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NOS: 232-243.
Wild-type human IFN-a mature proteins:
IFNa2
CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFS TKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYS PCAWEVVRAEIMRSFSLSTNLQESLRSKE (SEQ ID NO: 232).
IFNal
CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLF TTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKY SPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO: 233).
IFNa4
CDLPQTHSLGNRRALILLAQMGRISHFSCLKDRHDFGFPEEEFDGHQFQKTQAISVLHEMIQQTFNLF STEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLTEKKY SPCAWEVVRAEIMRSLSFSTNLQKRLRRKD (SEQ ID NO: 234).
IFNa5 CDLPQTHSLSNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLF STKDSSATWDETLLDKFYTELYQQLNDLEACMMQEVGVEDTPLMNVDSILTVRKYFQRITLYLTEKKY SPCAWEVVRAEIMRSFSLSANLQERLRRKE (SEQ ID NO: 235).
IFNa6
CDLPQTHSLGHRRTMMLLAQMRRISLFSCLKDRHDFRFPQEEFDGNQFQKAEAISVLHEVIQQTFNLF STKDSSVAWDERLLDKLYTELYQQLNDLEACVMQEVWVGGTPLMNEDSILAVRKYFQRITLYLTEKKY SPCAWEVVRAEIMRSFSSSRNLQERLRRKE (SEQ ID NO: 236).
IFNa7
CDLPQTHSLRNRRALILLAQMGRISPFSCLKDRHEFRFPEEEFDGHQFQKTQAISVLHEMIQQTFNLF STEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDFILAVRKYFQRITLYLMEKKY SPCAWEVVRAEIMRSFSFSTNLKKGLRRKD (SEQ ID NO: 237).
IFNa8
CDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFEFPQEEFDDKQFQKAQAISVLHEMIQQTFNLF STKDSSAALDETLLDEFYIELDQQLNDLESCVMQEVGVIESPLMYEDSILAVRKYFQRITLYLTEKKY SSCAWEVVRAEIMRSFSLSINLQKRLKSKE (SEQ ID NO: 238).
IFNalO
CDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQFQKAQAISVLHEMIQQTFNLF STEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLIERKY SPCAWEVVRAEIMRSLSFSTNLQKRLRRKD (SEQ ID NO: 239).
IFNa14
CNLSQTHSLNNRRTLMLMAQMRRISPFSCLKDRHDFEFPQEEFDGNQFQKAQAISVLHEMMQQTFNLF STKNSSAAWDETLLEKFYIELFQQMNDLEACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLMEKKY SPCAWEVVRAEIMRSLSFSTNLQKRLRRKD (SEQ ID NO: 240).
IFNa16
CDLPQTHSLGNRRALILLAQMGRISHFSCLKDRYDFGFPQEVFDGNQFQKAQAISAFHEMIQQTFNLF STKDSSAAWDETLLDKFYIELFQQLNDLEACVTQEVGVEEIALMNEDSILAVRKYFQRITLYLMGKKY SPCAWEVVRAEIMRSFSFSTNLQKGLRRKD (SEQ ID NO: 241).
IFNa17 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGLPQEEFDGNQFQKTQAISVLHEMIQQTFNLF STEDSSAAWEQSLLEKFSTELYQQLNNLEACVIQEVGMEETPLMNEDSILAVRKYFQRITLYLTEKKY SPCAWEVVRAEIMRSLSFSTNLQKILRRKD (SEQ ID NO: 242).
In certain aspects, the IFN-a or IFN-b variant polypeptide has an amino acid sequence that has at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has greater than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity to SEQ ID NOS: 232-243, respectively.
In some embodiments, the wild type or modified signaling agent is a modified interferon-a having decreased binding affinity for its receptor, particularly IFNAR2. In such embodiments, the modified IFNal , IFNa2, IFNa4, IFNa5, IFNa6, IFNa7, IFNa8, IFNa10, IFNa12, IFNa14, IFNa16 or IFNa17 agent has reduced affinity for and/or induction of signaling at IFNAR (IFNAR1 and/or IFNAR2 chains).
With the exception of wild-type IFNal, wild-type IFNs bind to IFNAR2 at affinities (KD), e.g., as determined by microcal or SPR, between 0.1 nM and 5nM and to IFNAR1 at an affinity of about 1 mM. IFNal binds to IFNAR2 with a KD of about 200 nM. In some embodiments, an IFN is modified so as to have an affinity for IFNAR1 and/or IFNAR2 that is equal or less than that of the NKp30 ABD for NKp30. In some embodiments, an IFN is modified so as to have an affinity for IFNAR1 and/or IFNAR2 that is at least 1-log less than that of the NKp30 ABD for NKp30.
For example, in the exemplary NKp30 ABDs shown herein, the NKp30 ABD has a KD for NKp30 binding of about 15 nM. An IFN can thus be modified by introduction of a modification that causes a reduction of binding affinity of between 10-fold (1-log) and 1000- fold (3-log) (an increase in KD of between 1-log and 3-log). An IFN can include any of the amino acid substitutions shown in the table below. The table below shows exemplary single amino acid substitutions that decrease binding affinity of IFN-a polypeptides to IFNAR2, with a cut-off of a decrease in affinity (higher KD) of at least 1 log compared to the wild-type counterpart and no more than 3-log compared to the wild-type counterpart. The table below shows the relative affinity based on KD values for IFNAR2 of the mutated cytokine compared to the wild-type cytokine.
Figure imgf000114_0001
Figure imgf000115_0001
The table below shows exemplary single amino acid substitutions decreasing binding affinity of IFN-a polypeptides to IFNAR1, with an at least 2-fold decrease in affinity. The table shows the relative affinity based on KD values for IFNAR1 of the mutated cytokine compared to the wild-type cytokine.
Figure imgf000115_0002
Mutant forms of IFNa2 are also described for example in PCT publication nos. W02008/124086, WO2010/030671 , WO2013/059885, WO2013/107791, W02015/007520 and W02020/198661, the disclosures of which are incorporated hereby by reference.
In some embodiments, said IFNa2 mutant (IFNa2a or IFNa2b) is mutated at one or more amino acids at positions 144-154, such as amino acid positions 148, 149 and/or 153. In some embodiments, the I FNa2 mutant comprises one or more mutations selected from L153A, R149A, and M148A, described in WO2013/107791. In some embodiments, the IFNa2 mutants have reduced affinity and/or activity for IFNAR1. In some embodiments, the IFNa2 mutant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A, as described in WO2010/030671. In some embodiments, the IFNa2 mutant comprises one or more mutations selected from K133A, R144A, R149A, and L153A as described in W02008/124086. In some embodiments, the IFNa2 mutant comprises one or more mutations selected from R120E and R120E/K121E, as described in W02015/007520 and WO20 10/030671. In one embodiment, the mutant human IFNa2 comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 232, wherein the mutant IFNa2 has one or more mutations at positions L15, A19, R22, R23, L26, F27, L30, L30, K31, D32, R33, H34, D35, Q40, H57, E58, Q61 , F64, N65, T69, L80, Y85, Y89, D 114, L117, R120, R125, K 133, K 134, R144, A145, M 148, R149, S 152, L153, and N156 with respect to SEQ ID NO: 232. In some embodiments, the human IFNa2 mutant comprises one or more mutations selected from, L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, T106A, T106E, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A as disclosed in WO 2013/059885, for example in some embodiments, the human IFNa2 mutant comprises the mutations H57Y, E58N, Q61S, and/or L30A; the mutations H57Y, E58N, Q61S, and/or R33A; the mutations H57Y, E58N, Q61S, and/or M148A; the mutations H57Y, E58N, Q61S, and/or L153A; the mutations N65A, L80A, Y85A, and/or Y89A; or the mutations N65A, L80A, Y85A, Y89A, and/or D114A.
In embodiments, the wild type or modified signaling agent is a modified interferon-a having decreased binding affinity for its receptor, particularly IFNAR2. In such embodiments, the modified IFNa2 agent has reduced affinity for and/or induction of signaling at IFNAR (IFNAR1 and/or IFNAR2 chains).
In some embodiments, the IFNal interferon is modified to have a mutation at one or more amino acids at positions L15, A19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154, and N157 with reference to SEQ ID NO: 233. The mutations can optionally be a hydrophobic mutation and can be, e.g., selected from alanine, valine, leucine, and isoleucine. In some embodiments, the FNa1 interferon is modified to have a one or more mutations selected from L15A, A19W, R23A, S25A, L30A, L30V, D32A, R33K, R33A, R33Q, H34A, Q40A, C86S, C86A, D115R, L118A, K121A, K121 E, R126A, R126E, E133A, K134A, K135A, R145A, R145D, R145E,
R145G, R145H, R145I, R145K, R145L, R145N, R145Q, R145S, R145T, R145V, R145Y,
A146D, A146E, A146G, A146H, A146I, A146K, A146L, A146M, A146N, A146Q, A146R,
A146S, A146T, A146V, A146Y, M149A, M149V, R150A, S153A, L154A, and N157A with reference to SEQ ID NO: 233. In some embodiments, the FNa1 mutant comprises one or more multiple mutations selected from L30A/H58Y/E59N/Q62S, R33A/H58Y/E59N/Q62S,
M 149A/H 58Y/E59N/Q62S, L154A/H58Y/E59N/Q62S, R145A/H58Y/E59N/Q62S, D115A/R121A, L118A/R121A, L118A/R121 A/K122A, R121A/K122A, and R121 E/K122E with reference to SEQ ID NO: 233. In some embodiments, the IFN-a1 is a variant that comprises one or more mutations which reduce undesired disulphide pairings wherein the one or more mutations are, e.g., at amino acid positions C1, C29, C86, C99, or C139 with reference to SEQ ID NO: 233. In some embodiments, the mutation at position C86 can be, e.g., C86S or C86A or C86Y.
In embodiments, the wild type or modified signaling agent is IFN-b. In some embodiments, the IFN-b is human having a sequence as shown below:
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFA IFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAK EYSHCAWTIVRVEILRNFYFINRLTGYLRN (SEQ ID NO: 243).
In some embodiments, the human IFN-b is a non-glycosylated form of human IFN-b that has a Met-1 deletion and a Cys-17 to Ser mutation. In various embodiments, the modified IFN-b has one or more mutations that reduce its binding to or its affinity for the IFNAR1 subunit of IFNAR. In one embodiment, the modified IFN-b has reduced affinity and/or activity at IFNAR1. In various embodiments, the modified IFN-b is human IFN-b and has one or more mutations at positions F67, R71, L88, Y92, 195, N96, K123, and R124. In some embodiments, the one or more mutations are substitutions selected from F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G, and R124G.
In some embodiments, the modified IFN-b has one or more mutations that reduce its binding to or its affinity for the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFN-b has reduced affinity and/or activity at IFNAR2. In various embodiments, the modified IFN-b is human IFN-b and has one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155. In some embodiments, the one or more mutations are substitutions selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G.
Exemplary IFN-b mutations are described in PCT publication nos. W02020/198661 , W02000/023114 and US20150011732 the disclosures of which are incorporated hereby by reference.
In another embodiment where the cytokine-binding ABD binds an IL-7 receptor (IL- 7R)-binding ABD, the ABD can be or comprise a suitable interleukin-7 (IL-7) polypeptide such that the IL-7R ABD binds IL-7Ra on the surface of NK cells. In some embodiments, the cytokine molecule is an IL-7 molecule, e.g., a full length, a fragment or a variant of IL-7, e.g., human IL-7. In embodiments, the IL-7 molecule is a wild-type, human IL-7, e.g., having the amino acid sequence of SEQ ID NO: 244. In some embodiments, the IL-7 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild- type human IL-7 amino acid sequence of SEQ ID NO: 244. In other embodiments, the IL-7 molecule is a variant of human IL-7, e.g., having one or more amino acid modifications. Optionally the IL-7 comprises a fragment of a human IL-7 polypeptide, wherein the fragment has an amino sequence is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 244.
Wild-type mature human IL-7:
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDF
DLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK
EH (SEQ ID NO: 244).
Wild-type IL-7 bind to IL-7Ra with an affinity (KD), e.g., as determined by microcal or SPR, of between about 50-100 nM. In some embodiments, an IL-7 is modified so as to have an affinity for IL-7Ra that is equal or less than that of the NKp30 ABD for NKp30. In some embodiments, an IL-7 is modified so as to have an affinity for IL-7Ra that is at least 1-log less than that of the NKp30 ABD for NKp30. In some embodiments, an IL-7 is modified so as to have an affinity for IL-7Ra that is at least 1-log less than that of the NKp30 ABD for NKp30, but no more than 3-log, or optionally 2-log, less than that of the NKp30 ABD for NKp30. For example, in the exemplary NKp30 ABDs shown herein, the NKp30 ABD has a KD for NKp30 binding of about 15 nM. An IL-7 can thus be modified by introduction of a modification such as amino acid substitutions Q22A, D74A and/or K81A (with reference to the wild-type mature IL-7 amino acid sequence) that causes a reduction of affinity between IL-7 and IL-7Ra.
In another embodiment where the cytokine-binding ABD binds an IL-27 receptor (IL- 27R)-binding ABD, the ABD can be or comprise a suitable interleukin-27 (IL-27) polypeptide such that the IL-27R ABD binds IL-27R (WSX-1 and/or gp130) on the surface of NK cells. In some embodiments, the cytokine molecule is an IL-27 molecule, e.g., a full length, a fragment or a variant comprising the P28 and EBI3 subunits, e.g., human single chain or heterodimeric IL-27 comprising the P28 and EBI3 subunits, optionally wherein the EBI3 and p28 subunits of IL-27 are linked by a domain linker (e.g., a flexible polypeptide linker, a linker containing glycine a serine residues, a (G4S)2 or (G4S)3 linker) into a single-chain format. Single-chain forms of IL-27 can be generated consisting of the p28 subunit linked to the EBI3 subunit by a flexible linker, either through the C-terminus of p28 linked to the N-terminus of EBI3 or vice versa. In embodiments, the IL-27 molecule is a wild-type, human IL-27, e.g., a single chain fusion product or a heterodimer comprising the amino acid sequences of SEQ ID NOS: 245 and 246 or a IL27R-binding fragment of any of the SEQ ID NOS: 245 and 246. In some embodiments, the IL-27 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-27 p28 subunit amino acid sequence of SEQ ID NO: 245 and/or an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-27 EBI3 subunit amino acid sequence of SEQ ID NO: 246. In other embodiments, the IL-27 molecule is a variant of human IL-27, e.g., having one or more amino acid modifications. Optionally the IL-27 comprises a fragment of a human IL-27 p28 subunit polypeptide, wherein the fragment has an amino sequence that is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 245, and/or a fragment of a human IL-27 EBI3 subunit polypeptide, wherein the fragment has an amino sequence that is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 246. The p28 subunit can be specified as being linked at its N-terminus to the multispecific protein (or to the NKp30 ABD thereof). The EBI3 subunit can be specified as being linked, at its N-terminus, to the C- terminus of the p28 subunit, optionally via a domain linker, or can be specified as being placed on a separate polypeptide that associates with the p28 subunit.
Wild-type mature human IL-27 p28 subunit:
FPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQA WRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLDLRDLQRHLRFQVLAAGFNLPE EEEEEEEEEEEERKGLLPGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVW PLGFPTLSPQP (SEQ ID NO: 245).
Wild-type mature human IL-27 EBI3 subunit:
RKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTS CTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWP FPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATAT MSLGK (SEQ ID NO: 246).
In some embodiments, an IL-27 is modified so as to have an affinity for WSX-1 and/or gp130 that is equal or less than that of the NKp30 ABD for NKp30. In some embodiments, an IL-27 is modified so as to have an affinity for WSX-1 and/or gp130 that is at least 1-log less than that of the NKp30 ABD for NKp30. In some embodiments, an IL-27 is modified so as to have an affinity for WSX-1 and/or gp130 that is at least 1-log less than that of the NKp30 ABD for NKp30, but no more than 3-log, or optionally 2-log, less than that of the NKp30 ABD for NKp30.
In another embodiment where the cytokine-binding ABD binds an IL-12 receptor (IL- 12R)-binding ABD, the ABD can be or comprise a suitable interleukin-12 (IL-12) polypeptide such that the IL-12R ABD binds IL-12R (II_-^b1 and/or II_-^b2) on the surface of NK cells. In some embodiments, the cytokine molecule is an IL-12 molecule, e.g., a full length, a fragment or a variant comprising the P35 and P40 subunits, e.g., human single chain or heterodimeric IL-12 comprising the P35 and P40 subunits, optionally wherein the p40 and p35 subunits of IL-12 are linked by a domain linker (e.g., a flexible polypeptide linker, a linker containing glycine a serine residues, a (G4S)2 or (G4S)3 linker) into a single-chain format. Single-chain forms of IL-12 can be generated consisting of the p35 subunit linked to the p40 subunit by a flexible linker, either through the C-terminus of p35 linked to the N-terminus of p40 or vice versa. In embodiments, the IL-12 molecule is a wild-type, human IL-12, e.g., a single chain fusion product or a heterodimer comprising the amino acid sequences of SEQ ID NOS: 247 and 248 or a IL12R-binding fragment of any of the SEQ ID NOS: 247 or 248. In some embodiments, the IL-12 molecule comprises an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-12 p35 subunit amino acid sequence of SEQ ID NO: 247 and/or an amino sequence at least 70%, 80%, 90%, 95%, 98% or 99% identical to a mature wild-type human IL-12 p40 subunit amino acid sequence of SEQ ID NO: 248. In other embodiments, the IL-12 molecule is a variant of human IL-12, e.g., having one or more amino acid modifications. Optionally the IL-12 comprises a fragment of a human IL-12 p35 subunit polypeptide, wherein the fragment has an amino sequence that is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 247, and/or a fragment of a human IL-12 p40 subunit polypeptide, wherein the fragment has an amino sequence that is identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NO: 248. The p35 (alpha) and P40 (beta) can be specified as being linked by a disulphide bridge between Cys74 of the P35 subunit and the Cys177 of the P40 subunit. The p35 subunit can be specified as being linked at its N-terminus to the multispecific protein (or to the NKp30 ABD thereof). The p40 subunit can be specified as being linked, at its N-terminus, to the C-terminus of the p35 subunit, optionally via a domain linker, or can be specified as being placed on a separate polypeptide that associates with the p35 subunit.
Wild-type mature human IL-12 p35 subunit:
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLEL TKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQN MLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 247).
Wild-type mature human IL-12 p40 subunit: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTC HKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKS SRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSS FFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT SATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 248).
Wild-type IL-12 dimer binds to II_-12Eb1 and \LA2R^2 with an affinity (KD), e.g., as determined by microcal or SPR, of about 5-7 nM and 5 nM, respectively, and IL-12 dimer binds to Iί12 b1 :Iί-12 b2 dimers with a KD of about 50 pM. In some embodiments, an IL-12 is modified so as to have an affinity for Iί-12 b1 and/or Iί-12 b2 that is equal or less than that of the NKp30 ABD for NKp30. In some embodiments, an IL-12 is modified so as to have an affinity for I L-12 b1 and/or \L 2R^>2 that is at least 1-log less than that of the NKp30 ABD for NKp30. In some embodiments, an IL-12 is modified so as to have an affinity for Iί-12 b1 and/or Iί-12 b2 that is at least 1-log lower (1-log higher KD) than that of the NKp30 ABD for NKp30, but no more than 3-log, or optionally 2-log, lower than that of the NKp30 ABD for NKp30.
Activity testing
A multispecific protein can be assessed for biological activity, e.g., antigen binding, ability to elicit proliferation of NK cells, ability to elicit target cell lysis by NK and/or elicit activation of NK cells, including any specific signaling activities elicited thereby, for example cytokine production or cell surface expression of markers of activation. In one embodiment, provided are methods of assessing the biological activity, e.g., antigen binding, ability to elicit target cell lysis and/or specific signaling activities elicited thereby, of a multispecific protein of the disclosure. It will be appreciated that when the specific contribution or activity of one of the components of the multispecific protein is to be assessed (e.g. an NKp30 binding ABD, antigen-of-interest binding ABD, an Fc domain, cytokine receptor ABD, etc.), the multispecific format can be produced in a suitable format which allows for assessment of the component (e.g. domain) of interest. The present disclosure also provides such methods, for use in testing, assessing, making and/or producing a multispecific protein. For example, where the contribution or activity of an cytokine is assessed, the multispecific protein can be produced as a protein having the cytokine and another protein in which the cytokine is modified to delete it or otherwise modulate its activity (e.g., wherein the two multispecific proteins otherwise have the same or comparable structure), and tested in an assay of interest. For example, where the contribution or activity of an anti-NKp30 ABD is assessed, the multispecific protein can be produced as a protein having the ABD and another protein in which the ABD is absent or is replaced by an ABD that does not bind NKp30 (e.g., an ABD that binds an antigen not present in the assay system), wherein the two multispecific proteins otherwise have the same or comparable structure, and the two multispecific proteins are tested in an assay of interest. In another example, where the contribution or activity of an anti-antigen of interest (e.g. tumor antigen) ABD is assessed, the multispecific protein can be produced as a protein having the ABD and another protein in which the ABD is absent or is replaced by an ABD that does not bind the tumor antigen or anti-antigen of interest (e.g., an ABD that binds an antigen not present in the assay system, an ABD that bind to a different tumor antigen), wherein the two multispecific proteins otherwise have the same or comparable structure, and the two multispecific proteins are tested in an assay of interest.
In one aspect of any embodiment described herein, the multispecific protein is capable of inducing activation of an NKp30-expressing cell (e.g. an NK cell, a reporter cell) when the protein is incubated in the presence of the NKp30-expressing cell (e.g. purified NK cells) and a target cell (e.g. tumor cell) that expresses the antigen of interest (e.g. tumor antigen).
In one aspect of any embodiment described herein, the multispecific protein is capable of inducing NKp30 signaling in an NKp30-expressing cell (e.g. an NK cell, a reporter cell) when the protein is incubated in the presence of an NKp30-expressing cell (e.g. purified NK cells) and a target cell that expresses the antigen of interest). In one aspect of any embodiment described herein, the multispecific protein is capable of inducing CD16A signaling in an CD16A and NKp30-expressing cell (e.g. an NK cell, a reporter cell) when the protein is incubated in the presence of a CD16A and NKp30-expressing cell (e.g. purified NK cells) and a target cell that expresses the antigen of interest).
Optionally, NK cell activation or signaling in characterized by the increased expression of a cell surface marker of activation, e.g. CD107, CD69, Sca-1 or Ly-6A/E, KLRG1, etc.
In one aspect of any embodiment described herein, the multispecific protein is capable of inducing an increase of CD137 present on the cell surface of an NKp30- and/or a CD16- expressing cell (e.g. an NK cell, a reporter cell) when the protein is incubated in the presence of the NKp30- and/or a CD16-expressing cell (e.g. purified NK cells), optionally in the absence of target cells.
In one aspect of any embodiment described herein, the multispecific protein is capable of activating or enhancing the proliferation of NK cells by at least 10-fold, at least 50-fold, or at least 100-fold compared to the same multispecific protein lacking the cytokine receptor ABD (e.g. the CD122 ABD). Optionally the multispecific protein displays an EC50 for activation or enhancing the proliferation of NK cells that is at least 10-fold, 50-fold or 100-fold lower than its EC50 for activation or enhancing the proliferation of CD25-expressing T cells.
In one aspect of any embodiment described herein, the multispecific protein is capable of activating or enhancing the proliferation of NK cells over CD25-expressing T cells, by at least 10-fold, at least 50-fold, or at least 100-fold. Optionally, the CD25 expressing T cells are CD4 T cells, optionally Treg cells, or CD8 T cells.
Activation or enhancement of proliferation via cytokine receptor in cells (e.g. NK cells, CD4 T cells, CD8 T cells or T reg cells) by the cytokine receptor ABD-containing protein can be determined by measuring the expression of pSTAT or the cell proliferation markers (e.g. Ki67) in said cells following the treatment with the multispecific protein. Activation or enhancement of proliferation via the IL-2R pathway in cells (e.g. NK cells, CD4 T cells, CD8 Tcells or Treg cells) by the CD122 ABD-containing protein can be determined by measuring the expression of pSTAT5 or the cell proliferation marker Ki67 in said cells following the treatment with the multispecific protein. IL-2 and IL-15 lead to the phosphorylation of the STAT5 protein, which is involved in cell proliferation, survival, differentiation and apoptosis. Phosphorylated STAT5 (pSTAT5) translocates into the nucleus to regulate transcription of the target genes including the CD25. STAT5 is also required for NK cell survival and NK cells are tightly regulated by the JAK-STAT signaling pathway. In one aspect of any embodiment described herein, the multispecific protein is capable of inducing STAT5 signaling in an NKp30-expressing cell (e.g. an NK cell) when the protein is incubated in the presence of an NKp30-expressing cell (e.g. purified NK cells). In one aspect of any embodiment described herein, the multispecific protein is capable of causing an increase of expression of pSTAT5 in NK cells over CD25-expressing T cells, by at least 10-fold, at least 50-fold, or at least 100-fold. Optionally the multispecific protein displays an EC50 for induction of expression of pSTAT5 in NK cells that is at least 10- fold, 50-fold or 100-fold lower than its EC50 for induction of expression of pSTAT5 in CD25- expressing T cells. Similarly, cytokine receptor signal transduction can also be assessed for other cytokine/cytokine receptor pairs, such as IL-15 (STAT5), IL-21 (STAT3), IL-27 (STAT1), IL-12 (STAT4), etc.
Activity can be measured for example by bringing NKp30-expressing cells (or CD25- expressing cells, depending on the assay) into contact with the multispecific polypeptide, optionally further in presence of target cells (e.g. tumor cells). In some embodiments, activity is measured for example by bringing target cells and NK cells (i.e. NKp30-expressing cells) into contact with one another, in presence of the multispecific polypeptide. The NKp30- expressing cells may be employed either as purified NK cells or NKp30-expressing cells, or as NKp30-expressing cells within a population of peripheral blood mononuclear cell (PBMC). The target cells can be cells expressing the antigen of interest, optionally tumor cells.
In one example, the multispecific protein can be assessed for the ability to cause a measurable increase in any property or activity known in the art as associated with NK cell activity, respectively, such as marker of cytotoxicity (CD107) or cytokine production (for example IFN-g orTNF-a), increases in intracellular free calcium levels, the ability to lyse target cells, for example in a redirected killing assay, etc.
In the presence of target cells (target cells expressing the antigen of interest) and NK cells that express NKp30, the multispecific protein will be capable of causing an increase in a property or activity associated with NK cell activity (e.g. activation of NK cell cytotoxicity, CD107 expression, IFNy production, killing of target cells) in vitro. For example, a multispecific protein according to the invention can be selected based on its ability to increase an NK cell activity by more than about 20%, preferably by least about 30%, at least about 40%, at least about 50%, or more compared to that achieved with the same effector: target cell ratio with the same NK cells and target cells that are not brought into contact with the multispecific protein, as measured by an assay that detects NK cell activity, e.g., an assay which detects the expression of an NK activation marker or which detects NK cell cytotoxicity, e.g., an assay that detects CD107 or CD69 expression, IFNy production, or a classical in vitro chromium release test of cytotoxicity. Examples of protocols for detecting NK cell activation and cytotoxicity assays are described in the Examples herein, as well as for example, in Pessino et al, J. Exp. Med, 1998, 188 (5): 953-960; Sivori et al, Eur J Immunol, 1999. 29:1656-1666; Brando et al, (2005) J. Leukoc. Biol. 78:359-371; El-Sherbiny et al, (2007) Cancer Research 67(18):8444-9; and Nolte-'t Hoen et al, (2007) Blood 109:670-673). In a classical in vitro chromium release test of cytotoxicity, the target cells are labeled with 51Cr prior to addition of NK cells, and then the killing is estimated as proportional to the release of 51 Cr from the cells to the medium, as a result of killing. Optionally, a multispecific protein according to the invention can be selected for or characterized by its ability to have greater ability to induce NK cell activity towards target cells, i.e., lysis of target cells compared to a conventional human lgG1 antibody that binds to the same antigen of interest, as measured by an assay of NK cell activity (e.g. an assay that detects NK cell-mediated lysis of target cells that express the antigen of interest).
As shown herein, a multispecific protein, the different ABDs contribute to the overall activity of the multispecific protein that ultimately manifests itself in potent anti-tumor activity in vivo. Testing methods exemplified herein allow the in vitro assessment of the activities of the different individual ABDs of the multispecific protein by making variants of the multispecific protein that lack a particular ABD and/or using cells that lack receptors for the particular ABD. As shown herein, a multispecific protein according to the disclosure, when it does not comprise the cytokine receptor ABD (e.g. the CD122 ABD) and when it possesses an Fc domain that does not bind CD16, does not, substantially induce NKp30 signaling (and/or NK activation that results therefrom) of NK cells when the protein is not bound to the antigen of interest on target cells (e.g. in the absence of the antigen of interest and/or target cells). Thus, the monovalent NKp30 binding component of the multispecific protein does not itself cause NKp30 signaling. Accordingly, in the case of multispecific proteins possessing an Fc domain that binds CD16, such multispecific protein can be produced in a configuration where the cytokine receptor ABD (e.g. CD122 ABD) is inactivated (e.g. modified, masked or deleted, thereby eliminating its ability to binds IL-2Rs) and the protein can be assessed for its ability to elicit NKp30 signaling or NKp30-mediated NK cell activation by testing the effect of this multispecific protein on NKp30 expression, by CD16-negative NK cells. The multispecific protein can optionally be characterized as not substantially causing (or increasing) NKp30 signaling by an NKp30- expressing, CD16-negative cell (e.g. a NKp30+CD16_ NK cell, a reporter cell) when the multispecific protein is incubated with such NKp30-expressing, CD16-negative cells (e.g., purified NK cells or purified reporter cells) in the absence of target cells.
In one aspect of any embodiment herein, a multispecific protein can for example be characterized by:
(a) capable of inducing cytokine receptor (e.g. CD122) signaling (e.g., as determined by assessing ST AT signaling, for example assessing ST AT phosphoylation) in an NKp30-expressing cell (e.g. an NK cell) when the multispecific protein is incubated in the presence of an NKp30-expressing cell (e.g. purified NK cells);
(b) being capable of inducing NK cells that express NKp30 (and optionally further CD16) to lyse target cells, when incubated in the presence of the NK cells and target cells; and
(c) lack of NK cell activation or cytotoxicity and/or lack of agonist activity at NKp30 when incubated with NK cells (optionally CD16-negative NK cells, NKp30- expressing NK cells that do not express CD16), in the absence of target cells, optionally wherein the NK cells are purified NK cells, when the multispecific protein is modified to lack the cytokine receptor ABD (e.g. CD122 ABD) or comprises an inactivated cytokine receptor ABD.
Uses of compounds
In one aspect, provided is the use of any of the multispecific proteins and/or cells which express the proteins (or a polypeptide chain thereof) for the manufacture of a pharmaceutical preparation for the treatment, prevention or diagnosis of a disease in a mammal in need thereof. Provided also are the use any of the compounds defined above as a medicament or an active component or active substance in a medicament. In a further aspect the invention provides methods for preparing a pharmaceutical composition containing a compound as defined herein, to provide a solid or a liquid formulation for administration (e.g., by subcutaneous or intravenous injection). Such a method or process at least comprises the step of mixing the compound with a pharmaceutically acceptable carrier.
In one aspect, provided is a method to treat, prevent or more generally affect a predefined condition in an individual or to detect a certain condition by using or administering a multispecific protein or antibody described herein, or a (pharmaceutical) composition comprising same.
For example, in one aspect, the invention provides a method of restoring or potentiating the activity and/or proliferation of NKp30-expressing cells, particularly NKp30+ NK cells (e.g. NKp30+CD16+ NK cells, NKp30+CD16_ NK cells) in a patient in need thereof (e.g. a patient having a cancer, or a viral or bacterial infection), comprising the step of administering a multispecific protein described herein to said patient. In one aspect, the invention provides a method of selectively restoring or potentiating the activity and/or proliferation of NK cells of over CD25-expressing lymphocytes, e.g. CD4 T cells, CD8 T cells, Treg cells. In one embodiment, the method is directed at increasing the activity of NKp30+ lymphocytes (e.g. NKp30+CD16+ NK cells, NKp30+CD16 NK cells) in patients having a disease in which increased lymphocyte (e.g. NK cell) activity is beneficial or which is caused or characterized by insufficient NK cell activity, such as a cancer, or a viral or microbial/bacterial infection.
In one aspect, the invention provides a method of restoring or potentiating the activity and/or proliferation of tumor-infiltrating NK cells or intra-tumoral NKp30-expressing cells, particularly NKp30+ NK cells (e.g. NKp30+CD16+ NK cells, NKp30+CD16- NK cells) in a patient in need thereof (e.g. a patient having a solid tumor), comprising the step of administering a multispecific protein described herein to said patient.
In one aspect, the invention provides a method of increasing the number of tumor- infiltrating NK cells or intra-tumoral NKp30-expressing cells, particularly activated NKp30- expressing cells, particularly NKp30+ NK cells (e.g. NKp30+CD16+ NK cells, NKp30+CD16- NK cells) in a patient in need thereof (e.g. a patient having a solid tumor), comprising the step of administering a multispecific protein described herein to said patient.
In another aspect, the invention provides a method of restoring or potentiating the activity and/or proliferation of NKp30+ NK cells (e.g. NKp30+CD16+ NK cells, NKp30+CD16- NK cells) in a patient in need thereof (e.g. a patient having a cancer, or a viral, parasite or bacterial infection), comprising the step of contacting cells derived from the patient, e.g., immune cells and optionally target cells expressing an antigen of interest with a multispecific protein according to the invention and reinfusing the multispecific protein treated cells into the patient. In one embodiment, this method is directed at increasing the activity of NKp30+ lymphocytes (e.g. NKp30+CD16+ NK cells) in patients having a disease in which increased lymphocyte (e.g. NK cell) activity is beneficial or which is caused or characterized by insufficient NK cell activity, such as a cancer, or a viral or microbial, e.g., bacterial or parasite infection.
In another embodiment the subject multispecific proteins may be used or administered in combination with immune cells, particularly NK cells, derived from a patient who is to be treated or from a different donor, and these NK cells administered to a patient in need thereof such as a patient having a disease in which increased lymphocyte (e.g. NK cell) activity is beneficial or which is caused or characterized by insufficient NK cell activity, such as a cancer, or a viral or microbial, e.g., bacterial or parasite infection. As NK cells (unlike CAR-T cells) do not express TCRs, these NK cells, even those derived from different donors will not induce a GVHD reaction (see e.g., Glienke et al., “Advantages and applications of CAR-expressing natural killer cells”, Front. Pharmacol. 6, Art. 21:1-6 (2015); Hermanson and Kaufman, Front. Immunol. 6, Art. 195:1-6 (2015)).
In one embodiment, the multispecific protein disclosed herein that mediates NK cell activation, proliferation, tumor infiltration and/or target cell lysis via multiple activating receptors of effector cells, including NKp30, CD16 and CD122, can be used advantageously for treatment of individuals whose effector cells or tumor-infiltrating effector cells (e.g. NKp30+ NK cells) cells are hypoactive, exhausted or suppressed, for example a patient who has a significant population of effector cells characterized by the expression and/or upregulation of one or multiple inhibitory receptors (e.g. TIM-3, PD1 , CD96, TIGIT, etc.), or the downregulation or low level of expression of CD16 (e.g., presence of elevated proportion of NKp30+CD16_ NK cells).
The multispecific polypeptides described herein can be used to prevent or treat disorders that can be treated with antibodies, such as cancers, solid and non-solid tumors, hematological malignancies, infections such as viral infections, and inflammatory or autoimmune disorders.
In one embodiment, the antigen of interest (the non-NKp30 antigen) is an antigen expressed on the surface of a malignant cell of a type of cancer selected from the group consisting of: carcinoma, including that of the bladder, head and neck, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, hairy cell lymphoma and Burkett’s lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma, hematopoietic tumors of lymphoid lineage, for example T-cell and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T- PLL), including of the small cell and cerebriform cell type; large granular lymphocyte leukemia (LGL) preferably of the T-cell type; Sezary syndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinal T-cell lymphoma; T-lymphoblastic; and lymphoma/leukemia (T-Lbly/T-ALL).
In one embodiment, a multispecific protein is used to prevent or treat a cancer selected from the group consisting of: carcinoma, including that of the bladder, head and neck, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, hairy cell lymphoma and Burkett’s lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma. Other exemplary disorders that can be treated according to the invention include hematopoietic tumors of lymphoid lineage, for example T-cell and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including of the small cell and cerebriform cell type; large granular lymphocyte leukemia (LGL) preferably of the T-cell type; Sezary syndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal) T- cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinal T-cell lymphoma; T- lymphoblastic; and lymphoma/leukaemia (T-Lbly/T-ALL).
In one example, the tumor antigen is an antigen expressed on the surface of a lymphoma cell or a leukemia cell, and the multispecific protein is administered to, and/or used for the treatment of, an individual having a lymphoma or a leukemia. Optionally, the tumor antigen is selected from HER2, CD19, CD20, CD22, CD30 or CD33.
In one embodiment, the inventive multispecific polypeptides described herein can be used to prevent or treat a cancer characterized by tumor cells that express the antigen of interest (e.g. tumor antigen) to which the multispecific protein of the disclosure specifically binds.
In one aspect, the methods of treatment comprise administering to an individual a multispecific protein described herein in a therapeutically effective amount, e.g., for the treatment of a disease as disclosed herein, for example any of the cancers identified above. A therapeutically effective amount may be any amount that has a therapeutic effect in a patient having a disease or disorder (or promotes, enhances, and/or induces such an effect in at least a substantial proportion of patients with the disease or disorder and substantially similar characteristics as the patient).
The multispecific protein may be used with our without a prior step of detecting the expression of the antigen of interest (e.g. tumor antigen) on target cells in a biological sample obtained from an individual (e.g. a biological sample comprising cancer cells, cancer tissue or cancer-adjacent tissue). In another embodiment, the disclosure provides a method for the treatment or prevention of a cancer in an individual in need thereof, the method comprising: a) detecting cells (e.g. tumor cells) in a sample from the individual that express an antigen of interest (e.g. the antigen of interest to which the multispecific protein specifically binds via its antigen of interest ABD), and b) upon a determination that cells which express an antigen of interest are comprised in the sample, optionally at a level corresponding at least to a reference level (e.g. corresponding to an individual deriving substantial benefit from a multispecific protein, or optionally at a level that is increased compared to a reference level (e.g. corresponding to a healthy individual or an individual not deriving substantial benefit from a protein described herein), administering to the individual a multispecific protein of the disclosure that binds to an antigen of interest, to NKp30, to cytokine receptor (e.g. CD122), and optionally to CD16A (e.g., via its Fc domain).
In some embodiments, the multispecific proteins are used to treat a tumor characterized by low levels of surface expression of the antigen of interest. Accordingly, in, the tumor or cancer can be characterized by cells expressing a low level of tumor antigen. Optionally, the level of the tumor antigen is less than 100,000 tumor antigen copies per cancer cell. In some aspects, the level of the tumor antigen is less than 90,000, less than 75,000, less than 50,000, or less than 40,000 tumor antigen copies per cancer cell. The uses optionally further comprise detecting the level of tumor antigen of one or more cancer cells of the subject. The multispecific protein may be used with our without a prior step of detecting or characterizing NK cells from an individual to be treated. Optionally, in one embodiment, the invention provides a method for the treatment or prevention of a cancer in an individual in need thereof, the method comprising: a) detecting NK cells (e.g. tumor-infiltrating NK cells) in a tumor sample from an individual (or within the tumor and/or within adjacent tissue), and b) upon a determination that the tumor or tumor sample is characterized by a low number or activity of NK cells, optionally at a level or number that is decreased compared to a reference level (e.g. at a level corresponding to an individual deriving no, low or insufficient benefit from a conventional IgG antibody therapy such a conventional lgG1 antibody that binds to the same cancer antigen), administering to the individual a multispecific protein that binds to a cancer antigen, to NKp30 (e.g., monovalently), to cytokine receptor (e.g., CD122) and optionally to CD16A.
In some embodiments, an individual has a tumor characterized by a CD16 (e.g. CD16A) deficient tumor microenvironment. Optionally, the methods of treatment using a multispecific protein comprise a step of detecting the expression level of CD16 in a sample (e.g. a tumor sample) from the individual. Detecting the CD16 optionally comprises detecting the level of CD16A or CD16B. In some aspects, the CD16 deficient microenvironment is assessed in a patient having undergone a hematopoietic stem cell transplantation. Optionally, the CD16 deficient microenvironment comprises a population of infiltrating NK cells, and the infiltrating NK cells have less than 50% expression of CD16 as compared to a control NK cell. In some aspects, the infiltrating NK cells have less than 30%, less than 20%, or less than 10% expression of CD16 as compared to a control NK cell. Optionally, the CD16 deficient microenvironment comprises a population of infiltrating NK cells, and at least 10% of the infiltrating NK cells have reduced expression of CD16 as compared to a control NK cell. In some aspects, at least 20%, at least 30%, or at least 40% of the infiltrating NK cells have reduced expression of CD16 as compared to a control NK cell.
Optionally, in one embodiment, provided is a method for the treatment or prevention of a cancer in an individual in need thereof, the method comprising: a) detecting CD16 expression in cells (e.g. in tumor-infiltrating NK cells) from a tumor or tumor sample (e.g., tumor and/or within adjacent tissue) from an individual, and b) upon a determination that the tumor or tumor sample is characterized by a CD16 deficient microenvironment, administering to the individual a multispecific protein that binds to a cancer antigen, to NKp30, and to the cytokine receptor (e.g. CD122) (and optionally further to CD16A).
Optionally, in one embodiment, provided is a method for the treatment or prevention of a cancer in an individual in need thereof, the method comprising: a) detecting CD16 expression at the surface of NK cells (e.g. tumor-infiltrating NK cells) in a tumor sample from an individual (or within the tumor and/or within adjacent tissue), and b) upon a determination that the tumor or tumor sample is characterized by an elevated proportion of CD16- NK cells, optionally at a level or number that is increased compared to a reference level, administering to the individual a multispecific protein that binds to a cancer antigen, to NKp30, and to the cytokine receptor (e.g. CD122) (and optionally further to CD16A).
In one embodiment, the disclosure provides a method for the treatment or prevention of a disease (e.g. a cancer) in an individual in need thereof, the method comprising: a) detecting cell surface expression of one or a plurality inhibitory receptors on immune effector cells (e.g. NK cells, T cells) in a sample from the individual (e.g. in circulation or in the tumor environment), and b) upon a determination of cell surface expression of one or a plurality inhibitory receptors on immune effector cells, optionally at a level that is increased compared to a reference level (e.g. at a level that is increased compared to a healthy individual, an individual not suffering from immune exhaustion or suppression, or an individual not deriving substantial benefit from a protein described herein), administering to the individual a multispecific protein (e.g. a multispecific protein) that binds to an antigen of interest (e.g. a cancer antigen), to NKp30 (e.g., monovalently), and to the cytokine receptor (e.g. CD122) (and optionally further to CD16A).
In some embodiments, the multispecific proteins are used to treat an individual having a gastric cancer or a prostate cancer. Decreased cell surface expression of NKG2D on immune effector cells has been observed in gastric cancer and prostate cancer.
In some embodiments, an individual has NK cells and/or T cells characterized by decreased expression of NKG2D, e.g. decreased cell surface expression. The level of expression can be for example compared to a reference value, for example a reference value corresponding to NKG2D levels observed on NK and/or T cells in healthy individuals. In some embodiments, an individual has NK and/or T cell characterized by decreased expression of NKG2D on NK and/or T cells in the tumor microenvironment. In some embodiments, an individual has NK and/or T cell characterized by presence (e.g. at increased levels) of soluble ligands of NKG2D in the tumor microenvironment, for example soluble MICA, MICB or ULBP proteins.
In one embodiment, the disclosure provides a method for the treatment or prevention of a disease (e.g. a cancer) in an individual in need thereof, the method comprising: a) detecting cell surface expression of NKG2D polypeptides on immune effector cells (e.g. NK cells, T cells) in a sample from the individual (e.g. in circulation or in the tumor environment), and b) upon a determination of decreased cell surface expression of one or a plurality inhibitory receptors on immune effector cells, optionally at a level that is decreased compared to a reference level (e.g. at a level that is increased compared to a healthy individual, an individual not suffering from immune exhaustion or suppression, or an individual not deriving substantial benefit from a protein described herein), administering to the individual a multispecific protein (e.g. a multispecific protein) that binds to an antigen of interest (e.g. a cancer antigen), to NKp30 (e.g., monovalently), and to the cytokine receptor (e.g. CD122) (and optionally further to CD16A).
In one embodiment, a multispecific protein may be used as a monotherapy (without other therapeutic agents), or in combined treatments with one or more other therapeutic agents. In one embodiment, a multispecific protein is administered in the absence of combined treatment with an IL-2, IL-15, IL-21 , IL-7, IL-27, IL-12, IL-18, IFN-a or lFN-b polypeptide.
The multispecific proteins can also be included in kits. The kits may optionally further contain any number of polypeptides and/or other compounds, e.g., 1, 2, 3, 4, or any other number of multispecific proteins and/or other compounds. It will be appreciated that this description of the contents of the kits is not limiting in any way. For example, the kit may contain other types of therapeutic compounds. Optionally, the kits also include instructions for using the polypeptides, e.g., detailing the herein-described methods such as in the detection or treatment of specific disease conditions.
Also provided are pharmaceutical compositions comprising the subject multispecific proteins and optionally other compounds as defined above. A multispecific protein and optionally another compound may be administered in purified form together with a pharmaceutical carrier as a pharmaceutical composition. The form depends on the intended mode of administration and therapeutic or diagnostic application. The pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver the compounds to the patient. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as (sterile) water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters, alcohol, fats, waxes, and inert solids. A pharmaceutically acceptable carrier may further contain physiologically acceptable compounds that act for example to stabilize or to increase the absorption of the compounds Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like, may also be incorporated into the pharmaceutical compositions. Non-limiting examples of such adjuvants include by way of example inorganic and organic adjuvants such as alum, aluminum phosphate and aluminum hydroxide, squalene, liposomes, lipopolysaccharides, double stranded (ds) RNAs, single stranded(s-s) DNAs, and TLR agonists such as unmethylated CpG’s.
Multispecific proteins according to the invention can be administered parenterally. Preparations of the compounds for parenteral administration must be sterile. Sterilization is readily accomplished by filtration through sterile filtration membranes, optionally prior to or following lyophilization and reconstitution. The parenteral route for administration of compounds is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial, or intralesional routes. The compounds may be administered continuously by infusion or by bolus injection. A typical composition for intravenous infusion could be made up to contain 100 to 500 ml of sterile 0.9% NaCI or 5% glucose optionally supplemented with a 20% albumin solution and 1 mg to 10 g of the compound, depending on the particular type of compound and its required dosing regimen. Methods for preparing parenterally administrable compositions are well known in the art.
Examples
Preparation of multispecific proteins
The amino acid sequences of the three chains of the GA101-T5-Com_mAb8-IL2v protein which bears an Fc domain capable of binding to CD16A are shown in SEQ ID NOS: 251 , 252 and 253. The amino acid sequences of the three chains of the GA101-T6- Com_mAb8-IL2v protein which bears an Fc domain that substantially lacks binding to CD16A are shown in SEQ ID NOS: 254, 255 and 256. Figure 2B shows the topology of the exemplary “T5” format multispecific proteins used in the Examples; the T6 format share the structure of the T5 format but differs from the T5 format by introduction of amino acid substitution in the Fc domain that reduces binding to CD16A (and other Fc gamma receptors). The T5 and T6 formats have one Fab structure located topologically N-terminal, and a cytokine topologically C-terminal, with the dimeric Fc domain interposed between Fab on the N-terminal side and the NKp30-binding Fab on the C-terminal side of the Fc dimer, and the cytokine on the C- terminal side of the NKp30 binding Fab. The domain structure of the exemplary “T5” format protein used in the Examples is shown in Figure 2A. Figure 2A shows domain linkers (such as hinge and glycine-serine linkers) of different lengths, and interchain disulfide bridges. The Fc domain has Fc gamma receptor binding site amino acid sequences of wild-type human lgG1 and thus retains binding to CD16A.
The sequences encoding each polypeptide chain for each multispecific antigen binding protein were inserted into the pTT-5 vector between the Hindlll and BamHI restriction sites. The three vectors (prepared as endotoxin-free midipreps or maxipreps) were used to cotransfect EXPI-293F cells (Life Technologies) in the presence of PEI (37°C, 5% CO2, 150 rpm). The cells were used to seed culture flasks at a density of 1 x 106 cells per ml (EXPI293 medium, Gibco). Valproic Acid (final concentration 0.5 mM), glucose (4 g/L) and tryptone N1 (0.5%) were added. The supernatant was harvested after six days after and passed through a Stericup filter with 0.22 pm pores.
The multispecific antigen-binding proteins were purified from the supernatant following harvesting using rProtein A Sepharose Fast Flow (GE Healthcare, reference 17- 1279-03). Size Exclusion Chromatography (SEC) purifications were then performed and the proteins eluted at the expected size were finally filtered on a 0.22 pm device. The amino acid sequence of the polypeptide chains of the multispecific proteins produced are shown below in Table 6.
Table 6
Figure imgf000134_0001
Figure imgf000135_0001
Example 1: IL2v limits IL2R activation on Treg
A heterotrimeric Fc-domain-containing protein containing one C-terminal moiety of mutant IL-2 was assessed for its ability to activate Treg cells. The protein incorporates a variant IL-2 polypeptide (IL-2v) in which a human IL-2 polypeptide is modified by introducing the mutations T3A, F42A, Y45A, L72G and C125A, conferring decreased binding affinity for CD25 compared to wild-type human IL-2.
The heterotrimeric had an IL2v fused to the C-terminus of one of the chains of an isotype control (IC) Fab, which in turn was fused to the C-terminus of an Fc domain mutated to substantially eliminate CD16A binding, in turn fused to another IC Fab. The IC Fabs have a VH/VL pair which does not bind to any protein in the test system. This heterotrimeric protein (IL2v immunoconjugate) was compared to an identical heterotrimeric protein in which IL2v was replaced by a wild-type human IL-2 polypeptide (IL2pWT immunoconjugate), and to recombinantly produced full-length wild-type IL-2 (rec hulL-2).
Briefly, 1 M/well of purified PBMC were seeded in 96-well plate and treated with increasing doses of recombinant hulL-2, IC-T6-IC-IL2 (IL2pWT immunoconjugate) or IC-T6- IC-IL2v (IL2v immunoconjugate) (dose from 133nM to 0,0000013nM) for 20min at 37°C, 5.5%C02 in incubator. STAT5 phosphorylation was then analysed by flow cytometry on Tregs (gated on CD3+ CD4+ CD25+ FoxP3+).
Results are shown in Figure 3. The IL2v immunoconjugate resulted in an approximately 3-log decrease in percent of pSTAT5+ cells among the Treg, compared to IL2pWT immunoconjugate and rec hulL-2. The IL2v immunoconjugate protein incorporating a mutated human IL-2 therefore displays a strongly decreased ability to activate Treg cells compared to wild-type IL-2.
Example 2: CD20 x NKp30 binding NKCE-IL2v promotes potent NK cell-mediated cytotoxicity
Different heterotrimeric proteins were compared that all bound tumor antigen (CD20, indicated interchangeable also as “GA101” referring to the anti-CD20 VH/VL pair), NKp30 and CD122 (by inclusion of an IL2v molecule), and that either did or did not bind CD16 (by inclusion of a dimeric Fc domain that retains wild-type Fc gamma receptor binding in the “T5” protein or a dimeric Fc domain with amino acid substitution to substantially eliminate CD16A binding in the “T6” protein).
The proteins tested were:
GA101-T5-Com_mAb8-IL2v
GA101-T6-Com_mAb8-IL2v
Conventional full-length human lgG1 antibody having GA101 VH/VL domains obinutuzmab (GA101 VH/VL containing antibody with glycoengineered Fc domain and enhanced CD16A binding).
In this experiment, the different proteins were assessed for their ability to induce killing of RAJ I tumor cells (CD20+) by NK cells from two human donors at effectortarget ratio of 10:1 in a standard 4-hour cytotoxicity assay using calcein release as readout.
Briefly, freshly purified NK cells were co-cultured with Raji tumor cells previously loaded with calcein, in a 10 to 1 ratio. Cells were incubated with test proteins described above (doses from 61 nM to 0.00000061 nM) for 4h at 37°C, 5.5% CO2 in incubator.
Results are shown in Figure 4, showing % specific lysis induced by NK cells on the y- axis and concentration of test protein on the x-axis. All proteins were highly potent in ability to mediate NK cell cytotoxicity toward tumor target cells. However, at concentrations consistent with use in treatment, the GA101-T5-Com_mAb8-IL2v provided a significant improvement in potency compared to the gold-standard Fc-engineered antibody obinutuzmab. GA101-T6- Com_mAb8-IL2v which engages NKp30 but not CD16A was as potent as conventional full- length human lgG1 antibody having GA101 VH/VL domains despite the ability of the latter to bind CD20 bivalently. However, GA101-T5-Com_mAb8-IL2v was more potent that all other proteins, including the gold-standard Fc-engineered antibody obinutuzmab, despite the ability of obinutuzumab to bind CD20 bivalently and to bind CD16 with greater affinity. Monovalent co-engagement of NKp30, CD16A and cytokine receptor (CD122) on NK cells therefore provides an important advantage in mediating NK cell lysis of tumor cells.
Example 3: NKCE-IL2v promotes IL2R activation selectively in NK cells
The heterotrim eric Fc-domain-containing GA101-T5-Com_mAb8-IL2v and GA101-T6- Com_mAb8-IL2v proteins were assessed for its ability to activate Treg cells, NK cells, CD4 T cells and CD8 T cells, in comparison to the GA101-T6-IC-IL2v protein which is a heterotrimer of the same structure in which the NKp30 binding domain is replaced by an isotype control (IC) domain in which the VH/VL pair does not bind to any protein of the test system, and wherein substitutions the Fc domain substantially eliminate binding to CD16.
Briefly, 1 M/well of purified PBMC were seeded in 96-well plate and treated with increasing doses of test protein (dose from 133nM to 0,0000013nM) for 20min at 37°C, 5.5%C02 in incubator. STAT5 phosphorylation was then analysed by flow cytometry on NK cells (CD3-CD56+), CD8 T cells (CD3+ CD8+), CD4 T cells (CD3+ CD4+ FoxP3-) and Tregs (gated on CD3+ CD4+ CD25+ FoxP3+).
Results are shown in Figure 5 showing % of pSTAT5 cells among NK cells on the y- axis and concentration of test protein on the x-axis. The GA101-T5-Com_mAb8-IL2v proteins were about 100-fold more potent than GA101-T6-Com_mAb8-IL2v proteins and GA101-T6- IC-IL2v in the ability to activate NK cells.
Results are shown in Figure 6 showing % of pSTAT5 cells among CD4 T cells on the y-axis and concentration of test protein on the x-axis. GA101-T5-Com_mAb8-IL2v, GA101- T6-Com_mAb8-IL2v and GA101-T6-IC-IL2v were all equally potent in the ability to activate CD4 T cells.
Results are shown in Figure 7 showing % of pSTAT5 cells among CD8 T cells on the y-axis and concentration of test protein on the x-axis. GA101-T5-Com_mAb8-IL2v, GA101- T6-Com_mAb8-IL2v and GA101-T6-IC-IL2v were all equally potent in the ability to activate CD8 T cells.
Results are shown in Figure 8 showing % of pSTAT5 cells among Treg cells on the y- axis and concentration of test protein on the x-axis. GA101-T5-Com_mAb8-IL2v, GA101-T6- Com_mAb8-IL2v and GA101-T6-IC-IL2v were all equally potent in the ability to activate Treg cells.
Overall, all proteins displayed comparable level of activation of Treg cells, CD4 T cells and CD8 T cells. However, the GA101-T5-Com_mAb8-IL2v was far more potent (lower EC50) in increasing pSTAT5+ cells among the NK cells. The GA101-T5-Com_mAb8-IL2v proteins permitted a potent and selective activation of NK cells over Treg cells, CD4 T cells and CD8 T cells.
Example 4: NKCE-IL2v redirects IL2v activity to promote IFNy production by NK cells
In this experiment, IL2v-containing NK cell engager proteins GA101-T5-Com_mAb8- IL2v and GA101-T6-Com_mAb8-IL2v were assessed for their ability to promote interferon- gamma (IFN-g) production by NK cells. Effector cells as PBMC and Raji tumor target cells at an E/T ratio of 2.5/1 (80000 Raji/well) were treated with doses of NK cell engagers GA101- T5-Com_mAb8-IL2v, GA101-T6-Com_mAb8-IL2v and comparator protein from 20pg/ml (133nM) to 0.000002 pg/ml (0.00001nM) (10-fold dilution). After 4 hours of incubation and treatment with Golgi stop, cytokine was monitored by intracellular flow cytometry. The percentage of IFNy+ cells among gated cell type and MIR1b MedFI on gated cells were determined by flow cytometry.
Figure 9 shows percentage of IFNy+ cells, with % of IFN-y expressing NK cells on the y-axis and concentration of test protein on the x-axis. GA101-T5-Com_mAb8-IL2v, GA101- T6-Com_mAb8-IL2v induced an increase in percentage of IFNy+ cells compared to IC-T6-IC- IL2v that did not bind CD16A or NKp30 on NK cells. The GA101-T5-DF_A49-IL2v had highest potency, and was more potent than thw human IgG isotype antibody having the GA101 VH/VL domains (HuG1), despite the HuG1 binding CD20 bivalently. Figure 10 shows MIR1b MedFI on NK cells on the y-axis and concentration of test protein on the x-axis. GA101-T5- Com_mAb8-IL2v, GA101-T6-Com_mAb8-IL2v induced an increase MIR1b MedFI compared to IC-T6-IC-IL2v that did not bind CD16A or NKp30 on NK cells. Once again GA101-T5- Com_mAb8-IL2v showed high potency.
Example 5: NKCE-IL2v induces NK cell-specific cytokine production
In this experiment, IL2v-containing NK cell engager proteins GA101-T5-Com_mAb8- IL2v and GA101-T6-Com_mAb8-IL2v were assessed for their ability to promote interferon- gamma (IFN-y) production by NK cells, CD4 T cells, CD8 T cells of gamma-delta T cells (gd T cells). Effector cells as PBMC and Raji tumor target cells at an E/T ratio of 2.5/1 (80000 Raji/well) were treated with doses of NK cell engagers GA101-T5-Com_mAb8-IL2v, GA101- T6-Com_mAb8-IL2v and comparator protein from 20pg/ml (133nM) to 0.000002 pg/ml (0.00001nM) (10-fold dilution), along with PMA/iono as positive control (PMA: 100 ng/mL, ionomycin: 1 pg/mL). After 4 hours of incubation and treatment with Golgi stop, cytokine was monitored by intracellular flow cytometry. The percentage of IFNy+ cells among gated cell type and MIR1b MedFI on gated cells were determined by flow cytometry.
Results are shown in Figure 11A and B. GA101-T5-Com_mAb8-IL2v and GA101-T6- Com_mAb8-IL2v induced IFN-g production (Figure 11 A) and MIP1 b MedFI (Figure 11 B) in NK cells, but not in CD4 T cells, CD8 T cells or in gd T cells.
All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e. g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by “about,” where appropriate). All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.
The description herein of any aspect or embodiment of the invention using terms such as reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that “consists of,” “consists essentially of” or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context). This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.
All publications and patent applications cited in this specification are herein incorporated by reference in their entireties as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. A multispecific protein that is capable of potentiating NK cell cytotoxicity toward a target cell expressing an antigen of interest, the multispecific protein comprising:
(a) an antigen binding domain (ABD) that binds to an antigen of interest;
(b) an ABD that binds to a human NKp30 polypeptide;
(c) an Fc domain, or a portion an Fc domain capable of binding FcRn; and
(d) an ABD that binds a human cytokine receptor present on NK cells, wherein the ABD that binds to a human NKp30 polypeptide is connected to the Fc domain, optionally via an Ig-derived or non-lg-derived polypeptide linker, and wherein the the ABD that binds a human cytokine receptor is connected, via a polypeptide linker, to the ABD that binds to a human NKp30 polypeptide or to the Fc domain.
2. The protein of claim 1, wherein the protein has only one ABD that binds to an antigen of interest, such that the protein binds to the antigen of interest monovalently, optionally further wherein the protein has only one ABD that binds to a human NKp30 polypeptide, only one ABD that binds a cytokine receptor, and only one Fc domain dimer.
3. The protein of claims 1 or 2, wherein the ABD that binds the cytokine receptor is connected via a polypeptide linker to the ABD that binds NKp30.
4. The protein of claims 1 , 2 or 3, wherein the Fc domain is capable of binding CD16A, and wherein the protein is capable of co-engaging NKp30 the cytokine receptor and CD16A, on the surface of an NK cell.
5. The protein of any one of the above claims, wherein the ABD that binds to a human NKp30 polypeptide and the ABD that binds a human cytokine receptor are configured to be capable of adopting a membrane planar binding confirmation.
6. The protein of any one of the above claims, wherein the ABD that binds a human cytokine is connected to the ABD that binds to a human NKp30 polypeptide or the Fc domain by a linker peptide having 20 or less than 20 amino acid residues, optionally less than 15 amino acid residues, optionally less than 10 amino acid residues, optionally between 5 and 15 residues, optionally between 5 and 10 residues, optionally between 3 and 5 residues.
7. The protein of any one of the above claims, wherein the ABD that binds to a human NKp30 polypeptide is connected to the Fc domain by a linker peptide having 15 or less than 15 amino acid residues, optionally less than 10 amino acid residues, optionally between 4 and 15 residues, optionally between 3 and 10 residues, optionally between 4 and 5 residues.
8. The protein of any one of the above claims, wherein the ABD that binds to a human NKp30 polypeptide is connected to the Fc domain by an immunoglulin-derived linker peptide having 20 or less than 20 amino acid residues, optionally less than 15 amino acid residues, optionally between 4 and 15 residues, optinally between 4 and 10 residues, optionally between 3 and 5 residues, optionally wherein the linker comprises the amino acid sequence RTVA.
9. The protein of any one of the above claims, wherein the ABD that binds a human cytokine receptor comprises a wild-type cytokine or a fragment or variant thereof that substantially retains the binding affinity of the wild-type cytokine for its NK cell-expressed receptor, as determined by surface plasmon resonance (SPR).
10. The protein of any one of the above claims, wherein the Fc domain is an Fc dimer that comprises two Fc monomers and binds to human CD16A.
11. The protein any one of the above claims, wherein the multispecific protein is capable of directing an NKp30-expressing NK cell to lyse a target cell expressing the antigen of interest, wherein said lysis of the target cell is mediated by NKp30-signaling.
12. The protein of any one of the foregoing claims, wherein the ABD that bind sthe antigen of interest and the ABD that binds NKp30 each comprise a VH and a VL domain.
13. The protein of any one of the foregoing claims, wherein the protein comprises two or more polypeptide chains and comprises a NKp30 ABD-cytokine unit having a domain arrangement:
Va-2 - (CH1 or CL)a (polypeptide chain 1)
I
Vb-2 - (CH1 or CL)b- L- Cyt (polypeptide chain 2) wherein:
Va-2 and Vb-2 are each a VH domain or a VL domain, wherein one of Va-2 and Vb-2 is a VH and the other is a VL and wherein Va-2 and Vb-2 form an ABD that binds NKp30;
CH1 is a human immunoglobulin CH1 domain and CL is a human light chain constant domain; one of (CH1 or CL)a and (CH1 or CL)b is a CH1 and the other is a CL such that a (CH1/CL) pair is formed;
L is an amino acid domain linker; and
Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21 , IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
14. The protein of any one of claims 1-12, wherein the protein is a heterotrimer comprising: a. a first polypeptide chain comprising from N- to C-terminus: a first variable domain (Va-2), a domain linker, a second variable domain (Vb-2), a domain linker or hinge amino acid sequence, a CH2 domain and a CH3 domain, wherein one of Va-2 and Vb-2 is a VH and the other is a VL and wherein Va-2 and Vb-2 form an scFv that binds NKp30; b. a second polypeptide chain comprising from N- to C-terminus: a variable domain (Va-i), a CH1 domain, a hinge sequence, a CH2 domain and a CH3 domain, wherein the CH3 domains of the second polypeptide chain dimerizes or associates with the CH3 domain of the first polypeptide chain; c. a third polypeptide chain that dimerizes with the second polypeptide chain, the third polypeptide chain comprising from N- to C-terminus: a variable domain (VM) and a CL domain; wherein one of Va-i and VM is a VH and the other is a VL, and the Vw and Vb-2 domains associate to form an ABD that binds an antigen of interest; wherein one of the first or second polypeptide chains further comprises a cytokine, optionally an IL-2, IL-15, IL-21 , IL-7, IL-27, IL-12, IL-18, IFN-a or IFN-b polypeptide, optionally an IL-2v or IL15v, covalently attached, optionally via a domain linker, to the C-terminus of the CH3 domain.
15. The protein of any one of claims 1-12, wherein the ABD that binds NKp30 comprises a VH and VL positioned within a tandem variable region comprising a VH and VL separated by a polypeptide linker.
16. The protein of any one of claims 1-12 or 15, wherein the protein comprises a NKp30 ABD-cytokine unit having a domain arrangement
- L1 — Va-2 - L2 - Vb-2 - L3- Cyt wherein: Va-2 and Vb-2 are each a VH domain or a VL domain, wherein one of Va-2 and Vb-2 is a VH and the other is a VL and wherein Va-2and Vb-2 form a second ABD that binds NKp30;
L1, L2 and L3 are each an amino acid domain linker, wherein L1, L2 and L3 can be different or the same, wherein L1 is a linker connecting the NKp30 ABD-cytokine unit to the rest of the protein; and Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL- 15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or lFN-b polypeptide.
17. The multispecific protein of claims 1-13, wherein the protein is a heterotrimer having a polypeptide chain 1, 2 and 3:
Vb-i - (CH1 or CL)c - Hinge - CH2 - CH3 (chain 2)
I
Va-i - (CH1 or CL)a- Hinge - CH2 - CH3- L1 -Va.2 - (CH1 or CL) (chain 1)
I
Vb-2 - (CH1 or CL)d- L2- Cyt (chain 3) wherein:
Va-i, Vb-i, Va-2 and Vb-2 are each a VH domain or a VL domain, wherein one of Va-i and Vb-i is a VH and the other is a VL and wherein Va-i and Vw form a first antigen binding domain (ABD) that binds an antigen of interest, wherein one of Va-2and Vb-2 is a VH and the other is a VL and wherein Va-2 and Vb-2 form a second ABD that binds NKp30;
CH1 is a human heavy chain constant domain 1 and CL is a human light chain constant domain; one of (CH1 or CL)a and (CH1 or CL)c is a CH1 and the other is a CL such that a (CH1/CL) pair is formed; one of (CH1 or CL)b and (CH1 or CL)d is a CH1 and the other is a CL such that a (CH1/CL) pair is formed;
Hinge is an immunoglobulin hinge region or portion thereof;
L1 and L2 are each an amino acid domain linker, wherein L1 and L2 can be different or the same;
CH2 and CH3 are human immunoglobulin CH2 and CH3 domains, respectively; and
Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21, IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
18. The protein of any one of claims 1-12, 15 or 16, wherein the protein is a heterodimer heterotrimer or heterotetramer, and wherein the ABD that binds to a human NKp30 polypeptide is an scFv and the ABD that binds to an antigen of interest is a Fab.
19. The protein of any one of claims 1-12, 15, 16 or 18 wherein the protein is a heterodimer having a polypeptide chain 1 and 2:
Vai - (CH1 or CK)a- (hinge or L1) - CH2 - CH3 - Va2 - L2 - \ 2- L3 -Cyt (chain 1)
I I
Vbi - (CH1 or CK) - (hinge or L4) - CH2 - CH3 (chain 2). wherein:
Va-i, Vb-i, Va-2 and Vb-2 are each a VH domain or a VL domain, wherein one of Va-i and Vb-i is a VH and the other is a VL and wherein Va-i and Vn form a first antigen binding domain (ABD) that binds an antigen of interest, wherein one of Va-2and Vb-2 is a VH and the other is a VL and wherein Va-2 and Vb-2 form a second ABD that binds NKp30;
CH1 is a heavy chain constant domain 1 and CL is a light chain constant domain; one of (CH1 or CL)a and (CH1 or CL)b is a CH1 and the other is a CL such that a (CH1/CL) pair is formed;
Hinge is an immunoglobulin hinge region or portion thereof;
L1, L2, L3 and L4 are each an amino acid domain linker, wherein L1, L2, L3 and L4 can be different or the same;
CH2 and CH3 are human immunoglobulin CH2 and CH3 domains, respectively; and Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21, IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
20. The protein of any one of claims 1-12, wherein the protein is a heterotrimer having a polypeptide chain 1, 2 and 3:
Va-2 - L1 - Vb-2 - (Hinge or L3) - CH2 - CH3 - L2 - Cyt (chain 2)
I (Hinge or L3) - CH2 - CH3 (chain 1)
Figure imgf000145_0001
(chain 3) wherein:
Va-i, Vb-i, Va-2 and Vb-2 are each a VH domain or a VL domain, wherein one of Va-i and Vb-i is a VH and the other is a VL and wherein Va-i and Vw form a first antigen binding domain (ABD) that binds an antigen of interest, wherein one of Va-2and Vb-2 is a VH and the other is a VL and wherein Va-2and Vb-2 form a second ABD that binds NKp30;
CH1 is a heavy chain constant domain 1 and CL is a light chain constant domain; one of (CH1 or CL)a and (CH1 or CL)b is a CH1 and the other is a CL such that a (CH1/CL) pair is formed; Hinge is an immunoglobulin hinge region or portion thereof;
L1, L2 and L3 are each an amino acid domain linker, wherein L1, L2 and L3 can be different or the same;
CH2 and CH3 are human immunoglobulin CH2 and CH3 domains, respectively; and
Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21, IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
21. The protein of any one of claims 1-12, wherein the protein is a heterotrimer comprising: a. a first polypeptide chain comprising from N- to C-terminus: a first variable domain (Va-2), a domain linker, a second variable domain (Vb-2), a domain linker or hinge amino acid sequence , a CH2 domain and a CH3 domain, wherein one of Va-2 and Vb-2 is a VH and the other is a VL and wherein Va-2 and Vb-2 form an scFv that binds an antigen of interest; b. a second polypeptide chain comprising from N- to C-terminus: a variable domain (Va-i), a CH1 domain, a hinge sequence, a CH2 domain and a CH3 domain, wherein the CH3 domains of the second polypeptide chain dimerizes or associates with the CH3 domain of the first polypeptide chain; c. a third polypeptide chain that dimerizes with the second polypeptide chain, the third polypeptide chain comprising from N- to C-terminus: a variable domain (VM) and a CL domain; wherein one of Va-i and Vn is a VH and the other is a VL, and the Va-i and Vn domains associate to form an ABD that binds NKp30; and wherein one of the first or second polypeptide chains further comprises a cytokine, optionally an IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or lFN-b polypeptide, optionally an IL-2v or IL15v, covalently attached, optionally via a domain linker, to the C-terminus of the CH3 domain.
22. The protein of any one of claims 1-12 or 21, wherein the protein is a heterotrimer having a polypeptide chain 1 , 2 and 3:
Va_2 - L1 - Vb-2 - (Hinge or L3) - CH2 - CH3 (chain 2)
I
Va-i - (CH1 or Ci_)a - (Hinge or L3) - CH2 - CH3 - L2 - Cyt (chain 1)
I
Vb-i - (CH1 or Ci_)b (chain 3) wherein: Va-i, Vb-i, Va-2 and Vb-2 are each a VH domain or a VL domain, wherein one of Va-i and Vb-i is a VH and the other is a VL and wherein Va-i and Vw form a first antigen binding domain (ABD) that binds NKp30, wherein one of Va-2and Vb-2 is a VH and the other is a VL and wherein Va-2 and Vb-2 form a second ABD that an binds antigen of interest;
CH1 is a heavy chain constant domain 1 and CL is a light chain constant domain; one of (CH1 or CL)a and (CH1 or CL)b is a CH1 and the other is a CL such that a (CH1/CL) pair is formed;
Hinge is an immunoglobulin hinge region or portion thereof;
L1, L2 and L3 are each an amino acid domain linker, wherein L1, L2 and L3 can be different or the same;
CH2 and CH3 are human immunoglobulin CH2 and CH3 domains, respectively; and
Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21 , IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
23. The protein of any one of claims 1-12, wherein the protein is a heterotetramer, and wherein the ABD that binds to a human NKp30 polypeptide is a Fab and the ABD that binds to an antigen of interest is a Fab.
24. The protein of any one of claims 1-12 or 23, wherein the protein is a heterotetramer comprising: a. a first polypeptide chain comprising from N- to C-terminus: a variable domain (Va-i ) , a CH1 domain, a hinge sequence, a CH2 domain and a CH3 domain; b. a second polypeptide chain comprising from N- to C-terminus: a variable domain (Va-2), a CH1 domain, a hinge sequence, a CH2 domain and a CH3 domain, wherein the CH3 domain of the second polypeptide chain dimerizes or associates with the CH3 domain of the first polypeptide chain; c. a third polypeptide chain that dimerizes with the first polypeptide chain, the third polypeptide chain comprising from N- to C-terminus: a variable domain (VM) and a CL domain; and d. a fourth polypeptide chain that dimerizes with the second polypeptide chain, the fourth polypeptide chain comprising from N- to C-terminus: a variable domain (Vb-2) and a CL domain; wherein one of Va-2 and Vb-2 is a VH and the other is a VL, and the Va-i and Vn domains associate to form an ABD that binds NKp30; wherein one of Va-i and Vn is a VH and the other is a VL, and the Va-2 and Vb-2 domains associate to form an ABD that binds an antigen of interest; wherein one of the first or second polypeptide chains further comprises a cytokine, optionally an IL-2, IL-15, IL-21, IL-7, IL-27, IL-12, IL-18, IFN-a or lFN-b polypeptide, optionally an IL-2v or IL15v, covalently attached, optionally via a domain linker, to the C-terminus of the CH3 domain.
25. The protein of any one of claims 1-12 or 23-24, wherein the protein is a heterotetramer having a polypeptide chain 1, 2, 3 and 4:
Vb-2 - (CH1 or Ci_)d (chain 4) (Hinge or L1) - CH2 - CH3 - L2 - Cyt (chain 2)
I (Hinge or L3) - CH2 - CH3 (chain 1)
Figure imgf000148_0001
(chain 3) wherein:
Va-i, Vb-i, Va-2 and Vb-2 are each a VH domain or a VL domain, wherein one of Va-i and Vb-i is a VH and the other is a VL and wherein Va-i and Vw form a first antigen binding domain (ABD) that binds an antigen of interest, wherein one of Va-2and Vb-2 is a VH and the other is a VL and wherein Va-2 and Vb-2 form a second ABD that binds NKp30;
CH1 is a heavy chain constant domain 1 and CL is a light chain constant domain; one of (CH1 or CL)a and (CH1 or CL)c is a CH1 and the other is a CL such that a (CH1/CL) pair is formed; one of (CH1 or CL)b and (CH1 or CL)d is a CH1 and the other is a CL such that a (CH1/CL) pair is formed;
Hinge is an immunoglobulin hinge region or portion thereof;
L1, L2 and L3 are each an amino acid domain linker, wherein L1, L2 and L3 can be different or the same;
CH2 and CH3 are human immunoglobulin CH2 and CH3 domains, respectively; and
Cyt is a cytokine polypeptide or portion thereof that binds to a cytokine receptor present on NK cells, optionally wherein Cyt is a wild-type or variant human IL-2, IL-15, IL-21, IL-7, IL- 27, IL-12, IL-18, IFN-a or IFN-b polypeptide.
26. The protein of any one of the above claims, wherein the ABD that binds to an antigen of interest comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH region comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or any of 45- 122, and wherein the VL comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or any of 123-212.
27. The protein of any one of the above claims, wherein the ABD that binds to NKp30 comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein the VH region comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence of any of SEQ ID NOS: 45-122 and 249, and wherein the VL comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity, to the amino acid sequence of any of SEQ ID NOS: 123-212 and 250.
28. The protein of any one of the above claims, wherein an Fc domain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the Fc polypeptide of any of SEQ ID NOS: 30-35.
29. The protein of any one of the above claims, wherein a CH1 domain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the CH1 polypeptide of any of SEQ ID NO: 26.
30. The protein of any one of the above claims, wherein a CK or CL domain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the CK polypeptide of any of SEQ ID NO: 29.
31. The protein of any one of the above claims, wherein a hinge domain comprises an amino acid sequence having at least about 80% identity to a hinge polypeptide of a human lgG1 , lgG2, lgG3 or lgG4 hinge domain.
32. The protein of any one of the foregoing claims, wherein the protein or the ABD that binds to a human NKp30 polypeptide binds human NKp30 polypeptide with a KD of between 1 and 100 nM, as determined by SPR.
33. The protein of any one of the foregoing claims, wherein the protein or the cytokine binds the human cytokine receptor with a KD of between 10 nM and 1 mM, as determined by SPR.
34. The protein of any one of the above claims, wherein the multispecific polypeptide binds to a human CD16A polypeptide, with an affinity for monovalent binding, as assessed by surface plasmon resonance, that is substantially equivalent to that of a full-length wild type human lgG1 antibody.
35. The protein of any one of the above claims, wherein the antigen of interest is a cancer antigen.
36. A pharmaceutical composition comprising a multispecific protein of any one of the above claims, and a pharmaceutically acceptable carrier or adjuvant.
37. A recombinant cell which expresses at one, two, three of four of (or all of) the polypeptide chains of a multispecific protein according to any one of the above claims.
38. Use of a protein or composition of any one of the above claims as a medicament for the treatment of disease.
39. A method of treating a disease in a subject comprising administering to the subject a multispecific protein or composition according to any of claims 1-36.
40. A method of potentiating NK cell activation, cytotoxicity and/or proliferation of NKp30+CD16+ NK cells and/or NKp30+CD16 NK cells in a subject having a disease, the method comprising administering to the subject a multispecific protein according to any of claims 1-35.
41. A method delivering or bringing a highly potent cytokine to NK cells and/or to a tumor in a subject having a disease, optionally further with reduced toxicity, the method comprising administering to the subject a multispecific protein according to any of claims 1- 35.
42. The method or use of claims 38-41 , wherein the cytokine is a variant or wild- type cytokine or cytokine fragment thereof that retains at least 70%, 80% or 90% of the affinity for its cytokine receptor present on NK cells, compared to the wild-type cytokine counterpart.
43. The method or use of claims 38-42, wherein the disease is a cancer.
44. The method or use of claims 38-43, wherein treatment with the multispecific protein comprises a plurality of administrations, wherein the multispecific protein is administered between 1 and 4 times per month, optionally wherein treatment is for a period of at least 3 months, 6 months or 12 months.
45. A method of making a heteromultimeric protein, comprising: a) providing a first nucleic acid encoding at least a first polypeptide chain according to any of claims 13, 18, 19 or 26-35; b) providing a second nucleic acid encoding at least a second polypeptide chain according to any of claims 13, 18, 19 or 26-35; and c) expressing said first and second nucleic acids in a host cell to produce a protein comprising said first and second polypeptide chains, respectively; loading the protein produced onto an affinity purification support, optionally a Protein-A support, and recovering the heteromultimeric protein.
46. A method of making a heteromultimeric protein, comprising:
(a) providing a first nucleic acid encoding a first polypeptide chain according to any of claims 13, 14, 17, 18, 20-22 or 26-35;
(b) providing a second nucleic acid encoding a second polypeptide chain according to any of claims 13, 14, 17, 18, 20-22 or 26-35;
(c) providing a third nucleic acid comprising a third polypeptide chain according to any of claims 13, 14, 17, 18, 20-22 or 26-35; and
(d) expressing said first, second and third nucleic acids in a host cell to produce a protein comprising said first, second and third polypeptide chains, respectively; loading the protein produced onto an affinity purification support, optionally a Protein-A support, and recovering the heteromultimeric protein.
47. A method of making a heteromultimeric protein, comprising:
(a) providing a first nucleic acid encoding a first polypeptide chain according to any of claims 18 or 23-35;
(b) providing a second nucleic acid encoding a second polypeptide chain according to any of claims 18 or 23-35;
(c) providing a third nucleic acid comprising a third polypeptide chain according to any of claims 18 or 23-35; providing a fourth nucleic acid comprising a fourth polypeptide chain according to any of claims 18 or 23-35; and (d) expressing said first, second and third nucleic acids in a host cell to produce a protein comprising said first, second and third polypeptide chains, respectively; loading the protein produced /onto an affinity purification support, optionally a Protein-A support, and recovering the heteromultimeric protein.
48. A method for identifying or evaluating a polypeptide, comprising the steps of:
(a) providing nucleic acid(s) encoding the polypeptides of a protein of any of claims
1-35;
(b) expressing said nucleic acid(s) in a host cell to produce said polypeptides, respectively; and recovering said protein; and
(c) evaluating the protein produced for a biological activity of interest.
49. The method of claim 48, wherein evaluating the protein comprises: testing the ability of the protein to potentiate NK cell activation, cytotoxicity, proliferation and/or cytokine receptor signaling, optionally in NKp30+CD16+ NK cells and/or NKp30+CD16 NK cells, when incubated with such NK cells in the presence of target cells (that express antigen of interest).
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