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WO2023089079A1 - Nouvelle protéine de fusion spécifique à ox40 et pd-l1 - Google Patents

Nouvelle protéine de fusion spécifique à ox40 et pd-l1 Download PDF

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WO2023089079A1
WO2023089079A1 PCT/EP2022/082368 EP2022082368W WO2023089079A1 WO 2023089079 A1 WO2023089079 A1 WO 2023089079A1 EP 2022082368 W EP2022082368 W EP 2022082368W WO 2023089079 A1 WO2023089079 A1 WO 2023089079A1
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seq
nos
tyr
fusion protein
asp
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Marina PAVLIDOU
Aizea MORALES KASTRESANA
Karoline UTSCHICK
Benjamin WEICHE
Lucia PATTARINI
Catherine GALLOU-HARNOIS
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Pieris Pharmaceuticals GmbH
Les Laboratoires Servier SAS
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Pieris Pharmaceuticals GmbH
Les Laboratoires Servier SAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Novel fusion protein specific for 0X40 and PD-L1 Novel fusion protein specific for 0X40 and PD-L1
  • Programmed death-ligand 1 or PD-L1 (also known as cluster of differentiation 274 or CD274 and B7 homolog 1 or B7-H1) is a single pass type I membrane protein belonging to the B7 family of co-stimulatory/co-inhibitory molecules of antigen presentation.
  • the extracellular portion of PD-L1 contains two domains, an N-terminal IgV-type domain and an IgC-type domain.
  • PD-L1 has a short cytoplasmatic domain without any obvious signal transduction motif, which led to the initial belief that there is no intrinsic signaling by PD-L1 as receptor.
  • cytoplasmatic domain of PD-L1 contains non- classical conserved signal transduction motifs capable of inhibiting interferon (IFN) transduction and protecting cancer cells from IFN cytotoxicity (Gato-Canas et al., Cell Rep, 2017).
  • IFN interferon
  • PD-L1 plays a crucial role in the suppression of the immune system during pregnancy, chronic infections, tissue allografts, autoimmune diseases, and cancer.
  • PD-L1 is expressed on a variety of cell types including B cells, T cells, macrophages, myeloid dendritic cells, mast cells, epithelial, and vascular endothelial cells. It is also expressed in several cancer types including but not limited to melanoma, lung, bladder, colon, and breast cancer.
  • High PD-L1 expression levels are associated with increased tumor aggressiveness by mediating the exhaustion and anergy of tumor infiltrating T cells, the secretion of immuno- suppressive cytokines, and protection from lysis by cytotoxic T cells.
  • PD-L1 is a ligand of the programmed cell death protein 1 (PD-1), a key immune checkpoint inhibitory receptor that is primarily expressed on activated T cells but also on other cells of the immune system including B cells and monocytes.
  • PD-1 is a member of the immunoglobulin family containing an IgV-like extracellular domain, a transmembrane domain and a cytoplasmatic tail with an ITIM (immunoreceptor tyrosine- based inhibitory motif) and an ITSM (immunoreceptor tyrosine-based switch motif).
  • SHP 1 and 2 src homology 2 domain-containing tyrosine phosphatases 1 and 2
  • SHP 1 and 2 src homology 2 domain-containing tyrosine phosphatases 1 and 2
  • E3 ubiquitin ligases of the CBL family.
  • These ubiquitin ligases subsequently ubiquitinate and inactivate key TCR signal transduction mediators leading to the removal of TCR’s from the cell surface (Karwacz et al., EMBO Mol Med, 2011).
  • the SHP 1 and SHP 2 phosphatases inhibit TCR signaling directly by terminating ZAP70 and PI3K phosphorylation.
  • PD-L1 engaged PD-1 can cause inhibition of TCR signaling pathways by affecting the expression and activity of CK2 and cyclin-dependent kinases (CDKs) (Arasanz et al., Oncotarget, 2017). It was also shown that PD-1 engagement leads to re-programming of the T cell metabolism from increased glycolysis, which is required to produce energy for effector functions, to fatty acid (3-oxidation, which is associated with long- lived cells. This may also explain the survival and persistence of high PD-1 expressing cells in patients with chronic infections and cancer (Patsoukis et al., Nat Commun, 2015).
  • Blocking the PD-1/PD-L1 interaction by anti-PD-1 or anti-PD-L1 targeting agents can reverse the immune checkpoint function and release the brake on T cell responses.
  • three PD-L1 antibodies atezolizumab (TECENTRIQ, MPDL3280A, RG7466), avelumab (BAVENCIO, MSB0010718C), and durvalumab (IMFINZI, MEDI4736), are approved for the treatment of cancer.
  • Several successful clinical trials with these antibodies have shown high objective response rates, durability of response, or improved survival rates in bladder cancer, skin cancer and lung cancer (Xu-Monette et al., Front Immunol, 2017).
  • OX40 also known as CD134 or tumor necrosis factor receptor superfamily member 4 (TNFRSF4), is a TNF receptor impacting many aspects of immune function and is one of the most prominent co-stimulatory receptors to control T cells (Croft et al., Immunol Rev, 2009). OX40 is predominantly expressed on activated T cells, preferentially on CD4 + T cells while also on CD8 + cells at tower level (Croft, Annu Rev Immunol, 2010), but not on naive cells.
  • CD134 tumor necrosis factor receptor superfamily member 4
  • TNFRSF4 tumor necrosis factor receptor superfamily member 4
  • dendritic cells B cells, macrophages, and endothelial cells
  • activated T cells Flynn et al., J Exp Med, 1998, Ohshima et al., J Immunol, 1997, Stuber et al., Immunity, 1995, Chen et al., Immunity, 1999).
  • Co-stimulatory signals from OX40 engagement by OX40L or anti-OX40 agonistic antibodies increase proliferation, effector function and survival of T cells (Weinberg et al., Immunol Rev, 2011, Croft et al., Immunol Rev, 2009).
  • OX40 signalling also influences Tregs function, resulting in either impaired suppressing ability of Tregs or Treg expansion depending on the microenvironment (Aspeslagh et al., Eur J Cancer, 2016).
  • OX40 and OX40L regulate cytokine production from T cells leading to generation and maintenance of memory CD4 + and memory CD8 + cells.
  • OX40 is expressed on tumor infiltrating T cells in different tumor settings including breast cancer, melanoma, B-cell lymphoma and head and neck cancer (Marabelle et al., J Clin Invest, 2013, Morris et al., Nat Med, 2009, Vetto et al., Am J Surg, 1997, Xie et al., Pathol Res Pract, 2010, Sarff et al., Am J Surg, 2008).
  • OX40 agonists including OX40L, OX40 fusion proteins, OX40 antibodies, and RNA aptamers
  • Vaccine 2004, Kjaergaard et al., Cancer Res, 2000, Morris et al., Breast Cancer Res Treat, 2001, Piconese et al., J Exp Med, 2008, Gough et al., J Immunother, 2010, Redmond et al., Crit Rev Immunol, 2009.
  • some anti-OX40 agonistic monoclonal antibodies e.g., MEDI0562, Medlmmune LLC
  • MEDI0562 e.g., MEDI0562, Medlmmune LLC
  • OX40 agonists mainly all reported effects of OX40 agonists involve directly regulating CD8 + T cell survival (Vetto et al., Am J Surg, 1997), promoting CD4 + T cell help for CD8 + T cells (Lee et al., J Immunol, 2004), and/or suppressive action on Tregs (Aspeslagh et al., Eur J Cancer, 2016).
  • the efficacy of OX40 agonists may be affected by many factors including tumor microenvironment and tumor burden.
  • OX40 and OX40L are typically upregulated at sites of inflammation or autoimmunity.
  • Blockade of OX40 and/or OX40L have been examined for most of the major animal models and showed significantly attenuated symptoms for diseases including allergic asthma (Arestides et al., Eur J Immunol, 2002, Jember et al., J Exp Med, 2001), experimental allergic encephalomyelitis (EAE) (Weinberg et al., J Immunol, 1999), multiple sclerosis (MS) (Weinberg et al., Nat Med, 1996), colitis (Higgins et al., J Immunol, 1999), diabetes (Pakala et al., Eur J Immunol, 2004), arthritis (Yoshioka et al., Eur J Immunol, 2000), atherosclerosis (van Wanrooij et al., Arterioscler Thromb Vase Bio
  • T cells weak CD4 + or CD8 + T cell responses
  • T cell- derived cytokines T cell- derived cytokines
  • the present disclosure provides, among other things, novel approaches for simultaneously engaging OX40 and PD-L1 via one or more fusion proteins having the properties of binding specificity for OX40 and binding specificity for PD-L1.
  • OX40 means human OX40 (huOX40).
  • Human OX40 means a full-length protein defined by UniProt P43489 (version 177 of 7 April 2021), a fragment thereof, or a variant thereof.
  • Human OX40 is encoded by the gene TNFRSF4.
  • OX40 is also known as cluster of differentiation 134 (CD134) or tumor necrosis factor receptor superfamily member 4 (TNFRSF4), which are used interchangeably.
  • Cynomolgus OX40 refers to the OX40 of cynomolgus monkeys.
  • OX40 of non-human species e.g., cynomolgus OX40 and mouse OX40, is used.
  • “programmed cell death 1 ligand 1” or “PD-L1” means human PD-L1 (huPD-LI).
  • Human PD-L1 means a full-length protein defined by UniProt Q9NZQ7 (version 184 of 2 June 2021), a fragment thereof, or a variant thereof.
  • Human PD-L1 is encoded by the CD274 gene.
  • PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1).
  • PD- L1 of non-human species e.g., cynomolgus PD-L1 and mouse PD-L1, is used.
  • binding affinity describes the ability of a biomolecule (e.g., a polypeptide or a protein) of the disclosure (e.g., a lipocalin mutein, an antibody, a fusion protein, or any other peptide or protein) to bind a selected target (and form a complex). Binding affinity is measured by a number of methods known to those skilled in the art including, but not limited to, fluorescence titration, enzyme-linked immunosorbent assay (ELISA)-based assays, including direct and competitive ELISA, calorimetric methods, such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR).
  • a biomolecule e.g., a polypeptide or a protein of the disclosure
  • ELISA enzyme-linked immunosorbent assay
  • ITC isothermal titration calorimetry
  • SPR surface plasmon resonance
  • Binding affinity is thereby reported as a value of the dissociation constant (K D ), half maximal effective concentration (EC 50 ), or half maximal inhibitory concentration (IC 50 ) measured using such methods.
  • K D dissociation constant
  • EC 50 half maximal effective concentration
  • IC 50 half maximal inhibitory concentration
  • the term “comparable to”, “about the same,” “substantially the same” or “substantially similar” means one biomolecule has a binding affinity reported as a K D , an EC 50 , or an IC 50 value that is identical or similar to that of another molecule within the experimental variability of the binding affinity measurement.
  • “comparable to”, “about the same,” “substantially the same” or “substantially similar” relate to a value that is within 50% deviation to a given reference value, more preferably within 20% deviation, most preferably within 10% deviation.
  • the experimental variability of the binding affinity measurement is dependent upon the specific method used and is known to those skilled in the art.
  • the term “substantially” may also refer to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • detect As used herein, the term “detect,” “detection,” “detectable,” or “detecting” is understood both on a quantitative and a qualitative level, as well as a combination thereof. It thus includes quantitative, semi-quantitative, and qualitative measurements performed on a biomolecule of the disclosure.
  • “detectable affinity” generally means the binding ability between a biomolecule and its target, reported by a K D , EC 50 , or IC 50 value, is at most about 10- 5 M or lower. A binding affinity, reported by a K D , EC 50 , or IC 5 o value, higher than 10- 5 M is generally no longer measurable with common methods such as ELISA and SPR and is therefore of secondary importance.
  • “detectable affinity” may refer to a K D value of about 10‘ 5 M or lower as determined by ELISA or SPR, preferably SPR.
  • binding affinity e.g., fluorescence titration, competitive ELISA (also called competition ELISA), and surface plasmon resonance
  • binding affinity reported by a Kg, EC 50 , or IC 50 value may vary within a certain experimental range, depending on the method and experimental setup.
  • binding specificity relates to the ability of a biomolecule to discriminate between the desired target (for example, OX40 and PD-L1) and one or more reference targets (for example, cellular receptor for neutrophil gelatinase-associated lipocalin). It is understood that such specificity is not an absolute but a relative property and can be determined, for example, by means of SPR, western blots, ELISA, fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), ImmunoHistoChemistry (IHC), and peptide scans.
  • desired target for example, OX40 and PD-L1
  • reference targets for example, cellular receptor for neutrophil gelatinase-associated lipocalin
  • the term “specific for,” “specific binding,” “specifically bind,” or “binding specificity” means that the fusion protein binds to, reacts with, or is directed against OX40 and PD-L1, as described herein, but does not essentially bind another protein.
  • the term “another protein” includes any proteins that are not OX40 or PD-L1 or proteins closely related to or being homologous to OX40 or PD-L1.
  • OX40 or PD-L1 from species other than human and fragments and/or variants of OX40 or PD-L1 are not excluded by the term “another protein.”
  • the term “does not essentially bind” means that the fusion proteins of the present disclosure bind another protein with lower binding affinity than OX40 and/or PD- L1, i.e., show a cross-reactivity of less than 30%, preferably 20%, more preferably 10%, particularly preferably less than 9, 8, 7, 6, or 5%.
  • fusion protein specifically reacts as defined herein above can easily be tested, inter alia, by comparing the reaction of a fusion protein of the present disclosure with OX40 and/or PD-L1 and the reaction of said fusion protein with (an)other protein(s).
  • lipocalin refers to a monomeric protein of approximately 18-20 kDa in weight, having a cylindrical p-pleated sheet supersecondary structural region comprising a plurality of P-strands (preferably eight p-strands designated A to H) connected pair-wise by a plurality of (preferably four) loops at one end to thereby comprise a ligand-binding pocket and define the entrance to the ligand-binding pocket.
  • the loops comprising the ligand-binding pocket used in the present disclosure are loops connecting the open ends of p-strands A and B, C and D, E and F, and G and H, and are designated loops AB, CD, EF, and GH.
  • lipocalin As used herein include, but are not limited to, human lipocalins including tear lipocalin (Tic, Lcn1), Lipocalin-2 (Lcn2) or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D (ApoD), apolipoprotein M, cq-acid glycoprotein 1, cq-acid glycoprotein 2, ⁇ 1 -microglobulin, complement component 8y, retinol-binding protein (RBP), the epididymal retinoic acid- binding protein, glycodelin, odorant-binding protein Ila, odorant-binding protein lib, lipocalin- 15 (Lcn15), and prostaglandin D synthase.
  • Tic tear lipocalin
  • Lcn2 Lipocalin-2
  • NGAL neutrophil gelatinase-associated lipocalin
  • ApoD apolipoprotein D
  • apolipoprotein M
  • Lipocalin-2 or “neutrophil gelatinase-associated lipocalin” refers to human Lipocalin-2 (hLcn2) or human neutrophil gelatinase-associated lipocalin (hNGAL) and further refers to the mature human Lipocalin-2 or mature human neutrophil gelatinase-associated lipocalin.
  • the term “mature” when used to characterize a protein means a protein essentially free from the signal peptide.
  • a “mature hNGAL” of the instant disclosure refers to the mature form of human neutrophil gelatinase-associated lipocalin, which is free from the signal peptide.
  • Mature hNGAL is described by residues 21-198 of the sequence deposited with the SWISS-PROT Data Bank under Accession Number P80188, and its amino acid sequence is shown in SEQ ID NO: 1.
  • a “native sequence” refers to a protein or a polypeptide having a sequence that occurs in nature or having a wild-type sequence, regardless of its mode of preparation. Such native sequence protein or polypeptide can be isolated from nature or can be produced by other means, such as by recombinant or synthetic methods.
  • the “native sequence lipocalin” refers to a lipocalin having the same amino acid sequence as the corresponding polypeptide derived from nature.
  • a native sequence lipocalin can have the amino acid sequence of the respective naturally occurring (wild-type) lipocalin from any organism, in particular, a mammal.
  • the terms “native sequence lipocalin” and “wild-type lipocalin” are used interchangeably herein.
  • a “mutein,” a “mutated” entity (whether protein or nucleic acid), or “mutant” refers to the exchange, deletion, or insertion of one or more amino acids or nucleotides, compared to the naturally occurring (wild-type) protein or nucleic acid. Said term also includes fragments of a mutein as described herein.
  • the present disclosure explicitly encompasses lipocalin muteins, as described herein, having a cylindrical p-pleated sheet supersecondary structural region comprising eight p-strands connected pair-wise by four loops at one end to thereby comprise a ligand-binding pocket and define the entrance of the ligand-binding pocket, wherein at least one amino acid located within said four loops has been mutated as compared to the native sequence lipocalin.
  • Lipocalin muteins of the present disclosure preferably have the function of binding OX40 as described herein.
  • fragment in connection with the lipocalin muteins of the disclosure, refers to proteins or polypeptides derived from full-length mature hNGAL or lipocalin muteins that are N-terminally and/or C-terminally truncated, i.e., lacking at least one of the N-terminal and/or C-terminal amino acids.
  • fragments may include at least 10 or more, such as 20 or 30 or more consecutive amino acids of the primary sequence of mature hNGAL or the lipocalin mutein it is derived from and are usually detectable in an immunoassay of mature hNGAL.
  • Such a fragment may lack up to 2, up to 3, up to 4, up to 5, up to 10, up to 15, up to 20, up to 25, or up to 30 (including all numbers in between) of the N- terminal and/or C-terminal amino acids.
  • the fragment is preferably a functional fragment of mature hNGAL or the lipocalin mutein from which it is derived, which means that it preferably retains the binding specificity, preferably to OX40, of mature hNGAL or the lipocalin mutein it is derived from.
  • a functional fragment may comprise at least amino acids at positions 13-157, 15-150, 18-141 , 20-134, 25-134, or 28-134 corresponding to the linear polypeptide sequence of mature hNGAL.
  • a “fragment” with respect to the corresponding target OX40 or PD-L1 of a fusion protein of the disclosure refers to N-terminally and/or C-terminally truncated OX40 or PD-L1 or protein domains of OX40 or PD-L1. Fragments of OX40 or fragments of PD-L1 as described herein retain the capability of the full-length OX40 or PD-L1 to be recognized and/or bound by a fusion protein of the disclosure. As an illustrative example, the fragment may be an extracellular domain of OX40 or PD-L1.
  • such an extracellular domain of human OX40 may comprise residues 29-214 of UniProt P43489 or residues 1-186 of SEQ ID NO: 4.
  • Such an extracellular domain may comprise, consist essentially of, or consist of amino acids of the extracellular subdomains of OX40, such as the individual or combined amino acid sequences of domain 1 (residues 30-65 of UniProt P43489), domain 2 (residues 66-107 of UniProt P43489), domain 3 (residues 108-126 of UniProt P43489) and domain 4 (residues 127-167 of UniProt P43489).
  • An extracellular domain of cynomolgus OX40 may comprise residues 1-192 of SEQ ID NO: 6.
  • such an extracellular domain of human PD-L1 may comprise, consist essentially of, or consist of amino acid residues 19-238 of UniProt Q9NZQ7.
  • variant relates to derivatives of a protein or polypeptide that include mutations, for example by substitutions, deletions, insertions, and/or chemical modifications of an amino acid sequence or nucleotide sequence. In some embodiments, such mutations and/or chemical modifications do not reduce the functionality of the protein or peptide. Such substitutions may be conservative, i.e., an amino acid residue is replaced with a chemically similar amino acid residue.
  • Examples of conservative substitutions are the replacements among the members of the following groups: 1 ) alanine, serine, threonine, and valine; 2) aspartic acid, glutamic acid, glutamine, asparagine, and histidine; 3) arginine, lysine, glutamine, asparagine, and histidine; 4) isoleucine, leucine, methionine, valine, alanine, phenylalanine, threonine, and proline; and 5) isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.
  • variants include proteins or polypeptides, wherein one or more amino acids have been substituted by their respective D- stereoisomers or by amino acids other than the naturally occurring 20 amino acids, such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline.
  • variants also include, for instance, proteins or polypeptides in which one or more amino acid residues are added or deleted at the N- and/or C-terminus.
  • a variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or at least about 98% amino acid sequence identity with the native sequence protein or polypeptide.
  • a variant preferably retains the biological activity, e.g. binding the same target, of the protein or polypeptide it is derived from.
  • variant as used herein with respect to the corresponding protein ligand OX40 or PD-L1 of a fusion protein of the disclosure, relates to OX40 or PD-L1 or a fragment thereof, respectively, that has one or more such as 1, 2, 3, 4 ,5 ,6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 60, 70, 80 or more amino acid substitutions, deletions and/or insertions in comparison to the native sequence of OX40 or PD-L1 (wild- type OX40 or PD-L1), such as OX40 as deposited with UniProt P43489 or PD-L1 as deposited with UniProt Q9NZQ7 as described herein.
  • An OX40 variant or a PD-L1 variant has preferably an amino acid identity of at least 50%, 60%, 70%, 80%, 85%, 90% or 95% with a wild-type OX40 or PD-L1.
  • An OX40 variant or a PD-L1 variant as described herein retains the ability to bind fusion proteins specific to OX40 and PD-L1 disclosed herein.
  • variant relates to a lipocalin mutein or fragment thereof of the disclosure, wherein the sequence has mutations, including substitutions, deletions, and insertions, and/or chemical modifications.
  • a variant of a lipocalin mutein as described herein retains the biological activity, e.g., binding to OX40, of the lipocalin mutein from which it is derived.
  • a lipocalin mutein variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% amino acid sequence identity with the lipocalin mutein from which it is derived.
  • mutagenesis refers to the introduction of mutations into a polynucleotide or amino acid sequence. Mutations are preferably introduced under experimental conditions such that the amino acid naturally occurring at a given position of the protein or polypeptide sequence can be altered, for example substituted by at least one amino acid.
  • mutagenesis also includes the (additional) modification of the length of sequence segments by deletion or insertion of one or more amino acids. Thus, it is within the scope of the disclosure that, for example, one amino acid at a chosen sequence position is replaced by a stretch of three amino acids, leading to an addition of two amino acid residues compared to the length of the respective segment of the native protein or polypeptide amino acid sequence.
  • an insertion or deletion may be introduced independently from each other in any of the sequence segments that can be subjected to mutagenesis in the disclosure.
  • an insertion may be introduced into an amino acid sequence segment corresponding to the loop AB of the native sequence lipocalin (cf. International Patent Publication No. WO 2005/019256, which is incorporated by reference in its entirety herein).
  • random mutagenesis means that no predetermined mutation (alteration of an amino acid) is present at a certain sequence position but that at least two amino acids can be incorporated with a certain probability at a predefined sequence position during mutagenesis.
  • sequence identity denotes a property of sequences that measures their similarity or relationship.
  • sequence identity or “identity” as used in the present disclosure means the percentage of pair-wise identical residues - following (homologous) alignment of a sequence of a protein or polypeptide of the disclosure with a sequence in question - with respect to the number of residues in the longer of these two sequences. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.
  • sequence homology or “homology” has its usual meaning, and a homologous amino acid includes identical amino acids as well as amino acids which are regarded to be conservative substitutions at equivalent positions in the linear amino acid sequence of a protein or polypeptide of the disclosure (e.g., any fusion proteins or lipocalin muteins of the disclosure).
  • BLAST Altschul et al., Nucleic Acids Res, 1997)
  • BLAST2 Altschul et al., J Mol Biol, 1990
  • Smith-Waterman Smith and Waterman, J Mol Biol, 1981
  • the percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version 2.2.5 (November 16, 2002; (Altschul et al., Nucleic Acids Res, 1997).
  • the percentage of homology is based on the alignment of the entire protein or polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1; cutoff value set to 10- 3 ) including the propeptide sequences, preferably using the wild-type protein scaffold as reference in a pairwise comparison. It is calculated as the percentage of numbers of “positives” (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.
  • a skilled artisan can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as BLAST 2.0, which stands for Basic Local Alignment Search Tool, or ClustalW, or any other suitable program which is suitable to generate sequence alignments.
  • BLAST 2.0 which stands for Basic Local Alignment Search Tool, or ClustalW, or any other suitable program which is suitable to generate sequence alignments.
  • amino acid sequence of a reference (wild-type) lipocalin can serve as “subject sequence” or “reference sequence”, while the amino acid sequence of a lipocalin mutein serves as “query sequence.”
  • the terms “wild-type sequence,” “reference sequence,” and “subject sequence” are used interchangeably herein.
  • a preferred wild-type sequence of a lipocalin is the sequence of mature hNGAL as shown in SEQ ID NO: 1.
  • Gaps are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of sequence identity.
  • the term “position” means the position of either an amino acid within an amino acid sequence disclosed herein or the position of a nucleotide within a nucleic acid sequence disclosed herein. It is to be understood that when the term “correspond” or “corresponding” is used herein in the context of the amino acid sequence positions of one or more lipocalin muteins, a corresponding position is not only determined by the number of the preceding nucleotides or amino acids. Accordingly, the absolute position of a given amino acid in accordance with the disclosure may vary from the corresponding position due to deletion or addition of amino acids elsewhere in a (mutant or wild-type) lipocalin.
  • the absolute position of a given nucleotide in accordance with the present disclosure may vary from the corresponding position due to deletions or additional nucleotides elsewhere in a mutein or wild-type lipocalin 5’-untranslated region (UTR) including the promoter and/or any other regulatory sequences or gene regions (including exons and introns).
  • a "corresponding position” in accordance with the disclosure may be the sequence position that aligns to the sequence position it corresponds to in a pairwise or multiple sequence alignment according to the present disclosure.
  • nucleotides or amino acids may differ from adjacent nucleotides or amino acids but said adjacent nucleotides or amino acids which may have been exchanged, deleted, or added may be comprised by the same one or more “corresponding positions”.
  • a corresponding position in a lipocalin mutein based on a reference sequence in accordance with the disclosure, it is preferably to be understood that the positions of nucleotides or amino acids of a lipocalin mutein can structurally correspond to the positions elsewhere in a reference lipocalin (wild-type lipocalin) or another lipocalin mutein, even if they may differ in the absolute position numbers, as appreciated by the skilled person in light of the highly-conserved overall folding pattern among lipocalins.
  • conjugate refers to the joining together of two or more subunits, through all forms of covalent or non-covalent linkage, by means including, but not limited to, genetic fusion, chemical conjugation, coupling through a linker or a cross-linking agent, and non-covalent association.
  • fusion polypeptide or “fusion protein” as used herein refers to a polypeptide or protein comprising two or more subunits.
  • a fusion protein as described herein comprises two or more subunits, at least one of these subunits being capable of specifically binding to OX40, and a further subunit capable of specifically binding to PD-L1.
  • a fusion protein as described herein does not comprise a subunit capable of specifically binding to 4-1 BB (CD137).
  • the subunits may be linked by covalent or non-covalent linkage.
  • the fusion protein is a translational fusion between the two or more subunits.
  • the translational fusion may be generated by genetically engineering the coding sequence for one subunit in a reading frame with the coding sequence of a further subunit. Both subunits may be interspersed by a nucleotide sequence encoding a linker. However, the subunits of a fusion protein of the present disclosure may also be linked through chemical conjugation. The subunits forming the fusion protein are typically linked to each other as follows: C- terminus of one subunit to N-terminus of another subunit, or C-terminus of one subunit to C- terminus of another subunit, or N-terminus of one subunit to N-terminus of another subunit, or N-terminus of one subunit to C-terminus of another subunit.
  • the subunits of the fusion protein can be linked in any order and may include more than one of any of the constituent subunits. If one or more of the subunits are part of a protein (complex) that consists of more than one polypeptide chain, the term “fusion protein” may also refer to the protein comprising the fused sequences and all other polypeptide chain(s) of the protein (complex). As an illustrative example, where an antibody is fused to a lipocalin mutein via a heavy or light chain of the antibody, the term “fusion protein” may refer to the single polypeptide chain comprising the lipocalin mutein and the heavy or light chain of the antibody. The term “fusion protein” may also refer to the entire antibody (both light and heavy chains) and the lipocalin mutein fused to one or both of its heavy and/or light chains.
  • a preferred subunit of a fusion protein disclosed herein refers to a single protein or a separate polypeptide chain, which can form a stable folded structure by itself and may define a unique function of providing a binding motif towards a target.
  • a preferred subunit of the disclosure is a lipocalin mutein.
  • a preferred subunit of the disclosure is an antibody or an antigen-binding domain/fragment thereof.
  • a “linker” that may be comprised by a fusion protein of the present disclosure joins together two or more subunits of a fusion protein as described herein.
  • the linkage can be covalent or non-covalent.
  • a preferred covalent linkage is via a peptide bond, such as a peptide bond between amino acids.
  • a preferred linker is a peptide linker. Accordingly, in a preferred embodiment, said linker comprises one or more amino acids, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids.
  • Preferred peptide linkers are described herein, including glycine-serine (GS) linkers, glycosylated GS linkers, and proline-alanine-serine polymer (PAS) linkers.
  • GS linker such as a (G 4 S) 3 linker as described in SEQ ID NO: 10
  • PES proline-alanine-serine polymer
  • GS linker such as a (G 4 S) 3 linker as described in SEQ ID NO: 10
  • Other preferred linkers include chemical linkers.
  • albumin includes all mammalian albumins such as human serum albumin or bovine serum albumin or rat serum albumin.
  • organic molecule or “small organic molecule” denotes an organic molecule comprising at least two carbon atoms, but preferably not more than 7 or 12 rotatable carbon bonds, having a molecular weight in the range between 100 and 2,000 daltons, preferably between 100 and 1,000 daltons, and optionally including one or two metal atoms.
  • sample is defined as a biological sample taken from any subject. Biological samples include, but are not limited to, blood, serum, urine, feces, semen, or tissue, including tumor tissue.
  • a “subject” is a vertebrate, preferably a mammal, more preferably a human.
  • the term “mammal” is used herein to refer to any animal classified as a mammal, including, without limitation, humans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as cynomolgus monkeys, to name only a few illustrative examples.
  • the “mammal” used herein is human.
  • an “effective amount” is an amount sufficient to yield beneficial or desired results.
  • An effective amount can be administered in one or more individual administrations or doses.
  • antibody includes whole antibodies or any antigen-binding fragment (i.e., “antigen-binding portion”) or single chain thereof.
  • antibody and immunoglobulin are used interchangeably herein.
  • a whole antibody refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable domain (V H or HCVR) and a heavy chain constant region (C H ).
  • the heavy chain constant region is comprised of three domains, C H1 , C H2 and C H 3.
  • Each light chain is comprised of a light chain variable domain (V L or LCVR) and a light chain constant region (C L ).
  • the light chain constant region is comprised of one domain, C L .
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxy-terminus: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen (for example, PD-L1 ).
  • the constant regions of the antibodies may optionally mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • antigen-binding fragment (also referred to herein as “antigen-binding domain”) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-L1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment consisting of the V H , V L , C L and C H1 domains; (ii) a F(ab') 2 fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment consisting of the V H , V L , C L and C m domains and the region between the C H1 and C H2 domains; (iv) an Fd fragment consisting of the V H and C m domains; (v) a single-chain Fv fragment consisting of the V H and V L domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al., Nature, 1989) consisting of a V H domain; (vii) an isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker
  • Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, or multispecific). Antibodies may also be fully human.
  • framework or “FR” refers to the variable domain residues other than the hypervariable region (CDR) residues.
  • Fc region refers to the C-terminal region of an antibody heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an antibody heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof numbering according to EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000).
  • the C-terminal lysine (residue 447 according to EU index of Kabat) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • Suitable native-sequence Fc regions for use in the antibodies of the disclosure include human lgG1, lgG2 (lgG2A, lgG2B), lgG3, and lgG4.
  • Fc receptor refers to a receptor that binds to the Fc region of an antibody.
  • isolated antibody refers to an antibody that is substantially free of its natural environment. For instance, an isolated antibody is substantially free of cellular material and other proteins from the cell or tissue source from which it is derived. An “isolated antibody” further refers to an antibody that is substantially free of other antibodies having different antigenic specificities. In the present case, an isolated antibody that binds specifically PD-L1 is substantially free of antibodies that specifically bind antigens other than PD-L1. However, an isolated antibody that specifically binds PD-L1 may have cross-reactivity to other antigens, such as PD-L1 molecules from other species.
  • monoclonal antibody refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • humanized antibody refers to an antibody that consists of the CDRs of antibodies derived from mammals other than human, and the FR region and the constant region of a human antibody or derived from a human antibody.
  • a humanized antibody may comprise a variable domain that has a variable region amino acid sequence which, analyzed as a whole, is closer to human than to other species as assessed using the Immunogenetics Information System (IMGT) DomainGapAlign tool, as described by Ehrenmann et al. (2010).
  • IMGT Immunogenetics Information System
  • a humanized antibody may be useful as an effective component in a therapeutic agent due to the reduced antigenicity.
  • a therapeutic agent may be any agent for the prevention, amelioration, or treatment of a disease, a physiological condition, a symptom, or for the evaluation or diagnosis thereof.
  • human antibody includes antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • the human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody”, as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • Figure 1 provides an overview over the design of the representative fusion proteins described in this application that are bispecific for the targets OX40 and PD-L1.
  • Representative fusion proteins were made based on an antibody specific for PD-L1 (e.g., an antibody whereby heavy chains are provided by SEQ ID NO: 78, or comprise a heavy chain variable domain of SEQ ID NO: 67, or comprise the CDR sequences of GFSLSNYD (HCDR1, SEQ ID NO: 45), IWTGGAT (HCDR2, SEQ ID NO: 46), and VRDSNYRYDEPFTY (HCDR3; SEQ ID NO: 47), and light chains are provided by SEQ ID NO: 79, or comprise a light chain variable domain of SEQ ID NO: 73, or comprise the CDR sequences of QSIGTN (LCDR1, SEQ ID NO: 48), YAS (LCDR2), and QQSNSWPYT (LCDR3; SEQ ID NO: 49)) and one or more
  • One or more lipocalin muteins were genetically fused to the C- and/or the N-terminus of either the heavy chain or the light chain of a PD-L1 specific antibody as depicted in Figure 1A-1I, resulting in the fusion proteins of, e.g., SEQ ID NOs: 82) and 79, SEQ ID NOs: 78 and 83, SEQ ID NOs: 84 and 79, SEQ ID NOs: 78 and 85, SEQ ID NOs: 86 and 79, and SEQ ID NOs: 82) and 83.
  • the generated fusion proteins can be bivalent to OX40 (e.g., as depicted in Figure 1A-1D), or tetravalent to OX40 (e.g., as depicted in Figure 1E-1H), or have even higher valency to OX40 (e.g., as depicted in Figure 11).
  • Additional monospecific fusion proteins were generated by fusing one or more OX40-specific lipocalin muteins (e.g., as depicted in Figure 1J-1K) to the C-terminus of the Fc region of an antibody provided as described herein via a peptide linker.
  • the resulting monospecific fusion proteins are provided in, e.g., SEQ ID NO: 92 and SEQ ID NO: 42.
  • Figure 2 shows the results of ELISA experiments in which the binding to PD- L1 or OX40 of representative fusion proteins was determined as described in Example 4.
  • PD-L1 or OX40 (with C-terminal His or Fc tag) was coated on a microtiter plate, and the tested agents were titrated starting with the highest concentration of 100 nM.
  • Bound agents under study were detected via anti-human IgG Fc-HRP or anti-NGAL-HRP, respectively.
  • the data was fit with a 1:1 binding model with EC 50 value and the maximum signal as free parameters, and a slope that was fixed to one.
  • the resulting EC 50 values are provided in Table 5.
  • Figure 3 illustrates the results of an ELISA experiment in which the ability of representative fusion proteins to simultaneously bind both targets, PD-L1 and OX40, was determined as described in Example 5.
  • Recombinant huPD-L1-His or huOX40-His was coated on a microtiter plate, followed by a titration of the fusion proteins starting with the highest concentration of 100 nM.
  • Figure 4 shows the results of an assessment of the target binding of fusion proteins by flow cytometry using human or cynomolgus OX40 (Figure 4A) as well as human or cynomolgus PD-L1 ( Figure 4B) expressing Flp-ln-CHO cells as described in Example 6. No binding was observed when using mock transfected Flp-ln-CHO cells ( Figure 4C).
  • Figure 5 depicts the results of fluorescence-activated cell sorting (FACS) studies assessing the off-target or non-specific binding of fusion proteins to human endothelial cells (HUVEC) that are OX40 negative but PD-L1 positive, as described in Example 8. None of the tested fusion proteins show non-specific or off-target binding.
  • FACS fluorescence-activated cell sorting
  • Figure 6 shows that the fusion proteins compete with OX40 for binding to OX40L, depicted in competitive ELISA studies as described in Example 9.
  • a constant concentration of huOX40L-His was coated on a microtiter plate, followed by adding a mixture of tested molecules at different concentrations and tracer huOX40-Fc at a fixed concentration. Bound tracer was detected using an HRP-labelled anti-IgG Fc antibody. Dose- dependent inhibition of huOX40-Fc binding to OX40L by the OX40 and PD-L1 bispecific fusion proteins or OX40-specific antibodies was observed.
  • Figure 7 shows that the fusion proteins compete with PD-L1 for binding to PD-1, depicted in competitive ELISA studies as described in Example 10.
  • a constant concentration of huPD-1-His was coated on a microtiter plate, followed by adding a mixture of tested molecules at different concentrations and tracer huPD-L1-Fc at a fixed concentration. Bound tracer was detected using an HRP-labelled anti-IgG Fc antibody. Dose- dependent inhibition of huPD-L1-Fc binding to PD-1 by the OX40 and PD-L1 bispecific fusion proteins or PD-L1 specific antibodies was observed.
  • Figure 8 shows the potential of representative fusion proteins to block the inhibitory signal mediated by PD-1/PD-L1 interaction, evaluated using a PD-1/PD-L1 blockade bioassay as described in Example 11.
  • PD-1-NFAT-luc Jurkat T cells a Jurkat cell line expressing PD-1 and a NFAT-mediated luciferase gene under the NFAT promoter control
  • PD-L1 aAPC/CHO-K1 cells were co-cultured with PD-L1 aAPC/CHO-K1 cells in presence of various concentrations of tested molecules. After 6 hours, luciferase assay reagent was added, and luminescent signals were measured.
  • Background signal is PD-1-NFAT-luc Jurkat T cells co- cultured with only PD-L1 aAPC/CHO-K1 cells.
  • the fusion proteins of SEQ ID NOs: 82 and 79, 78 and 87, 88 and 79, 89 and 79, 93 and 90, and 91 and 94 block the PD-1/PD-L1 pathway, comparable to the building block PD-L1 antibody shown in SEQ ID NOs: 78 and 79 and the reference PD-L1/OX40 bispecific fusion protein shown in SEQ ID NOs: 43 and 44.
  • Figure 9 shows the results of a representative experiment in which the ability of selected fusion proteins to induce T cell activation was investigated.
  • human peripheral blood mononuclear cells PBMCs
  • PBMCs peripheral blood mononuclear cells
  • SEB staphylococcal enterotoxin B
  • IL-2 interleukin 2
  • A shows that the selected fusion proteins are capable of inducing a stronger T cell activation as measured by the secretion of IL-2 than the PD-L1 antibody used to build the fusion proteins.
  • Fc fusions of OX40-specific lipocalin muteins do not induce T cell activation indicating that binding to PD-L1 is a requirement of OX40-clustering and OX40-mediated activation of T cells.
  • B shows that the bispecific fusion protein depicted in the graph induces stronger T cell activation than the reference bispecific fusion protein, the anti-PD-L1/anti- OX40 antibody cocktail and the combination of building blocks used to build the bispecific fusion.
  • Figure 10 shows the ability of representative fusion proteins to co-stimulate T cell activation in the presence of tumor cells.
  • PD-L1 expressing human breast carcinoma cells MDA-MB-231 were seeded into anti-human CD3 coated plates.
  • Pan T cells and various concentrations of fusion proteins and single building blocks were added and incubated for 3 days.
  • Levels of secreted IL-2 were determined by an electrochemoluminescence-based assay, as described in Example 13.
  • A shows that bispecific fusion proteins built with lipocalin muteins and the reference bispecific induce a dose-dependent T cell activation.
  • the antibody building block does not induce T cell activation in this experimental set up.
  • B shows that a representative fusion protein induces strong T cell activation as measured by the secretion of IL-2 while neither the building blocks of this bispecific fusion protein alone or in combination or the cocktail of anti-PD-L1 and anti-OX40 antibodies induced T cell activation.
  • Figure 11 demonstrates the ability of representative fusion proteins to co- stimulate T cell activation in the presence of PD-L1.
  • the PD-L1 antibody building block, Fc fusions of OX40-specific lipocalin muteins and a reference bispecific were tested in parallel.
  • CHO cells either transfected with human PD-L1 (A) or mock transfected (human PD-L1 negative, B) were seeded into anti-human anti-CD3 coated plates.
  • Pan T cells as well as various concentrations of tested molecules were added and incubated for 3 days. Levels of secreted IL-2 in the supernatant were determined by an electrochemoluminescence-based assay, as described in Example 14.
  • the fusion proteins induce a strong dose-dependent increase in IL-2 secretion only in the presence of PD-L1.
  • Figure 12 shows the ability of representative fusion proteins to stimulate human T cells in the presence of PD-L1 expressed by human primary immune cells.
  • the PD- L1 building block (SEQ ID NOs: 78 and 79), an Fc fusion of an OX40-specific lipocalin mutein (SEQ ID NO:42), a reference anti-PD-L1 antibody (SEQ ID NOs: 80 and 81), a reference bispecific (SEQ ID NOs: 43 and 44) and a reference OX40 agonistic antibody (SEQ ID NOs: 25 and 26) were tested in parallel.
  • Human monocyte-derived dendritic cells were co-cultured with human primary CD4 T cells in a one-way mixed lymphocyte reaction.
  • IFNg interferon gamma
  • Figure 13 demonstrates the ability of representative fusion proteins to stimulate human CD3 T cells in the presence of PD-L1 expressed by human primary immune cells.
  • the PD-L1 building block (SEQ ID NOs: 78 and 79) and a tandem lipocalin-Fc fusion (SEQ ID NO: 92) were tested in parallel.
  • Human monocyte-derived dendritic cells were co-cultured with human primary CD3 T cells in a one-way mixed lymphocyte reaction. Tested molecules were added and incubated for 6 days at the fixed concentration of 10 pg/ml.
  • Levels of secreted IL-2, IFNg, IL-13, Granzyme A, Granzyme B and FasLigand were measured by a luminescence-based assay. The data demonstrate that the fusion proteins induce secretion of IL-2, IL-13, Granzyme A and Granzyme B at higher levels than the building blocks.
  • Figure 14 provides the results of pharmacokinetic analyses of the bispecific fusion proteins of SEQ ID NOs: 78 and 87, SEQ ID NOs: 88 and 79, SEQ ID NOs: 89 and 79, SEQ ID NOs: 82 and 79, SEQ ID NOs: 93 and 90, and SEQ ID NOs: 91 and 94, the reference bispecific (SEQ ID NOs: 43 and 44) and the building block PD-L1 antibody (SEQ ID NOs: 78 and 79) in mice, as described in Example 17.
  • CD-1 NUDE mice or male CD-1 mice (3 mice per timepoint) were injected intravenously with fusion proteins at a dose of 2 mg/kg.
  • Drug levels were detected using a Sandwich ECL assay detecting the full molecule via the targets PD-L1 and OX40 or the lipocalin scaffold.
  • the anti-PD-L1 antibody plasma levels were determined using a Sandwich ECL assay with the targets PD-L1 and human Fc. The data demonstrate that the fusion proteins have long, antibody-like terminal half-lives in mice.
  • Figure 15 provides the results of a pharmacokinetic analysis of bispecific fusion proteins (SEQ ID NOs: 82 and 79, SEQ ID NOs: 78 and 87, and SEQ ID NOs: 88 and 79) and the building block PD-L1 antibody (SEQ ID NOs: 78 and 79) in cynomolgus monkeys as described in Example 18. Cynomolgus monkeys (1-2 mokeys per timepoint) were injected intravenously with tested molecules at a dose of 7.6 - 10 mg/kg. Drug levels were detected using a Sandwich format at Gyrolab xP system at the indicated time points. The data were plotted in a time vs. concentration graph.
  • the PK profile is influenced by the formation of anti-drug antibodies (ADA) that, depending on the fusion protein, leads from 96 h to 168 h onwards to a decrease in exposure. At time points not influenced by ADA, the plasma levels are comparable to the anti-PD-L1 antibody.
  • ADA anti-drug antibodies
  • Figure 16 provides an SEC chromatogram of an exemplary fusion protein (SEQ ID NOs: 82 and 79) before heat stress (black trace), after 1 week at 40°C (blue trace) and after 2 weeks at 40°C (pink trace) as described in Example 20.
  • SEQ ID NOs: 82 and 79 an exemplary fusion protein before heat stress (black trace), after 1 week at 40°C (blue trace) and after 2 weeks at 40°C (pink trace) as described in Example 20.
  • Figure 17 provides an icIEF electropherogram of an exemplary fusion protein (SEQ ID NOs: 82 and 79) as described in Example 21.
  • Figure 18 provides the results obtained with a fusion protein (SEQ ID NOs: 82 and 79), an Fc fusion of an OX40-specific lipocalin mutein (SEQ ID NO: 42), an anti-PD- L1 antibody (SEQ ID NOs: 78 and 79) or an hlgG4 isotype control antibody (SEQ ID NOs: 21 and 22) in an assay as described in Example 22.
  • Figure 18A provides the results regarding the reversal of the suppression of Treg cells on CD4 Tresp cells. Human Tresp CD4 T cells were cultured at different ratios with induced Treg (iTreg).
  • Tested molecules at one fixed concentration of 200 nM were precoated on plastic to ensure crosslinking and incubated for 4 days in presence of a mix of iTreg and Tresp.
  • the proliferation of Tresp was quantified as percentage CFSE negative cells at day 4, and data were expressed as suppression of proliferation vs the control of Tresp alone.
  • Figure 18B provides the results of CD25 expression quantified by flow cytometry on the CD4 Tresp after 4 days of incubation with the tested items.
  • Figure 19 describes the median tumor volume of each group of mice at different times after administration of PBS solution (white circle), 20 mg/kg of fusion protein (SEQ ID NOs: 101 and 102) (black circle), 15.4 mg/kg of anti-PD-L1 antibody (SEQ ID NOs: 80 and 81) (equimolar dose of fusion protein) (grey circle) and 7.7 mg/kg of anti-OX40 antibody (SEQ ID NOs: 25 and 26) (black triangle). Data are expressed as median +/- IQR (Interquartile Range). Dotted lines have been drawn when there was less than 100% of mice but more than 50% of initial squad.
  • Figure 20 shows scatter dot plots of anti-KLH IgG ( Figure 20A) and anti-KLH IgM ( Figure 20B) quantification by ELISA on mouse plasma from a KLH model.
  • the PBS group is represented by a white circle
  • the anti-PD-L1 antibody (SEQ ID NOs: 80 and 81) group is represented by a grey circle
  • the fusion protein (SEQ ID NOs: 101 and 102) group is represented by a black circle.
  • Data are expressed as geometric mean +/- SD. ** p ⁇ 0.01**** p ⁇ 0.0001.
  • the present disclosure provides, among other things, novel approaches for simultaneously engaging OX40 and PD-L1 via one or more fusion proteins having binding specificity for OX40 and binding specificity for PD-L1.
  • Provided fusion proteins are designed to bridge OX40-positive T cells with PD-L1 expressed in the tumor microenvironment.
  • Such bispecific molecules may combine OX40-induced T cell activation and expansion with anti- PD-L1 mediated immune checkpoint blockade and thus may overcome certain limitations of single agent therapy and offer benefits to, for example, resistant or non-responsive patients.
  • the fusion proteins are also designed to provide potentials of a combinatorial therapy in one molecule and at the same time allow the localized induction of antigen-specific T cells in the tumor microenvironment, potentially reducing peripheral toxicity.
  • the present disclosure provides fusion proteins that bind OX40 and PD-L1, as well as methods and useful applications therefor.
  • the disclosure also provides methods of making OX40 and PD-L1 binding fusion proteins described herein as well as compositions comprising such proteins.
  • OX40 and PD-L1 binding fusion proteins of the disclosure as well as compositions thereof may be used in methods of detecting OX40 and/or PD-L1 in a sample, in methods of binding of OX40 and/or PD-L1 in a subject, or in methods of modulating immune responses in a subject. No such fusion proteins having these features attendant to the uses provided by present disclosure have been previously described.
  • Exemplary fusion proteins specific for OX40 and PD-L1 of the disclosure are examples of the disclosure.
  • a provided fusion protein contains at least two subunits in any order: (1) a first subunit that comprises a full-length antibody or an antigen-binding domain thereof specific for PD-L1 , and (2) a second subunit that comprises a lipocalin mutein specific for OX40.
  • a provided fusion protein also may contain at least one additional subunit, for example, a third subunit.
  • a fusion protein may contain a third subunit specific for OX40.
  • a third subunit may be or comprise a lipocalin mutein specific for OX40.
  • two lipocalin muteins may be fused to a first antibody subunit, one at the C-terminus and one at the N-terminus of the antibody.
  • lipocalin muteins may be fused to the heavy chain or light chain of an antibody.
  • provided fusion proteins may comprise one or more additional subunits (e.g., a fourth, fifth, or sixth subunit).
  • At least one subunit may be fused at its N-terminus and/or its C-terminus to another subunit.
  • a linker is a peptide linker, for example, an unstructured glycine-serine (GS) linker, a glycosylated GS linker, or a proline-alanine-serine polymer (PAS) linker.
  • GS linker is a (Gly 4 Ser) 3 linker ((G 4 S) 3 ) as shown in SEQ ID NO: 10.
  • Other exemplary linkers are shown in SEQ ID NOs: 11-20.
  • a peptide linker may have from 1 to 50 amino acids, such as 1 , 2, 3, 4, 5, 10, 11 , 12, 13, 14, 15, 16, 17 18, 19, 20, 25, 30, 35, 40, 45 or 50 amino acids.
  • a first subunit comprises an antibody, such as a full-length antibody
  • a second subunit may be linked via a peptide linker between the N-terminus of the second subunit and the C-terminus of a heavy chain constant region (CH) of said antibody.
  • a third subunit may be linked via a peptide linker between the N- terminus of the third subunit and the C-terminus of a light chain constant region (CL) of said antibody.
  • one subunit can be linked to another subunit as essentially described in Figure 1.
  • one subunit may be fused at its N-terminus and/or its C-terminus to another subunit.
  • a lipocalin mutein subunit can be fused at its N-terminus and/or its C-terminus to an antibody subunit.
  • one lipocalin mutein can be linked, preferably via a peptide linker, to the C-terminus of the antibody heavy chain domain (HC), the N-terminus of the HC, the C- terminus of the antibody light chain (LC), and/or the N-terminus of the LC ( Figure 1A-1D).
  • a lipocalin mutein subunit can be fused at its N- terminus and/or its C-terminus to an antibody fragment.
  • a lipocalin mutein may be linked, preferably via a peptide linker, at the C-terminus of a heavy chain constant region (CH) or the C-terminus of a light chain constant region (CL) of the antibody.
  • a second subunit when one subunit comprises an antibody, such as a full-length antibody, a second subunit may be linked between the N-terminus of the second subunit and the C-terminus of a heavy chain constant region (CH) of said antibody.
  • CH heavy chain constant region
  • a third subunit may be linked between the N-terminus of the third subunit and the C-terminus of a light chain constant region (CL) of said antibody.
  • the Fc function of the Fc region of the antibody may be preserved at the same time while the fusion protein is simultaneously engaging OX40 and PD-L1.
  • the Fc region of the antibody may still be capable of binding to an Fc receptor-positive cell at the same time while the fusion protein is simultaneously engaging OX40 and PD-L1.
  • the Fc function of the Fc region of the antibody may be reduced or fully suppressed by protein engineering while the fusion protein is capable of simultaneously engaging OX40 and PD-L1.
  • the capacity of the Fc region of the antibody to bind an Fc receptor positive cell may be reduced or fully suppressed by protein engineering while the fusion protein is capable of simultaneously engaging OX40 and PD-L1. In some embodiments, this may be achieved, for example, by switching from the lgG1 backbone to lgG4, as lgG4 is known to display reduced Fc-gamma receptor interactions compared to lgG1.
  • mutations may be introduced into the lgG4 backbone such as F234A and L235A.
  • an S228P mutation may also be introduced into the lgG4 backbone to minimize the exchange of lgG4 half-antibody (Silva et al., J Biol Chem, 2015).
  • F234A and L235A mutations may be introduced for decreased ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010) and/or M428L and N434S mutations or M252Y, S254T, and T256E mutations for extended serum half-life (Dall'Acqua et al., J Biol Chem, 2006, Zalevsky et al., Nat Biotechnol, 2010).
  • an additional N297A mutation may be present in the antibody heavy chain of the fusion protein in order to remove the natural glycosylation motif.
  • the Fc portion of an antibody included in a fusion protein of the disclosure may contribute to maintaining the serum levels of the fusion protein.
  • the Fc portion may become internalized and recycled back to the bloodstream, enhancing its half-life within the body.
  • fusion proteins of the disclosure bind OX40 with high affinity.
  • provided fusion proteins bind PD-L1 with high affinity.
  • provided fusion proteins simultaneously bind OX40 and PD-L1.
  • the simultaneous binding to OX40 and PD-L1 allows provided fusion proteins to exhibit a durable anti-tumor or anti-infection response.
  • a fusion protein of the disclosure may be able to bind PD-L1 with a K D value of at most about 2 nM or even lower, such as about 1.5 nM or lower, about 1 nM or lower, about 0.6 nM or lower, or about 0.4 nM or lower.
  • a fusion protein of the disclosure may be able to bind PD-L1 with a K D value comparable to or lower than the K D value of the antibody specific for PD-L1 as included in such fusion protein, such as the antibody having the heavy and light chains provided by SEQ ID NOs: 78 and 79.
  • the K D values of provided fusion proteins may be measured, for example, in a surface-plasmon-resonance (SPR) assay, such as an SPR assay as essentially described in Example 3.
  • SPR surface-plasmon-resonance
  • a fusion protein of the disclosure may be able to bind OX40 with a K D value of at most about 10 nM or even lower, such as about 7 nM, about 6 nM, or about 5 nM, about 4 nM, about 3 nM, about 2 nM or even lower.
  • a fusion protein of the disclosure may be able to bind OX40 with a K D value comparable to or lower than the K D value of the lipocalin mutein specific for OX40 that is included in a particular fusion protein, e.g., SEQ ID NOs: 33, 34, 35, 36, 95, 96, or 97, or the lipocalin mutein fused to the Fc region of an antibody, e.g., SEQ ID NOs: 39, 40, 41 , 42, 98, 99, or 100.
  • the K D values of provided fusion proteins may be measured, for example, in an SPR assay, such as an SPR assay as essentially described in Example 3.
  • a fusion protein of the disclosure may be able to bind PD-L1 with an EC 50 value of at most about 0.5 nM or even lower, such as about 0.3 nM or lower, about 0.2 nM or lower, about 0.15 nM or lower, or about 0.1 nM or lower.
  • a fusion protein of the disclosure may be able to bind PD-L1 with an EC 50 value comparable to or lower than the EC 50 value of the antibody specific for PD-L1 that is included in a particular fusion protein, such as the antibody having the heavy and light chains provided by SEQ ID NOs: 78 and 79.
  • the EC 50 values of provided fusion proteins may be measured, for example, in an enzyme-linked immunosorbent assay (ELISA) assay, such as an ELISA assay as essentially described in Example 4.
  • ELISA enzyme-linked immunosorbent assay
  • a fusion protein of the disclosure may be able to bind OX40 with an EC 50 value of at most about 0.6 nM or even lower, such as about 0.5 nM or lower, about 0.2 nM or lower, about 0.15 nM or lower, or about 0.1 nM or lower.
  • a fusion protein of the disclosure may be able to bind OX40 with an EC 50 value comparable to or lower than the EC 50 value of the lipocalin mutein specific for OX40 that is included in a particular fusion protein, e.g., SEQ ID NOs: 33, 34, 35, 36, 95, 96, or 97or the lipocalin mutein fused to the Fc region of an antibody, e.g., SEQ ID NOs: 39, 40, 41, 42, 98, 99, or 100.
  • the EC 50 values of provided fusion proteins may be measured, for example, in an ELISA assay, such as an ELISA assay as essentially described in Example 4.
  • fusion proteins of the disclosure are cross-reactive with cynomolgus PD-L1.
  • a provided fusion protein may be able to bind cynomolgus PD-L1 with an EC 50 value of at most about 0.5 nM or even lower, such as about 0.2 nM or lower, about 0.1 nM or lower, or about 0.05 nM or lower.
  • the EC 50 values of provided fusion proteins may be measured, for example, in an ELISA assay, such as an ELISA assay as essentially described in Example 4.
  • fusion proteins of the disclosure are cross-reactive with cynomolgus OX40.
  • a provided fusion protein may be able to bind cynomolgus OX40 with an EC 50 value of at most about 15 nM or even lower, such as about 10 nM or lower, about 8 nM or lower, about 6 nM or lower, about 3 nM or lower, about 1 nM or lower, about 0.5 nM or lower, about 3 nM or lower, or about 0.1 nM or lower.
  • the EC 50 values of provided fusion proteins may be measured, for example, in an ELISA assay, such as an ELISA assay as essentially described in Example 4.
  • fusion proteins of the disclosure may be able to simultaneously bind OX40 and PD-L1.
  • a provided fusion protein may be able to simultaneously bind OX40 and PD-L1 , with an EC 50 value of at most about 1 nM or even lower, such as 0.8 nM or lower, 0.6 nM or lower, or 0.4 nM or lower.
  • a provided fusion protein may be able to simultaneously bind OX40 and PD- L1 , with an EC 50 value of at most about 10 nM or even lower, such as 8 nM or lower, 6 nM or lower, 3 nM or lower, or 2 nM or lower.
  • the simultaneous binding may be determined, for example, in an ELISA assay, such as an ELISA assay as essentially described in Example 5.
  • a fusion protein of the disclosure may be able to bind OX40 expressed on a cell.
  • the ability of a provided fusion protein to bind OX40 expressed on a cell may be measured, for example, in a flow cytometric analysis as essentially described in Example 6.
  • the cell expressing OX40 may be, for example, a CHO cell transfected with human OX40 or cynomolgus OX40.
  • a fusion protein of the disclosure may be able to bind PD-L1 expressed on a cell.
  • the ability of a provided fusion protein to bind PD-L1 expressed on a cell may be measured, for example, in a flow cytometric analysis as essentially described in Example 6.
  • the cell expressing PD-L1 may be, for example, be a CHO cell transfected with human PD-L1 or cynomolgus PD-L1.
  • fusion proteins of the disclosure may be able to inhibit the binding of OX40 to OX40L.
  • fusion proteins of the disclosure may be able to bind OX40 in a similar mode as an anti-OX40 antibody having the heavy and light chains provided by SEQ ID NOs: 25 and 26 and/or the benchmark fusion protein of SEQ ID NOs: 43 and 44.
  • the inhibition of the binding of OX40 to OX40L of a fusion protein may be determined, for example, by an ELISA assay, such as an ELISA assay as essentially described in Example 9.
  • fusion proteins of the disclosure may be able to compete with PD-1 for binding to PD-L1.
  • a provided fusion protein may be able to compete with PD-1 for binding to PD-L1 with an IC 50 value of at most about 5 nM or even lower, such as about 3 nM or lower, about 2 nM or lower, or about 1 or even lower.
  • the inhibitory mode of action can be determined, for example, by an ELISA assay, such as an ELISA assay as essentially described in Example 10.
  • fusion proteins of the disclosure may be able to compete with an anti-OX40 antibody shown in SEQ ID NOs: 25 and 26 for binding to OX40.
  • a provided fusion protein may have an overlapping epitope with the anti-OX40 antibody shown in SEQ ID NOs: 25 and 26.
  • fusion proteins of the disclosure may be able to co- stimulate T cell responses.
  • provided fusion proteins lead to a comparable or stronger T cell activation as compared to a PD-L1 antibody, such as the building block PD-L1 antibody of SEQ ID NOs: 78 and 79 or the reference PD-L1 antibody of SEQ ID NOs: 80 and 81 , or an OX40 antibody, such as the reference antibody of SEQ ID NOs: 25 and 26.
  • provided fusion proteins lead to T cell activation with a comparable or better efficiency as compared to the combination of an anti-PD-L1 antibody and a OX40-targeting molecule such as an anti-OX40 antibody or an OX40-specific lipocalin mutein.
  • the stimulated T cell response or T cell activation may be measured, for example, in a PD-1/PD-L1 blockade bioassay as described in Example 11, or in a functional T cell activation assay as essentially described in Example 12, Example 13, Example 14, Example 15, and/or Example 16.
  • fusion proteins of the disclosure may be able to induce increased IL-2 secretion.
  • provided fusion proteins may be able to induce a concentration-dependent IL-2 secretion and/or demonstrate a tendency to induce enhanced IL-2 secretion at higher concentrations.
  • provided fusion proteins may lead to increased IL-2 secretion with a comparable or beter efficiency as compared to the combination of an anti-PD-L1 antibody and an OX40-targeting molecule such as an anti-OX40 antibody or an OX40-specific lipocalin mutein.
  • IL-2 secretion may be measured, for example, in a functional T cell activation assay as essentially described in Example 12, Example 13, and/or Example 14.
  • fusion proteins of the disclosure may be able to induce increased IFN-y (or IFNg) secretion of CD4 T cells.
  • provided fusion proteins may be able to induce concentration-dependent IFN-y secretion and/or demonstrate a tendency to induce enhanced IFN-y secretion at higher concentrations.
  • provided fusion proteins may lead to increased IFN-y secretion with a comparable or better efficiency as compared to the combination of an anti-PD-L1 antibody and an OX40-targeting molecule such as an anti-OX40 antibody or an OX40-specific lipocalin mutein.
  • IFN-y secretion of CD4 T cells may be measured, for example, in a mixed lymphocyte reaction assessment assay as essentially described in Example 15.
  • fusion proteins of the disclosure may be able to induce increased IL-13 secretion of CD4 T cells.
  • provided fusion proteins may be able to induce concentration-dependent IL-13 secretion and/or demonstrate a tendency to induce enhanced IL-13 secretion at higher concentrations.
  • provided fusion proteins may lead to increased IL-13 secretion with a comparable or better efficiency as compared to the combination of an anti-PD-L1 antibody and an OX40-targeting molecule such as an anti-OX40 antibody or an OX40-specific lipocalin mutein.
  • provided fusion proteins may lead to increased IL-13 secretion with a higher EC50 as compared to the reference bispecific fusion protein of SEQ ID NOs: 43 and 44.
  • IL-13 secretion of CD4 T cells may be measured, for example, in a mixed lymphocyte reaction assessment assay as essentially described in Example 15.
  • fusion proteins of the disclosure may be able to induce increased IL-2 and/or IL-13 secretion of CD3 T cells.
  • provided fusion proteins may lead to a higher secretion of IL-2 and/or IL-13 as compared to the building block PD-L1 antibody of SEQ ID NOs: 78 and 79.
  • IL-2 and/or IL-13 secretion of CD3 T cells may be measured, for example, in a mixed lymphocyte reaction assessment assay as essentially described in Example 16.
  • fusion proteins of the disclosure may be able to induce increased IL-2 and/or IL-13 secretion of CD3 T cells.
  • provided fusion proteins may lead to a higher secretion of IL-2 and/or IL-13 as compared to the building block PD-L1 antibody of SEQ ID NOs: 78 and 79.
  • IL-2 and/or IL-13 secretion of CD3 T cells may be measured, for example, in a mixed lymphocyte reaction assessment assay as essentially described in Example 16.
  • fusion proteins of the disclosure may be able to induce increased Granzyme A, Granzyme B, and/or soluble Fas ligand secretion of CD3 T cells.
  • provided fusion proteins may lead to a higher secretion of Granzyme A, Granzyme B, and/or soluble Fas ligand as compared to the building block PD-L1 antibody of SEQ ID NOs: 78 and 79.
  • Granzyme A, Granzyme B, and/or soluble Fas ligand secretion of CD3 T cells may be measured, for example, in a mixed lymphocyte reaction assessment assay as essentially described in Example 16.
  • fusion proteins of the disclosure may be able to co- stimulate T cell responses in a PD-L1 dependent manner.
  • provided fusion proteins may lead to local induction of the IL-2 production by T cells in the vicinity of PD-L1 -positive cells, such as PD-L1 transfected cells or PD-L1 positive tumor cells.
  • PD-L1 -positive cells such as PD-L1 transfected cells or PD-L1 positive tumor cells.
  • “In the vicinity of PD-L1 -positive cells” when used herein refers to a T cell and a PD-L1 -positive cell being brought close to each other through a provided fusion protein which binds OX40 and PD-L1 simultaneously.
  • the PD-L1 dependent activation of T cells by provided fusion proteins may be determined, for example, in a PD-1/PD-L1 blockade bioassay essentially described in Example 11, or in a functional T cell activation assay as essentially described in Example 12, Example 13, Example 14, Example 15, and/or Example 16.
  • provided fusion proteins may be able to co- stimulate T cell responses in the presence of PD-L1 expressing tumor cells and/or in a tumor microenvironment.
  • a provided fusion protein may be able to co- stimulate T cell responses in the presence of PD-L1 -positive tumor cells with an EC 50 value of about 1 nM or lower, about 0.5 nM or lower, about 0.3 nM or lower, about 0.1 nM or lower, or about 0.05 nM or lower.
  • the T cell activation by provided fusion proteins in the presence of PD-L1 expressing tumor cells and/or in a tumor microenvironment may be assessed, for example, in a functional T cell activation assay essentially described in Example 13.
  • provided fusion proteins are not able to co-stimulate T cell responses in the absence of PD-L1. In some embodiments, provided fusion proteins are not able to co-stimulate T cell responses in the absence of PD-L1 expressing cells. In some embodiments, a provided fusion protein may be able to discern the presence of PD-L1 and lead to corresponding T cell activation beter than an OX40 antibody shown in SEQ ID NOs: 25 and 26.
  • the PD-L1 dependent action of the fusion proteins may be determined, for example, in a PD-1/PD-L1 blockade bioassay essentially described in in Example 11, or in a functional T cell activation assay as essentially described in Example 12, Example 13, Example 14, Example 15, and/or Example 16.
  • provided fusion proteins may be able to block the inhibitory signal mediated by binding of PD-1 to PD-L1.
  • a provided fusion protein may be able to release a brake for T cell activation or lead to successful T cell activation by blocking the PD-1/PD-L1 interaction.
  • the blockade of PD-1 inhibitory signal may be measured, for example, in a PD-1/PD-L1 blockade bioassay as essentially described in Example 11.
  • fusion proteins of the disclosure may be able to stimulate T cell proliferation and/or activation.
  • provided fusion proteins may be able to stimulate CD4 + T cell proliferation and/or activation.
  • provided fusion proteins may be able to induce IL-13 and/or IFN-y secretion, preferably dose-dependent IL-13 and/or IFN-y secretion.
  • provided fusion proteins may be able to induce higher IL-13 and/or IFN-y secretion as compared to the combination of an anti-PD-L1 antibody and an OX40-targeting molecule such as an anti- OX40 antibody or an OX40-specific lipocalin mutein.
  • the IL-13 and/or IFN-y secretion as used herein may be a measure of T cell activation.
  • the CD4 + T cell proliferation and/or activation stimulated by provided fusion proteins may be assessed by, for example, a mixed lymphocyte reaction (MLR) assay as essentially described in Example 15.
  • MLR mixed lymphocyte reaction
  • fusion proteins of the disclosure may be able to stimulate CD3* T cell proliferation and/or activation.
  • provided fusion proteins may be able to induce the production of IL-2 and effector molecules, such as Granzyme A, Granzyme B, and/or soluble Fas ligand.
  • provided fusion proteins may be able to induce increased production of IL-2 and cytotoxic factors, such as Granzyme A, Granzyme B, and/or soluble Fas ligand, as compared to the building block PD- L1 antibody of SEQ ID NOs: 78 and 79and an OX40-targeting molecule such as an anti- OX40 antibody or an OX40-specific lipocalin mutein.
  • the CD3 + T cell proliferation and/or activation stimulated by provided fusion proteins may be assessed by, for example, an MLR assay as essentially described in Example 16.
  • provided fusion proteins have favorable stability and pharmacokinetics profiles.
  • a provided fusion protein has a comparable pharmacokinetics profile as the building block antibody of SEQ ID NOs: 78 and 79.
  • a provided fusion protein has antibody-like pharmacokinetics.
  • a provided fusion protein has a terminal half-life of about 200 hours or longer, about 250 hours or longer, about 300 hours or longer, about 350 hours or longer, about 400 hours or longer, or even longer.
  • a provided fusion protein has a more favorable pharmacokinetic profile than a benchmark fusion protein shown in SEQ ID NOs: 43 and 44.
  • a provided fusion protein is capable of binding PD-L1 with a K D value of at most about 1 nM. In some embodiments, a provided fusion protein is capable of binding OX40 with a K D value of at most about 100 nM. The K D value is preferably determined by a surface-plasmon-resonance (SPR) assay.
  • SPR surface-plasmon-resonance
  • a provided fusion protein is capable of binding PD-L1 with an EC 50 value of at most about 1 nM. In some embodiments, a provided fusion protein is capable of binding OX40 with an EC 50 value of at most about 2 nM.
  • the EC 50 value is preferably determined by an enzyme-linked immunosorbent assay (ELISA).
  • a provided fusion protein is cross-reactive with cynomolgus PD-L1. In some embodiments, a provided fusion protein is cross-reactive with cynomolgus OX40.
  • a provided fusion protein is capable of simultaneously binding OX40 and PD-L1 with an EC 50 value of at most about 10 nM (preferably measured in an ELISA assay).
  • a provided fusion protein is capable of binding PD-L1 expressing tumor cells.
  • a provided fusion protein is capable of inhibiting the binding of OX40 to OX40 ligand.
  • a provided fusion protein is capable of competing with PD-1 for binding to PD-L1.
  • a provided fusion protein is capable of competing with the antibody shown in SEQ ID NOs: 25 and 26 for binding to OX40.
  • a provided fusion protein binds to an epitope on OX40 overlapping with that of the antibody shown in SEQ ID NOs: 25 and 26.
  • a provided fusion protein is capable of stimulating T cell proliferation and/or responses.
  • a provided fusion protein is capable of stimulating CD4+ and/or CD8+ T cell proliferation.
  • a provided fusion protein is capable of inducing increased secretion of IL-2 and/or interferon gamma (IFNg or IFN-y).
  • IFNg interferon gamma
  • a provided fusion protein is capable of inducing increased secretion of cytotoxic factors.
  • a provided fusion protein is capable of co-stimulating T cell responses in a PD-L1 -dependent manner.
  • a provided fusion protein does not co-stimulate T cell responses in the absence of PD-L.1.
  • a provided fusion protein has a pharmacokinetics profile that is comparable to that of an antibody. In some embodiments, a provided fusion protein has a serum half life that is comparable to that of an antibody.
  • a provided fusion protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 82-91.
  • a provided fusion protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or even higher sequence identity to the amino acid sequences shown in any one of SEQ ID NOs: 82-91.
  • a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 78 and 87, SEQ ID NOs: 88 and 79, SEQ ID NOs: 89 and 79, SEQ ID NOs: 82 and 79, SEQ ID NOs: 93 and 90, or SEQ ID NOs: 91 and 94.
  • a provided fusion protein comprises amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequences shown in SEQ ID NOs: 78 and 87, SEQ ID NOs: 88 and 79, SEQ ID NOs: 89 and 79, SEQ ID NOs: 82 and 79, SEQ ID NOs: 93 and 90, or SEQ ID NOs: 91 and 94.
  • a provided fusion protein comprises amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequences shown in SEQ ID NOs: 78 and 87. In some embodiments, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 78 and 87.
  • a provided fusion protein comprises amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequences shown in SEQ ID NOs: 88 and 79. In some embodiments, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 88 and 79.
  • a provided fusion protein comprises amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequences shown in SEQ ID NOs: 89 and 79. In some embodiments, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 89 and 79.
  • a provided fusion protein comprises amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequences shown in SEQ ID NOs: 82 and 79. In some embodiments, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 82 and 79.
  • a provided fusion protein comprises amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequences shown SEQ ID NOs: 93 and 90. In some embodiments, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 93 and 90.
  • a provided fusion protein comprises amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequences shown in SEQ ID NOs: 91 and 94. In some embodiments, a provided fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 91 and 94.
  • a first subunit may be or comprise an antibody, such as a full-length antibody, or an antigen-binding domain thereof specific for PD-L1.
  • an antibody for example, may be lgG1, lgG2 or lgG4.
  • an antibody is or comprises lgG4.
  • an antibody is a monoclonal antibody against PD-L1.
  • Illustrative examples of PD-L1-binding antibodies of the disclosure may comprise an antigen-binding region which cross-blocks or binds to the same epitope as a PD-L1 -binding antibody comprising the heavy chain variable domain (V H ) and light chain variable domain (V L ) regions of a known antibody such as atezolizumab (also known as MPDL3280A or RG7446, trade name Tecentriq®), avelumab (also known as MSB0010718C, trade name Bavencio®), durvalumab (previously known as MEDI4736, trade name Imfinzi®), and BMS-936559 (also known as MDX-1105), 5C10 (including humanized 5010), 5F10 (including humanized 5F10), and 9F6 (including humanized 9F6).
  • V H heavy chain variable domain
  • V L light chain variable domain
  • a known antibody such as atezolizumab (also known as MPDL3280A or
  • a PD-L1 -binding antibody of the disclosure may comprise an antigen-binding region, such as any one of the three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and the three light chain CDRs (LCDR1 , LCDR2 and LCDR3) from an antibody selected from the group consisting of atezolizumab, avelumab, durvalumab, BMS- 936559, 5C10, 5F10, and 9F6.
  • CDRs three heavy chain complementarity determining regions
  • LCDR1 , LCDR2 and LCDR3 three light chain CDRs
  • a provided PD-L1 antibody or antigen-binding domain thereof may have a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 65-70, and/or a light chain variable region (LCVR) selected from the group consisting of SEQ ID NOs: 71-76.
  • HCVR heavy chain variable region
  • LCVR light chain variable region
  • a provided PD-L1 antibody or antigen-binding domain thereof may have a heavy chain that is any one of SEQ ID NOs: 66-67, and/or a light chain that is SEQ ID NO: 73.
  • the heavy chain and light chain pair of a provided PD- L1 antibody or antigen-binding domain thereof are or comprise a HCVR and LCVR, respectively, as follows: SEQ ID NOs: 65 and 71, SEQ ID NOs: 66 and 72, SEQ ID NOs: 67 and 73, SEQ ID NOs: 68 and 74, SEQ ID NOs: 69 and 75, or SEQ ID NOs:70 and 76.
  • the heavy chain and light chain pair of a provided PD- L1 antibody are or comprise the amino acid sequences as shown in SEQ ID NOs: 77 and 79, SEQ ID NOs: 78 and 79, SEQ ID NOs: 80 and 81, or SEQ ID NOs: 93 and 94.
  • a provided PD-L1 antibody or antigen-binding domain thereof may have a HCVR with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-70, and/or a LCVR with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-76.
  • a provided PD-L1 antibody or antigen-binding domain thereof may have a heavy chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 77-78, 80, and 93 and/or a light chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 79,81, and 94.
  • a provided PD-L1 antibody or antigen-binding domain thereof may have a heavy chain and a light chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to each of SEQ ID NO: 77 and SEQ ID NO: 79.
  • a provided PD-L1 antibody or antigen-binding domain thereof may have a heavy chain and a light chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to each of SEQ ID NO: 78 and SEQ ID NO: 79.
  • a provided PD-L1 antibody or antigen-binding domain thereof may have a heavy chain and a light chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to each of SEQ ID NO: 80 and SEQ ID NO: 81.
  • a provided PD-L1 antibody or antigen-binding domain thereof may have a heavy chain and a light chain with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to each of SEQ ID NO: 93 and SEQ ID NO: 94.
  • the heavy chain variable region of a provided PD-L1 antibody or antigen-binding domain thereof may have the three CDRs having following sequences: GFSLSNYD (HCDR1, SEQ ID NO: 45), IWTGGAT (HCDR2, SEQ ID NO: 46), VRDSNYRYDEPFTY (HCDR3; SEQ ID NO: 47).
  • the heavy chain variable region of a provided PD-L1 antibody or antigen-binding domain thereof may have the three CDRs having following sequences: GFDIKDTY (HCDR1, SEQ ID NO: 50), IDPADGNT (HCDR2, SEQ ID NO: 51), ARGLGAWFAS (HCDR3; SEQ ID NO: 52).
  • the heavy chain variable region of a provided PD-L1 antibody or antigen- binding domain thereof may have the three CDRs having following sequences: GFNIKDTY (HCDR1 , SEQ ID NO: 55), IDPANGNT (HCDR2, SEQ ID NO: 56), SRGPPGGIGEYIYAMDY (HCDR3; SEQ ID NO: 57).
  • the heavy chain variable region of a provided PD-L1 antibody or antigen-binding domain thereof may have the three CDRs having following sequences: GFTFSDSW (HCDR1, SEQ ID NO: 60), ISPYGGST (HCDR2, SEQ ID NO: 61), ARRHWPGGFDY (HCDR3; SEQ ID NO: 62).
  • the light chain variable region of a provided PD-L1 antibody or antigen-binding domain thereof may have the three CDRs having following sequences: QSIGTN (LCDR1 , SEQ ID NO: 48), YAS (LCDR2), QQSNSWPYT (LCDR3; SEQ ID NO: 49).
  • the light chain variable region of a provided PD-L1 antibody or antigen-binding domain thereof may have the three CDRs having following sequences: QDITNS (LCDR1 , SEQ ID NO: 53), YTS (LCDR2), QQGHTLPPT (LCDR3; SEQ ID NO: 54).
  • the light chain variable region of a provided PD-L1 antibody or antigen-binding domain thereof may have the three CDRs having following sequences: SSVSSSY (LCDR1, SEQ ID NO: 58), STS (LCDR2), HQYHRSPPT (LCDR3; SEQ ID NO: 59).
  • the light chain variable region of a provided PD-L1 antibody or antigen-binding domain thereof may have the three CDRs having following sequences: ); QDVSTA (LCDR1, SEQ ID NO: 63), SAS (LCDR2), QQYLYHPAT (LCDR3; SEQ ID NO: 64).
  • a provided PD-L1 antibody or antigen-binding domain thereof comprises a heavy chain variably region that has the three CDRs having following sequences: GFSLSNYD (HCDR1 , SEQ ID NO: 45), IWTGGAT (HCDR2, SEQ ID NO: 46), VRDSNYRYDEPFTY (HCDR3; SEQ ID NO: 47), and a light chain variably region that has the three CDRs having following sequences: QSIGTN (LCDR1, SEQ ID NO: 48), YAS (LCDR2), QQSNSWPYT (LCDR3; SEQ ID NO: 49).
  • GFSLSNYD HCDR1 , SEQ ID NO: 45
  • IWTGGAT HCDR2, SEQ ID NO: 46
  • VRDSNYRYDEPFTY HCDR3; SEQ ID NO: 47
  • QSIGTN LCDR1, SEQ ID NO: 48
  • YAS YAS
  • QQSNSWPYT LCDR3; SEQ ID NO: 49
  • a provided PD- L1 antibody or antigen-binding domain thereof comprises a heavy chain variably region that has the three CDRs having following sequences: GFDIKDTY (HCDR1, SEQ ID NO: 50), IDPADGNT (HCDR2, SEQ ID NO: 51), ARGLGAWFAS (HCDR3; SEQ ID NO: 52), and a light chain variably region that has the three CDRs having following sequences: QDITNS (LCDR1 , SEQ ID NO: 53), YTS (LCDR2), QQGHTLPPT (LCDR3; SEQ ID NO: 54).
  • a provided PD-L1 antibody or antigen-binding domain thereof comprises a heavy chain variably region that has the three CDRs having following sequences: GFNIKDTY (HCDR1, SEQ ID NO: 55), IDPANGNT (HCDR2, SEQ ID NO: 56), SRGPPGGIGEYIYAMDY (HCDR3; SEQ ID NO: 57), and a light chain variably region that has the three CDRs having following sequences: SSVSSSY (LCDR1, SEQ ID NO: 58), STS (LCDR2), HQYHRSPPT (LCDR3; SEQ ID NO: 59).
  • a provided PD-L1 antibody or antigen-binding domain thereof comprises a heavy chain variably region that has the three CDRs having following sequences: GFTFSDSW (HCDR1, SEQ ID NO: 60), ISPYGGST (HCDR2, SEQ ID NO: 61), ARRHWPGGFDY (HCDR3; SEQ ID NO: 62), and a light chain variably region that has the three CDRs having following sequences: QDVSTA (LCDR1, SEQ ID NO: 63), SAS (LCDR2), QQYLYHPAT (LCDR3; SEQ ID NO: 64).
  • GFTFSDSW HCDR1, SEQ ID NO: 60
  • ISPYGGST HCDR2, SEQ ID NO: 61
  • ARRHWPGGFDY HCDR3; SEQ ID NO: 62
  • QDVSTA LCDR1, SEQ ID NO: 63
  • SAS LCDR2
  • QQYLYHPAT LCDR3; SEQ ID NO: 64.
  • a provided PD-L1 antibody or antigen-binding domain thereof comprises a heavy chain variably region that has the three CDRs having following sequences: GFSLSNYD (HCDR1, SEQ ID NO: 45), IWTGGAT (HCDR2, SEQ ID NO: 46), VRDSNYRYDEPFTY (HCDR3; SEQ ID NO: 47), and a light chain variably region that has the three CDRs having following sequences: QSIGTN (LCDR1, SEQ ID NO: 48), YAS (LCDR2), QQSNSWPYT (LCDR3; SEQ ID NO: 49).
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 70% sequence identity to the sequences set forth in SEQ ID NOs: 65 and 71. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 75% sequence identity to the sequences set forth in SEQ ID NOs: 65 and 71. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 80% sequence identity to the sequences set forth in SEQ ID NOs: 65 and 71. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 85% sequence identity to the sequences set forth in SEQ ID NOs: 65 and 71.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 90% sequence identity to the sequences set forth in SEQ ID NOs: 65 and 71. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 92% sequence identity to the sequences set forth in SEQ ID NOs: 65 and 71. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 95% sequence identity to the sequences set forth in SEQ ID NOs: 65 and 71. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 97% sequence identity to the sequences set forth in SEQ ID NOs: 65 and 71.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 98% sequence identity to the sequences set forth in SEQ ID NOs: 65 and 71, In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 99% sequence identity to the sequences set forth in SEQ ID NOs: 65 and 71. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that comprise the sequences set forth in SEQ ID NOs: 65 and 71.
  • a provided PD-L1 antibody or antigen-binding domain thereof comprises a heavy chain variably region that has the three CDRs having following sequences: GFSLSNYD (HCDR1, SEQ ID NO: 45), IWTGGAT (HCDR2, SEQ ID NO: 46), VRDSNYRYDEPFTY (HCDR3; SEQ ID NO: 47), and a light chain variably region that has the three CDRs having following sequences: QSIGTN (LCDR1, SEQ ID NO: 48), YAS (LCDR2), QQSNSWPYT (LCDR3; SEQ ID NO: 49).
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 70% sequence identity to the sequences set forth in SEQ ID NOs: 66 and 72. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 75% sequence identity to the sequences set forth in SEQ ID NOs: 66 and 72. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 80% sequence identity to the sequences set forth in SEQ ID NOs: 66 and 72. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 85% sequence identity to the sequences set forth in SEQ ID NOs: 66 and 72.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 90% sequence identity to the sequences set forth in SEQ ID NOs: 66 and 72. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 92% sequence identity to the sequences set forth in SEQ ID NOs: 66 and 72. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 95% sequence identity to the sequences set forth in SEQ ID NOs: 66 and 72. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 97% sequence identity to the sequences set forth in SEQ ID NOs: 66 and 72.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 98% sequence identity to the sequences set forth in SEQ ID NOs: 66 and 72. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 99% sequence identity to the sequences set forth in SEQ ID NOs: 66 and 72. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that comprise the sequences set forth in SEQ ID NOs: 66 and 72.
  • a provided PD-L1 antibody or antigen-binding domain thereof comprises a heavy chain variably region that has the three CDRs having following sequences: GFSLSNYD (HCDR1, SEQ ID NO: 45), IWTGGAT (HCDR2, SEQ ID NO: 46), VRDSNYRYDEPFTY (HCDR3; SEQ ID NO: 47), and a light chain variably region that has the three CDRs having following sequences: QSIGTN (LCDR1, SEQ ID NO: 48), YAS (LCDR2), QQSNSWPYT (LCDR3; SEQ ID NO: 49).
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 70% sequence identity to the sequences set forth in SEQ ID NOs: 67 and 73. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 75% sequence identity to the sequences set forth in SEQ ID NOs: 67 and 73. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 80% sequence identity to the sequences set forth in SEQ ID NOs: 67 and 73.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 85% sequence identity to the sequences set forth in SEQ ID NOs: 67 and 73. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 90% sequence identity to the sequences set forth in SEQ ID NOs: 67 and 73. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 92% sequence identity to the sequences set forth in SEQ ID NOs: 67 and 73.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 95% sequence identity to the sequences set forth in SEQ ID NOs: 67 and 73. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 97% sequence identity to the sequences set forth in SEQ ID NOs: 67 and 73. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 98% sequence identity to the sequences set forth in SEQ ID NOs: 67 and 73.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 99% sequence identity to the sequences set forth in SEQ ID NOs: 67 and 73. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that comprise the sequences set forth in SEQ ID NOs: 67 and 73.
  • a provided PD-L1 antibody or antigen-binding domain thereof comprises a heavy chain variably region that has the three CDRs having following sequences: GFDIKDTY (HCDR1 , SEQ ID NO: 50), IDPADGNT (HCDR2, SEQ ID NO: 51), ARGLGAWFAS (HCDR3; SEQ ID NO: 52), and a light chain variably region that has the three CDRs having following sequences: QDITNS (LCDR1 , SEQ ID NO: 53), YTS (LCDR2), QQGHTLPPT (LCDR3; SEQ ID NO: 54).
  • GFDIKDTY HCDR1 , SEQ ID NO: 50
  • IDPADGNT HCDR2, SEQ ID NO: 51
  • ARGLGAWFAS HCDR3; SEQ ID NO: 52
  • QDITNS LCDR1 , SEQ ID NO: 53
  • YTS YTS
  • QQGHTLPPT LCDR3; SEQ ID NO: 54
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 70% sequence identity to the sequences set forth in SEQ ID NOs: 68 and 74. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 75% sequence identity to the sequences set forth in SEQ ID NOs: 68 and 74. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 80% sequence identity to the sequences set forth in SEQ ID NOs: 68 and 74.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 85% sequence identity to the sequences set forth in SEQ ID NOs: 68 and 74. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 90% sequence identity to the sequences set forth in SEQ ID NOs: 68 and 74. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 92% sequence identity to the sequences set forth in SEQ ID NOs: 68 and 74.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 95% sequence identity to the sequences set forth in SEQ ID NOs: 68 and 74. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 97% sequence identity to the sequences set forth in SEQ ID NOs: 68 and 74. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 98% sequence identity to the sequences set forth in SEQ ID NOs: 68 and 74.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 99% sequence identity to the sequences set forth in SEQ ID NOs: 68 and 74. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that comprise the sequences set forth in SEQ ID NOs: 68 and 74.
  • a provided PD-L1 antibody or antigen-binding domain thereof comprises a heavy chain variably region that has the three CDRs having following sequences: GFNIKDTY (HCDR1, SEQ ID NO: 55), IDPANGNT (HCDR2, SEQ ID NO: 56), SRGPPGGIGEYIYAMDY (HCDR3; SEQ ID NO: 57), and a light chain variably region that has the three CDRs having following sequences: SSVSSSY (LCDR1 , SEQ ID NO: 58), STS (LCDR2), HQYHRSPPT (LCDR3; SEQ ID NO: 59).
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 70% sequence identity to the sequences set forth in SEQ ID NOs: 69 and 75. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 75% sequence identity to the sequences set forth in SEQ ID NOs: 69 and 75. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 80% sequence identity to the sequences set forth in SEQ ID NOs: 69 and 75. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 85% sequence identity to the sequences set forth in SEQ ID NOs: 69 and 75.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 90% sequence identity to the sequences set forth in SEQ ID NOs: 69 and 75. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 92% sequence identity to the sequences set forth in SEQ ID NOs: 69 and 75. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 95% sequence identity to the sequences set forth in SEQ ID NOs: 69 and 75. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 97% sequence identity to the sequences set forth in SEQ ID NOs: 69 and 75.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 98% sequence identity to the sequences set forth in SEQ ID NOs: 69 and 75. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 99% sequence identity to the sequences set forth in SEQ ID NOs: 69 and 75. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that comprise the sequences set forth in SEQ ID NOs: 69 and 75.
  • a provided PD-L1 antibody or antigen-binding domain thereof comprises a heavy chain variably region that has the three CDRs having following sequences: GFTFSDSW (HCDR1, SEQ ID NO: 60), ISPYGGST (HCDR2, SEQ ID NO: 61), ARRHWPGGFDY (HCDR3; SEQ ID NO: 62), and a light chain variably region that has the three CDRs having following sequences: QDVSTA (LCDR1, SEQ ID NO: 63), SAS (LCDR2), QQYLYHPAT (LCDR3; SEQ ID NO: 64).
  • GFTFSDSW HCDR1, SEQ ID NO: 60
  • ISPYGGST HCDR2, SEQ ID NO: 61
  • ARRHWPGGFDY HCDR3; SEQ ID NO: 62
  • QDVSTA LCDR1, SEQ ID NO: 63
  • SAS LCDR2
  • QQYLYHPAT LCDR3; SEQ ID NO: 64.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 70% sequence identity to the sequences set forth in SEQ ID NOs: 70 and 76. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 75% sequence identity to the sequences set forth in SEQ ID NOs: 70 and 76. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 80% sequence identity to the sequences set forth in SEQ ID NOs: 70 and 76. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 85% sequence identity to the sequences set forth in SEQ ID NOs: 70 and 76.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 90% sequence identity to the sequences set forth in SEQ ID NOs: 70 and 76. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 92% sequence identity to the sequences set forth in SEQ ID NOs: 70 and 76. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 95% sequence identity to the sequences set forth in SEQ ID NOs: 70 and 76. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 97% sequence identity to the sequences set forth in SEQ ID NOs: 70 and 76.
  • said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 98% sequence identity to the sequences set forth in SEQ ID NOs: 70 and 76. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that have at least 99% sequence identity to the sequences set forth in SEQ ID NOs: 70 and 76. In some embodiments, said provided antibody comprises a heavy chain variable region and a light chain variable region that comprise the sequences set forth in SEQ ID NOs: 70 and 76.
  • CDR1 consists of positions 27 to 38
  • CDR2 consists of positions 56 to 65
  • CDR3 for germline V-genes consists of positions 105 to 116
  • CDR3 for rearranged V-J-genes or V-D-J- genes consists of positions 105 to 117 (position preceding J-PHE or J-TRP 118) with gaps at the top of the loop for rearranged CDR3-IMGT with less than 13 amino acids, or with additional positions 112.1, 111.1 , 112.2, 111.2, etc. for rearranged CDR3-IMGT with more than 13 amino acids.
  • the positions given in this paragraph are according to the IMGT numbering described in Lefranc, M.-P., The Immunologist, 7, 132-136 (1999).
  • Antibodies specifically binding to PD-L1 as included in fusion proteins of the disclosure may comprise an Fc part which allows for extending the in vivo half-life of the bispecific binding molecule of the disclosure.
  • Fc part is preferably from human origin, more preferably a human Fc part of an lgG1 or lgG4 antibody, even more preferably an engineered human Fc part of an lgG1 or lgG4 with activating or silencing effector functions.
  • silencing effector functions may be preferred over activating effector functions.
  • such an Fc part is engineered to silence effector functions with mutation(s) at positions 234 and/or 235, numbering according to EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000).
  • mutations in positions F234 and L235 of a provided anti-PD-L1 antibody may be introduced to silence effector functions.
  • mutations in positions D265 and P329 of a provided anti-PD-L1 antibody may be introduced to silence effector function. Numbering for both sets of these potential mutations is according to the EU index of Kabat (Shields et al., J Biol Chem, 2001).
  • polyclonal antibodies can be obtained from the blood of an animal following immunization with an antigen in mixture with additives and adjuvants and monoclonal antibodies can be produced by any technique which provides antibodies produced by continuous cell line cultures. Examples of such techniques are described, e.g., in Harlow and Lane (1999), (1988), and include the hybridoma technique originally described by Kohler and Milstein, 1975, the trioma technique, the human B cell hybridoma technique (see e.g.
  • recombinant antibodies may be obtained from monoclonal antibodies or can be prepared de novo using various display methods such as phage, ribosomal, mRNA, or cell display.
  • a suitable system for the expression of the recombinant (humanized) antibodies or fragments thereof may be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, e.g., US Patent No.
  • Lipocalins are proteinaceous binding molecules that have naturally evolved to bind ligands. Lipocalins occur in many organisms, including vertebrates, insects, plants, and bacteria. The members of the lipocalin protein family (Pervaiz and Brew, FASEB J, 1987) are typically small, secreted proteins and have a single polypeptide chain.
  • lipocalins fulfill a variety of physiological functions. These include roles in retinol transport, olfaction, pheromone signaling, and the synthesis of prostaglandins. Lipocalins have also been implicated in the regulation of the immune response and the mediation of cell homeostasis (reviewed, e.g., in Flower et al., Biochim Biophys Acta, 2000, Flower, Biochem J, 1996).
  • Lipocalins share unusually low levels of overall sequence conservation, often with sequence identities of less than 20%. In strong contrast, their overall folding patern is highly conserved.
  • the central part of the lipocalin structure consists of a single eight- stranded anti-parallel p-sheet closed back on itself to form a continuously hydrogen-bonded P-barrel. This P-barrel forms a central cavity.
  • One end of the barrel is sterically blocked by the N-terminal peptide segment that runs across its bottom as well as three peptide loops connecting the P-strands.
  • the other end of the p-barrel is open to the solvent and encompasses a target-binding site, which is formed by four flexible peptide loops (AB, CD, EF, and GH). It is the diversity of the loops in the otherwise rigid lipocalin scaffold that gives rise to a variety of different binding modes each capable of accommodating targets of different size, shape, and chemical character (reviewed, e.g., in Skerra, Biochim Biophys Acta, 2000, Flower et al., Biochim Biophys Acta, 2000, Flower, Biochem J, 1996).
  • a lipocalin mutein according to the present disclosure may be a mutein of any lipocalin.
  • suitable lipocalins also sometimes designated as “reference lipocalin,” “wild-type lipocalin,” “reference protein scaffolds,” or simply “scaffolds”
  • suitable lipocalins include, but are not limited to, tear lipocalin (lipocalin-1 , Tic, or von Ebner’s gland protein), retinol binding protein, neutrophil lipocalin-type prostaglandin D-synthase, ⁇ - lactoglobulin, bilin-binding protein (BBP), apolipoprotein D (APOD), neutrophil gelatinase- associated lipocalin (NGAL), ⁇ 2-microglobulin-related protein (A2m), 24p3/uterocalin (24p3), von Ebner’s gland protein 1 (VEGP 1), von Ebner’s gland protein 2 (VEGP 2), and
  • a lipocalin mutein is derived from the lipocalin group consisting of human tear lipocalin (hTIc), human neutrophil gelatinase- associated lipocalin (hNGAL), human apolipoprotein D (hAPOD) and the bilin-binding protein of Pieris brassicae.
  • the amino acid sequence of a lipocalin mutein according to the disclosure may have a high sequence identity to the reference (or wild-type) lipocalin from which it is derived, for example, hNGAL, when compared to sequence identities with another lipocalin (see also above).
  • the amino acid sequence of a lipocalin mutein according to the disclosure is at least substantially similar to the amino acid sequence of the corresponding reference (wild-type) lipocalin, with the proviso that there may be gaps (as defined herein) in an alignment that are the result of additions or deletions of amino acids.
  • a respective sequence of a lipocalin mutein of the disclosure being substantially similar to the sequences of the corresponding reference (wild-type) lipocalin, has, in some embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, or at least 90% identity, including at least 95% identity to the sequence of the corresponding lipocalin.
  • a lipocalin mutein of the disclosure of course may contain, in comparison with the wild-type lipocalin, substitutions as described herein which renders the lipocalin mutein capable of binding to OX40.
  • a lipocalin mutein of the disclosure contains one or more mutated amino acid residues - relative to the amino acid sequence of the wild-type or reference lipocalin, for example, hNGAL - in the four loops at the open end that comprise a ligand- binding pocket and define the entrance of the ligand-binding pocket (cf. above). As explained above, these regions are essential in determining the binding specificity of a lipocalin mutein for the desired target.
  • a lipocalin mutein of the disclosure may also contain mutated amino acid residues in regions outside of the four loops.
  • a lipocalin mutein of the disclosure may contain one or more mutated amino acid residues in one or more of the three peptide loops (designated BC, DE, and FG) connecting the ( ⁇ -strands at the closed end of the lipocalin.
  • a mutein derived from of tear lipocalin, NGAL or a homologue thereof may have 1, 2, 3, 4, or more mutated amino acid residues at any sequence position in the N-terminal region and/or in the three peptide loops BC, DE, and FG arranged at the end of the P-barrel structure that is located opposite to the natural lipocalin binding pocket.
  • any types and numbers of mutations are envisaged as long as the lipocalin mutein retains its capability to bind OX40, and/or it has a sequence identity of at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or higher identity to the amino acid sequence of the reference (wild-type) lipocalin, for example, mature hNGAL.
  • a substitution is a conservative substitution. In some embodiments, a substitution is a non-conservative substitution or one or more from the exemplary substitutions below.
  • Conservative substitutions are generally the following substitutions, listed according to the amino acid to be mutated, each followed by one or more replacement(s) that can be taken to be conservative: Ala Ser, Thr, or Vai; Arg Lys, Gin, Asn, or His; Asn Gin, Glu, Asp, or His; Asp ⁇ Glu, Gln, Asn, or His; Gln ⁇ Asn, Asp, Glu, or His; Glu ⁇ Asp, Asn, Gin, or His; His ⁇ Arg, Lys, Asn, Gin, Asp, or Glu; IIe ⁇ Thr, Leu, Met, Rhe, Vai, Trp, Tyr, Ala, or Pro; Leu ⁇ Thr, IIe ,Val Met, Ala, Phe, Pro, Tyr, or Trp; Lys ⁇ Arg, His, Gin, or Asn; Met ⁇ Thr, Leu, Tyr, IIe, Phe, Val , Ala, Pro, or Trp; Phe Thr, Met, Leu, Tyr
  • substitutions are also permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.
  • the following groups each contain amino acids that can typically be taken to define conservative substitutions for one another: a. Alanine (Ala), Serine (Ser), Threonine (Thr), Valine (Vai); b. Aspartic acid (Asp), Glutamic acid (Glu), Glutamine (Gin), Asparagine (Asn), Histidine (His); c. Arginine (Arg), Lysine (Lys), Glutamine (Gin), Asparagine (Asn), Histidine (His); d.
  • substantial modifications in the physical and biological properties of the lipocalin are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side- chain properties: (1) hydrophobic: methionine, alanine, valine, leucine, iso-leucine; (2) neutral hydrophilic: cysteine, serine, threonine, asparagine, glutamine; (3) acidic: aspartic acid, glutamic acid; (4) basic: histidine, lysine, arginine; (5) residues that influence chain orientation: glycine, proline; and (6) aromatic: tryptophan, tyrosine, phenylalanine.
  • substitutions may entail exchanging a member of one of these classes for a member of another class.
  • cysteine residue not involved in maintaining the proper conformation of the respective lipocalin also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the lipocalin to improve its stability.
  • a lipocalin is a polypeptide defined by its supersecondary structure, namely cylindrical Ppleated sheet supersecondary structural region comprising eight pstrands connected pair-wise by four loops at one end to define thereby a binding pocket.
  • the present disclosure is not limited to lipocalin muteins specifically disclosed herein.
  • the disclosure relates to a lipocalin mutein having a cylindrical ppleated sheet supersecondary structural region comprising eight ⁇ strands connected pair-wise by four loops at one end to define thereby a binding pocket, wherein at least one amino acid of each of at least three of said four loops has been mutated and wherein said lipocalin is effective to bind OX40 with detectable affinity.
  • a lipocalin mutein disclosed herein is a mutein of mature human neutrophil gelatinase-associated lipocalin (hNGAL).
  • hNGAL mutein A mutein of mature hNGAL may be designated herein as an “hNGAL mutein”.
  • the present disclosure includes any number of lipocalin muteins derived from a reference (wild-type) lipocalin, preferably derived from mature hNGAL, that bind OX40 with detectable affinity.
  • the disclosure includes various lipocalin muteins that are capable of activating the downstream signaling pathways of OX40 by binding to OX40.
  • OX40 can be regarded as a non-natural target of the reference (wild-type) lipocalin, preferably hNGAL, where “non-natural target” refers to a substance that does not bind to the reference (wild-type) lipocalins under physiological conditions.
  • a lipocalin mutein of the present disclosure may lack 1 , 2, 3, 4 or more amino acids at its N-terminal end and/or 1 , 2, 3, 4 or more amino acids at its C-terminal end, in comparison to the respective reference (wild-type) lipocalin.
  • a lipocalin mutein of the disclosure may include the wild-type (natural) amino acid sequence of the reference (wild-type) lipocalin, preferably hNGAL, outside the mutated amino acid sequence positions.
  • one or more mutated amino acid residues incorporated into a lipocalin mutein of the disclosure do, at least essentially, not hamper or not interfere with the binding activity to the designated target and the folding of the mutein.
  • Such mutations can be accomplished at the DNA level using established standard methods (Sambrook and Russell, 2001 , Molecular cloning: a laboratory manual).
  • mutated amino acid residue(s) at one or more sequence positions corresponding to the linear polypeptide sequence of the reference (wild-type) lipocalin, preferably hNGAL is/are introduced through random mutagenesis by substituting the nucleotide triplet(s) encoding the corresponding sequence positions of the reference lipocalin with a subset of nucleotide triplets.
  • a lipocalin mutein that binds OX40 with detectable affinity may include at least one amino acid substitution of a native cysteine residue by another amino acid, for example, a serine residue.
  • a lipocalin mutein that binds OX40 with detectable affinity may include one or more non-native cysteine residues substituting one or more amino acids of a reference (wild-type) lipocalin, preferably hNGAL.
  • a lipocalin mutein according to the disclosure includes at least two amino acid substitutions of a native amino acid by a cysteine residue, hereby to form one or more cysteine bridges.
  • said cysteine bridge may connect at least two loop regions. The definition of these regions is used herein in accordance with Skerra, Biochim Biophys Acta (2000), Flower (1996) and Breustedt et al. (2005).
  • a lipocalin mutein of the disclosure may have at least about 70%, including at least about 80%, such as at least about 85% amino acid sequence identity, with the amino acid sequence of mature hNGAL (SEQ ID NO: 1).
  • the present disclosure provides OX40-binding hNGAL muteins.
  • the disclosure provides one or more hNGAL muteins that are capable of binding OX40 with a detectable affinity, preferably with an affinity measured by a K D of about 10' 5 M or lower.
  • the preferred hNGAL muteins are capable of binding OX40 with an affinity measured by a K D of about 400 nM or lower, about 135 nM or lower, about 50 nM or lower, about 30 nM or lower, about 20 nM or lower, about 10 nM or lower, about 5 nM or lower, about 1 nM or lower, about 0.5 nM or lower, about 0.3 nM or lower, about 0.2 nM or lower, about 0.1 nM or lower, or about 0.05 nM or even lower.
  • the K D values can be, for example, measured in an SPR assay.
  • the OX40-binding hNGAL muteins may be cross- reactive with cynomolgus OX40 (cyOX40) and in some embodiments, capable of binding cyOX40 with an affinity measured by a K D of about 290 nM or lower, about 115 nM or lower, about 55 nM or lower, about 10 nM or lower, about 5 nM or lower, about 4 nM or lower, about 2 nM or lower, about 1 nM or lower, about 0.5 nM or lower, about 0.4 nM or lower, about 0.3 nM or lower, about 0.2 nM or even lower.
  • a K D of about 290 nM or lower, about 115 nM or lower, about 55 nM or lower, about 10 nM or lower, about 5 nM or lower, about 4 nM or lower, about 2 nM or lower, about 1 nM or lower, about 0.5 nM or lower, about 0.4 n
  • an hNGAL mutein of the disclosure may interfere or compete with the binding of OX40L to OX40.
  • an hNGAL mutein of the disclosure may be capable of binding OX40 in the presence of OX40L and/or binding OX40/OX40L complex.
  • a lipocalin mutein according to the disclosure may comprise at least one amino acid substitution of a native cysteine residue by, e.g., a serine residue.
  • an hNGAL mutein according to the disclosure may comprise an amino acid substitution of a native cysteine residue at positions corresponding to positions 76 and/or 175 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1) by another amino acid, such as a serine residue.
  • another amino acid such as a serine residue.
  • hNGAL muteins may provide hNGAL muteins that are not only stably folded but are also able to bind a given non-natural target with high affinity.
  • the elimination of the structural disulfide bond may provide the further advantage of allowing for the generation or deliberate introduction of non-natural disulfide bonds into muteins of the disclosure, thereby, increasing the stability of the muteins.
  • hNGAL muteins that bind OX40 and that have the disulfide bridge formed between Cys 76 and Cys 175 are also part of the present disclosure.
  • provided OX40-binding hNGAL muteins may comprise, at one or more positions corresponding to positions 25, 28, 36, 40, 41 , 44, 49, 50, 52, 59, 62, 63, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81 , 82, 83, 87, 93, 96, 100, 103, 106, 114, 118, 125, 127, 129, 132, 134, 143, and 164 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1), one or more of the following mutated amino acid residues: Asn 25 ⁇ Ser; Gin 28 ⁇ His; Leu 36 ⁇ Phe; Ala 40 ⁇ Tyr; He 41 Trp or Arg; Glu 44 ⁇ Gly; Gln 49 ⁇ Gly; Lys 50 ⁇ Glu or Thr; Tyr 52 Gln; Lys 59 ⁇ Arg; Lys
  • an hNGAL mutein of the disclosure comprises 2 or more, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, or 31 or more of the afore-mentioned mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 1).
  • an hNGAL mutein of the disclosure comprises 10 or more of the afore-mentioned mutated amino acid residues. In a preferred embodiment, an hNGAL mutein of the disclosure comprises 15 or more of the afore-mentioned mutated amino acid residues. In a preferred embodiment, an hNGAL mutein of the disclosure comprises 20 or more of the afore-mentioned mutated amino acid residues. In a preferred embodiment, an hNGAL mutein of the disclosure comprises 25 or more of the afore-mentioned mutated amino acid residues. In a preferred embodiment, an hNGAL mutein of the disclosure comprises 29 or more of the afore-mentioned mutated amino acid residues.
  • provided OX40-binding hNGAL muteins may comprise one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1 ):
  • an OX40-binding hNGAL mutein includes all but three, all but two, or all but one mutated amino acid residues of one of the aforementioned sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1).
  • an hNGAL mutein of the disclosure has at least 70% sequence identity or at least 70% sequence homology to the sequence of mature hNGAL (SEQ ID NO: 1).
  • an hNGAL mutein of the disclosure comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-36 and 95-97 or a fragment or variant thereof. [00197] In some embodiments, an hNGAL mutein of the disclosure has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-36 and 95-97.
  • the present disclosure also includes structural homologues of an hNGAL mutein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-36 and 95-97, which structural homologues have an amino acid sequence homology or sequence identity of more than about 60%, preferably more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92% and most preferably more than 95% in relation to said hNGAL mutein.
  • the present disclosure provides a lipocalin mutein that binds OX40 with an affinity measured by a K D of about 5 nM or lower, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity to the amino acid sequence of SEQ ID NO: 33.
  • the present disclosure provides a lipocalin mutein that binds OX40 with an affinity measured by a K D of about 5 nM or lower, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity to the amino acid sequence of SEQ ID NO: 34.
  • the present disclosure provides a lipocalin mutein that binds OX40 with an affinity measured by a K D of about 5 nM or lower, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity to the amino acid sequence of SEQ ID NO: 35.
  • the present disclosure provides a lipocalin mutein that binds OX40 with an affinity measured by a K D of about 5 nM or lower, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity to the amino acid sequence of SEQ ID NO: 36.
  • the present disclosure provides a lipocalin mutein that binds OX40 with an affinity measured by a K D of about 5 nM or lower, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity to the amino acid sequence of SEQ ID NO: 95.
  • the present disclosure provides a lipocalin mutein that binds OX40 with an affinity measured by a K D of about 5 nM or lower, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity to the amino acid sequence of SEQ ID NO: 96.
  • the present disclosure provides a lipocalin mutein that binds OX40 with an affinity measured by a K D of about 5 nM or lower, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity to the amino acid sequence of SEQ ID NO: 97.
  • a lipocalin mutein of the present disclosure can comprise a heterologous amino acid sequence at its N-or C-Terminus, preferably C- terminus, such as a Strep II tag (SEQ ID NO: 9) or a cleavage site sequence for certain restriction enzymes, without affecting the biological activity (binding to its target, e.g., OX40) of the lipocalin mutein.
  • a heterologous amino acid sequence at its N-or C-Terminus preferably C- terminus
  • SEQ ID NO: 9 a Strep II tag
  • a cleavage site sequence for certain restriction enzymes without affecting the biological activity (binding to its target, e.g., OX40) of the lipocalin mutein.
  • a lipocalin mutein may be introduced in order to modulate certain characteristics of the mutein, such as to improve folding stability, serum stability, protein resistance or water solubility or to reduce aggregation tendency, or to introduce new characteristics to the mutein.
  • modification(s) may result in two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) characteristics of a provided mutein being modulated.
  • mutate one or more amino acid sequence positions of a lipocalin mutein to introduce new reactive groups, for example, for the conjugation to other compounds, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins, or for the formation of non-naturally occurring disulphide linkages.
  • PEG polyethylene glycol
  • HES hydroxyethyl starch
  • biotin peptides or proteins
  • the conjugated compound for example, PEG and HES, can in some cases increase the serum half-life of the corresponding lipocalin mutein.
  • a reactive group of a lipocalin mutein may occur naturally in its amino acid sequence, such as naturally occurring cysteine residues in said amino acid sequence.
  • such reactive group may be introduced via mutagenesis.
  • a reactive group is introduced via mutagenesis, one possibility is the mutation of an amino acid at the appropriate position by a cysteine residue.
  • Exemplary possibilities of such a mutation to introduce a cysteine residue into the amino acid sequence of an hNGAL mutein include the introduction of a cysteine residue at one or more of the sequence positions that correspond to sequence positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wild-type sequence of mature hNGAL (SEQ ID NO: 1).
  • the generated thiol moiety may be used to PEGylate or HESylate the mutein, for example, in order to increase the serum half-life of a respective lipocalin mutein [00204]
  • artificial amino acids may be introduced to the amino acid sequence of a lipocalin mutein.
  • such artificial amino acids are designed to be more reactive and thus to facilitate the conjugation to the desired compound.
  • Such artificial amino acids may be introduced by mutagenesis, for example, using an artificial tRNA, such as para-acetyl-phenylalanine.
  • fusion proteins of the disclosure may produce synergistic effect through dual-targeting of OX40 and PD-L1. In some embodiments, fusion proteins of the disclosure may produce synergistic effect through OX40 co-stimulation and PD-1/PD-L1 pathway blockade. In some embodiments, fusion proteins of the disclosure may produce localized anti-tumor effect through dual-targeting of OX40 and PD-L1. Numerous possible applications for the fusion proteins of the disclosure, therefore, exist in medicine.
  • the present disclosure encompasses the use of one or more fusion proteins disclosed herein or of one or more compositions comprising such fusion proteins for simultaneously binding of OX40 and PD-L1.
  • the present disclosure also involves the use of one or more fusion proteins as described for complex formation with OX40 and/or PD-L1.
  • provided fusion proteins may be used for the detection of OX40 and PD-L1.
  • Such use may include the steps of contacting one or more said fusion proteins, under suitable conditions, with a sample suspected of containing OX40 and/or PD-L1 , thereby allowing formation of a complex between the fusion proteins and OX40 and/or PD-L1, and detecting the complex by a suitable signal.
  • the detectable signal can be caused by a label, as explained above, or by a change of physical properties due to the binding, i.e., the complex formation, itself.
  • One example is surface plasmon resonance, the value of which is changed during binding of binding partners from which one is immobilized on a surface such as a gold foil.
  • Fusion proteins of the disclosure may also be used for the separation of OX40 and/or PD-L1. Such use may include the steps of contacting one or more said fusion proteins, under suitable conditions, with a sample supposed to contain OX40 and/or PD-L1, thereby allowing the formation of a complex between the fusion proteins and OX40 and/or PD-L1 and separating the complex from the sample.
  • the present disclosure provides diagnostic and/or analytical kits comprising one or more fusion proteins according to the disclosure.
  • compositions comprising one or more fusion proteins of the disclosure and a pharmaceutically acceptable excipient.
  • the present disclosure provides fusion proteins that simultaneously bind OX40 and/or PD-L1 for use as anti-tumor and/or anti- infection agents, and immune modulators.
  • fusion proteins of the present disclosure are envisaged to be used in a method of treatment of human diseases, such as a variety of cancers, including PD-L1 -positive cancers.
  • a PD-L1 positive cancer is a cancer in which PD-L1 is expressed. Expression of PD-L1 can be measured by methods well known in the art, such as immunohistochemistry or flow cytometry.
  • a method of treating human diseases such as a variety of cancers, including PD-L1- positive cancer, in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of one or more fusion proteins of the disclosure.
  • the term “treat”, “treating”, or “treatment” as used herein covers any treatment of a disease state and includes preventing the disease state or condition from occurring in a subject, inhibiting the disease state or condition, i.e. arresting its development; and/or relieving the disease state or condition, i.e. causing its regression and/or amelioration of the disease state or condition or any of its symptoms.
  • PD-L1 positive cancers may, e.g., comprise breast cancer (e.g., breast carcinoma).
  • breast cancer e.g., breast carcinoma
  • cancer and “tumor” are used interchangeably herein.
  • a tumor is a solid tumor.
  • fusion proteins of the disclosure may simultaneously target tumor cells where PD-L1 is expressed and activate lymphocytes of the host immune system adjacent to such tumor cells.
  • fusion proteins of the disclosure may increase targeted anti-tumor T cell activity, enhance anti-tumor immunity and/or have a direct inhibiting effect on tumor growth, thereby producing synergistic anti-tumor results.
  • fusion proteins of the disclosure may activate immune responses in a tumor microenvironment.
  • fusion proteins of the disclosure may reduce side effects of effector lymphocytes towards healthy cells, i.e. off-target toxicity, for example, via locally inhibiting oncogene activity and inducing lymphocyte activation.
  • the present disclosure encompasses the use of a fusion protein of the disclosure, or a composition comprising a provided fusion protein, for inducing a localized lymphocyte response in the vicinity of PD-L1 -positive tumor cells. Accordingly, in some embodiments, the present disclosure provides methods of inducing a localized lymphocyte response in the vicinity of PD-L1 -positive tumor cells, comprising applying one or more fusion proteins of the disclosure or one or more compositions comprising such fusion proteins.
  • “Localized” means that upon simultaneous binding T cells via OX40 and engaging PD-L1 -positive tumor cells, T cells produce cytokines, particularly IL-2 and/or interferon gamma in vicinity of the PD-L1 -positive cells. Such cytokines reflect activation of T cells which may then be able to kill PD-L1 -positive cells, either directly or indirectly by attracting other killer cells, such as T cells or NK cells.
  • the present disclosure encompasses the use of a fusion protein of the disclosure, or a composition comprising such fusion protein, for co- stimulating T cells, and/or activating downstream signaling pathways of OX40.
  • a provided fusion protein co-stimulates T cells and/or activates downstream signaling pathways of OX40 when engaging tumor cells where PD-L1 is expressed.
  • the present disclosure provides methods of inducing T lymphocyte proliferation and/or activating downstream signaling pathways of OX40, preferably when engaging tumor cells where PD-L1 is expressed, comprising applying one or more fusion proteins of the disclosure and/or one or more compositions comprising such fusion proteins.
  • the present disclosure encompasses the use of a fusion protein of the disclosure, or a composition comprising such fusion protein, for inducing OX40 clustering and activation on T cells and directing such T cells to tumor cells where PD- L1 is expressed.
  • the present disclosure also encompasses the use of a fusion protein of the disclosure or a composition comprising such fusion protein for simultaneously activating downstream signaling pathways of OX40 and engaging PD-L1 -positive tumor cells.
  • the present disclosure also encompasses a method of simultaneously activating downstream signaling pathways of OX40 and engaging PD-L1 -positive tumor cells, comprising applying one or more fusion proteins of the disclosure or one or more compositions comprising such fusion protein to a tissue comprising a tumor.
  • the present disclosure also encompasses a method of simultaneously co- stimulating T cells and engaging PD-L1 -positive tumor cells, comprising applying one or more fusion proteins of the disclosure or one or more compositions comprising such fusion protein to a tissue comprising a tumor.
  • the present disclosure also encompasses a method of simultaneously inducing lymphocyte activity and engaging PD-L1 -positive tumor cells, comprising applying one or more fusion proteins of the disclosure or one or more compositions comprising such fusion protein to a tissue comprising a tumor.
  • the present disclosure also encompasses a method of inducing OX40 clustering and activation on T cells and directing said T cells to PD-L1 -positive tumor cells, comprising applying one or more fusion proteins of the disclosure or one or more compositions comprising such fusion protein to a tissue comprising a tumor.
  • the present disclosure also encompasses a method of inducing a localized lymphocyte response in the vicinity of PD-L1 -positive tumor cells, comprising applying one or more fusion proteins of the disclosure or one or more compositions comprising such fusion protein to a tissue comprising a tumor.
  • the present disclosure also encompasses a method of inducing increased secretion of IL-2, IL-13, TGF-y and/or one or more cytotoxic factors by T cells in the vicinity of PD-L1 -positive tumor cells, comprising applying one or more fusion proteins of the disclosure or one or more compositions comprising such fusion protein to a tissue comprising a tumor.
  • the one or more cytotoxic factors may be selected from the group consisting of Granzyme A, Granzyme B and soluble Fas ligand.
  • the present disclosure also encompasses a method of inducing increased secretion of one or more cytotoxic factors by T cells in the vicinity of PD-L1 -positive tumor cells, comprising applying one or more fusion proteins of the disclosure or one or more compositions comprising such fusion protein to a tissue comprising a tumor.
  • the one or more cytotoxic factors may be selected from the group consisting of Granzyme A, Granzyme B and soluble Fas ligand.
  • the present disclosure provides nucleic acid molecules (DNA and RNA) that include nucleotide sequences encoding provided fusion proteins.
  • the disclosure encompasses a host cell containing a provided nucleic acid molecule. Since the degeneracy of the genetic code permits substitutions of certain codons by other codons specifying the same amino acid, the disclosure is not limited to a specific nucleic acid molecule encoding a fusion protein as described herein, rather, encompassing all nucleic acid molecules that include nucleotide sequences encoding a functional fusion protein. In this regard, the present disclosure also relates to nucleotide sequences encoding provided fusion proteins.
  • a nucleic acid molecule such as DNA
  • An operable linkage is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression.
  • promoter regions necessary for gene expression may vary among species, but in general these regions include a promoter, which, in prokaryotes, contains both the promoter per se, i.e., DNA elements directing the initiation of transcription, as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation.
  • promoter regions normally include 5’ non-coding sequences involved in initiation of transcription and translation, such as the -35/-10 boxes and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5’-capping elements in eukaryotes.
  • These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native protein to a specific compartment of a host cell.
  • the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactorily functional in a particular host cell, then they may be substituted with signals functional in that cell.
  • a nucleic acid molecule of the disclosure may be “operably linked” to a regulatory sequence (or regulatory sequences), such as a promoter sequence, to allow expression of this nucleic acid molecule.
  • a nucleic acid molecule of the disclosure includes a promoter sequence and a transcriptional termination sequence.
  • Suitable prokaryotic promoters are, for example, the tet promoter, the lacllV5 promoter or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.
  • a nucleic acid molecule encoding a lipocalin mutein disclosed in this application may be “operably linked” to another nucleic acid molecule encoding an antibody or antibody chain of the disclosure to allow expression of a fusion protein disclosed herein.
  • muteins of the disclosure as included in the fusion proteins, in some embodiments, the naturally occurring disulfide bond between Cys 76 and Cys 175, respectively, may be removed. Accordingly, such muteins can be produced in a cell compartment having a reducing redox milieu, for example, in the cytoplasm of Gram-negative bacteria.
  • the disclosure also includes nucleic acid molecules encoding fusion proteins comprising such muteins which, in some embodiments, may include one or more additional mutations outside the indicated sequence positions of experimental mutagenesis. Such mutations are often tolerated or can even prove to be advantageous, for example, if they contribute to an improved folding efficiency, serum stability, thermal stability or ligand binding affinity of the lipocalin muteins and/or the fusion proteins.
  • provided nucleic acid molecules can also be part of a vector or any other kind of cloning vehicle, such as a plasmid, a phagemid, a phage, a baculovirus, a cosmid or an artificial chromosome.
  • a nucleic acid molecule is included in a phagemid.
  • a phagemid vector denotes a vector encoding the intergenic region of a temperate phage, such as M13 or f1 , or a functional part thereof fused to the cDNA of interest.
  • helper phage e.g., M13K07, VCS-M13 or R408
  • Such cloning vehicles can include, aside from the regulatory sequences described above and a nucleic acid sequence encoding a fusion protein as described herein, replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art and are commercially available.
  • the disclosure also relates, in some embodiments, to methods for the production of fusion proteins of the disclosure starting from a nucleic acid coding for a fusion protein or any subunits therein using genetic engineering methods.
  • a provided method can be carried out in vivo, wherein a provided fusion protein can, for example, be produced in a bacterial or eukaryotic host organism, and then isolated from this host organism or its culture. It is also possible to produce a fusion protein of the disclosure in vitro, for example, using an in vitro translation system.
  • a nucleic acid encoding such fusion protein may be introduced into a suitable bacterial or eukaryotic host organism using recombinant DNA technology well known in the art.
  • a DNA molecule encoding a fusion protein as described herein, and in particular a cloning vector containing the coding sequence of such a fusion protein can be transformed into a host cell capable of expressing the gene. Transformation can be performed using standard techniques.
  • the disclosure is also directed to host cells containing a nucleic acid molecule as disclosed herein.
  • the transformed host cells may be cultured under conditions suitable for expression of a nucleotide sequence encoding a fusion protein of the disclosure.
  • Suitable host cells can be prokaryotic, such as Escherichia co// (E. coli) or Bacillus subtilis, or eukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) or primary mammalian cells.
  • a lipocalin mutein of the disclosure including as comprised in a fusion protein disclosed herein, includes intramolecular disulfide bonds
  • an oxidizing environment may be provided by the periplasm of Gram-negative bacteria such as E. coli, in the extracellular milieu of Gram- positive bacteria or the lumen of the endoplasmic reticulum of eukaryotic cells and usually favors the formation of structural disulfide bonds.
  • a fusion protein of the disclosure in the cytosol of a host cell, preferably E. coli.
  • a provided fusion protein can either be directly obtained in a soluble and folded state or recovered in the form of inclusion bodies, followed by renaturation in vitro.
  • a further option is the use of specific host strains having an oxidizing intracellular milieu, which may thus allow the formation of disulfide bonds in the cytosol (Venturi et al., J Mol Biol, 2002).
  • a fusion protein as described herein may not necessarily be generated or produced only by use of genetic engineering. Rather, such protein can also be obtained by any of the many conventional and well-known techniques such as plain organic synthesis strategies, chemical synthesis such as Merrifield solid phase synthesis, commercially available automated synthesizers, or by in vitro transcription and translation. It is, for example, possible that promising fusion proteins or lipocalin muteins included in such fusion proteins are identified using molecular modeling, synthesized in vitro, and investigated for the binding activity for the target(s) of interest. Methods for the solid phase and/or solution phase synthesis of proteins are well known in the art (see e.g. Bruckdorfer et al., Curr Pharm Biotechnol, 2004).
  • a fusion protein of the disclosure may be produced by in vitro transcription/translation employing well-established methods known to those skilled in the art.
  • a fusion protein as described herein may also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques.
  • a fusion protein according to the present disclosure may be obtained by conjugating together individual subunits, e.g., antibodies and muteins as included in the fusion protein. Such conjugation can be, for example, achieved through all forms of covalent or non-covalent linkage using conventional methods.
  • modifications of the amino acid sequence include, e.g., directed mutagenesis of single amino acid positions to simplify sub-cloning of a protein gene or its parts by incorporating cleavage sites for certain restriction enzymes.
  • these mutations can be incorporated to further improve the affinity of a fusion protein for its targets (e.g., OX40 and PD-L1 ).
  • mutations can be introduced to modulate one or more characteristics of the protein such as to improve folding stability, serum stability, protein resistance or water solubility or to reduce aggregation tendency, if necessary.
  • Example 1 Expression and analysis of representative fusion proteins
  • representative antibody-lipocalin mutein fusion proteins were generated by fusing together a PD-L1 specific antibody having the heavy chain provided by SEQ ID NO: 77 or 78, or comprising a heavy chain variable domain of SEQ ID NO: 67, or comprising the CDRs of GFSLSNYD (HCDR1, SEQ ID NO: 45), IWTGGAT (HCDR2, SEQ ID NO: 46), VRDSNYRYDEPFTY (HCDR3; SEQ ID NO: 47), and having the light chain provided by SEQ ID NO: 79, or comprising a light chain variable domain of SEQ ID NO: 73, or comprising the CDRs of QSIGTN (LCDR1, SEQ ID NO: 48), YAS (LCDR2), QQSNSWPYT (LCDR3; SEQ ID NO: 49), and an OX40-specific lipocalin mutein of the disclosure such as any one of SEQ ID NOs: 33-36, via a linker, such as
  • fusion proteins e.g., the fusion proteins of SEQ ID NOs: 82 and 79, SEQ ID NOs: 88 and 79, SEQ ID NOs: 89 and 79, SEQ ID NOs: 91 and 79, and SEQ ID NOs: 78 and 87, were generated via fusing the one or more lipocalin muteins of any one of SEQ ID NOs: 33-36 to either one or more of the four termini of an antibody comprisingthe heavy chain provided by SEQ ID NO: 77 or 78, or comprising a heavy chain variable domain of SEQ ID NO: 67, or comprising the CDRs of GFSLSNYD (HCDR1 , SEQ ID NO: 45), IWTGGAT (HCDR2, SEQ ID NO: 46), VRDSNYRYDEPFTY (HCDR3; SEQ ID NO: 47), and having the light chain provided by SEQ ID NO: 79, or comprising a light
  • the generated fusion proteins can be bivalent to OX40 (e.g., as depicted in Figure 1A-1D) or tetravalent to OX40 (e.g., as depicted in Figure 1E-1H), or have even higher valency to OX40 (e.g., as depicted in Figure 11).
  • the PD-L1 specific antibodies as well as all antibody lipocalin mutein fusion proteins described in this Example had an engineered IgG 1 backbone or an engineered lgG4 backbone, which contained a S228P mutation to minimize lgG4 half-antibody exchange in- vitro and in-vivo (Silva et al., J Biol Chem, 2015).
  • the engineered lgG1 backbone contained L234A and L235A mutations. Additional mutations in the lgG4 backbones may also exist in all antibodies and fusion proteins described here, including any one or more of mutations F234A, L235A, M428L, N434S, M252Y, S254T, and T256E.
  • F234A and L235A mutations which may be introduced to decrease ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010).
  • M428L and N434S mutations or M252Y, S254T, and T256E mutations may be introduced for extended serum half-life (Dall'Acqua et al., J Biol Chem, 2006, Zalevsky et al., Nat Biotechnol, 2010). All antibodies were expressed without the carboxy-terminal lysine to avoid heterogeneity.
  • monospecific lipocalin mutein Fc fusions were generated by fusing one or more of the OX40-specific lipocalin mutein of any one of SEQ ID NOs: 33-36, via a linker, e.g., a (G4S)3 linker of SEQ ID NO: 10, to the C-terminus of the Fc region of an antibody provided in SEQ ID NO: 29 as depicted in Figure 1J-1K.
  • the resulting construct is provided in SEQ ID NOs: 39-42.
  • the present invention also embodies asymmetrical antibody-lipocalin mutein fusion formats where, for example, one light chain of the antibody may be fused with a lipocalin mutein while the other is not.
  • the constructs of the fusion proteins were generated by gene synthesis and cloned into a mammalian expression vector. They were then transiently expressed in Expi293FTM cells (Life Technologies). The concentration of fusion proteins in the cell culture medium was measured by HPLC (Agilent Technologies) employing a POROS® protein A affinity column (Applied Biosystems). The titers of the fusion proteins are summarized in Table 1.
  • the fusion proteins were purified using Protein A chromatography followed by size-exclusion chromatography (SEC) in phosphate-buffered saline (PBS). After SEC purification, the fractions containing monomeric protein were pooled and analyzed again using analytical SEC.
  • SEC Size-exclusion chromatography
  • Example 2 Stable expression of the fusion proteins
  • exemplary fusion proteins were generated by gene synthesis, including codon optimization, and cloned into a mammalian expression vector. They were then stably expressed in Chinese hamster ovary (CHO) cells. The concentration of fusion proteins in the cell culture medium was measured using Octet (ForteBio, Pall Corp.) with Protein-A sensors and quantified using human lgG1 standard. The titers of the fusion proteins are summarized in Table 2.
  • Example 3 Binding of fusion proteins towards PD-L1 or OX40 determined by surface plasmon resonance (SPR)
  • the anti-human IgG Fc antibody (GE Healthcare) was immobilized on a CM5 sensor chip using standard amine chemistry: the carboxyl groups on the chip were activated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Subsequently, anti-human IgG Fc antibody solution (GE Healthcare) at a concentration of 25 ⁇ g/mL in 10 mM sodium acetate (pH 5.0) was applied at a flow rate of 5 pL/min until an immobilization level of 4000-10000 resonance units (RU) was achieved.
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
  • NHS N-hydroxysuccinimide
  • Anti-PD-L1 antibodies including a reference antibody (SEQ ID NOs: 80 and 81) and an antibody as included in the fusion proteins (SEQ ID NOs: 78 and 79) and a reference anti-OX40 antibody (SEQ ID NOs: 25 and 26) were also tested as controls.
  • the binding assay was carried out with a contact time of 180 s, a dissociation time of 900 s and a flow rate of 30 pL/min. All measurements were performed at 25°C. Regeneration of the chip surface was achieved with injections of 3 M MgCI 2 for 120 s. Prior to the protein measurements, three startup cycles were performed for conditioning purposes. Data were evaluated with Biacore Inside Evaluation software (V1.0.5). Double referencing was used, and the 1:1 binding model was used to fit the raw data.
  • Example 4 Binding of fusion proteins towards PD-L1 or OX40 in enzyme-linked immunosorbent assay (ELISA) [00266] An enzyme-linked immunosorbent assay (ELISA) was employed to determine the binding potency of exemplary fusion proteins to human PD-L1 and human OX40.
  • ELISA enzyme-linked immunosorbent assay
  • huPD-L1-His or huOX40-His (human PD-L1 or OX40 with a C- terminal polyhistidine tag, R&D Systems or Sino Biologies) at the concentration of 1 ⁇ g/mL in PBS was coated overnight on microtiter plates at 4°C. After washing with PBS-0.05%T (PBS supplemented with 0.05% (v/v) Tween 20), the plates were blocked with 2% BSA (w/v) in PBS-0.1%T (PBS supplemented with 0.1% (v/v) Tween 20) for 1 h at room temperature.
  • exemplary fusion proteins SEQ ID NOs: 78 and 87, 88 and 79, 89 and 79, and 82 and 79
  • Fc fusions of OX40-specific lipocalin muteins SEQ ID NOs: 39-42
  • anti-PD-L1 antibodies SEQ ID NOs: 78 and 79, and 80 and 81
  • a reference anti-OX40 antibody SEQ ID NOs: 27 and 28
  • a reference bispecific fusion protein targeting PD-L1 and OX40 SEQ ID NOs: 43 and 44
  • Bound molecules under study were detected by incubation with 1 :5000 or 1 :25000 diluted anti-human IgG Fc- HRP (Jackson Laboratory) in PBS-0.1%T-2%BSA. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescence microplate reader.
  • cyPD-L1-His cynomolgus PD-L1 with C- terminal polyhistidine tag, Sino Biologicals
  • cyOX40-Fc cynomolgus OX40 C-terminally fused to Fc, Evitria
  • the tested agents were similarly titrated, and bound agents detected.
  • binding of the fusion proteins to cyOX40-Fc may be detected via anti-NGAL-HRP or anti-Fab-HRP.
  • the observed EC 50 values towards the two human targets of provided fusion proteins were very similar or comparable to tested PD-L1 antibodies (reference PD-L1 antibody of SEQ ID NOs: 80 and 81 and PD-L1 antibody of SEQ ID NOs: 78 and 79 as included in the fusion proteins), and/or Fc fusions of the OX40-specific lipocalin mutein as included in the bispecific fusion proteins (SEQ ID NO: 39-42).
  • All tested fusion proteins show cross-reactivity to cynomolgus PD-L1 , with comparable EC 50 values to the reference PD-L1 antibody (SEQ ID NOs: 80 and 81) or the PD-L1 antibody included in the fusion proteins (SEQ ID NOs: 78 and 79).
  • all tested bispecific lipocalin mutein fusion proteins show cross-reactivity to cynomolgus OX40, with comparable EC 50 values to the Fc fusions of the OX40-specific lipocalin muteins included in the bispecific fusion proteins.
  • Table 5 ELISA data for PD-L1 or OX40 binding
  • Example 5 Simultaneous binding of fusion proteins to PD-L1 and OX40 in ELISA
  • Different concentrations of tested fusion proteins (SEQ ID NOs: 78 and 87, 88 and 79, 89 and 79, 82 and 79, 93 and 90, and 91 and 94), Fc fusions of OX40-specific lipocalin mutein (SEQ ID NOs: 39-42), anti-PD-L1 antibodies (SEQ ID NOs: 78 and 79, and 80 and 81), a reference anti-OX40 antibody (SEQ ID NOs: 27 and 28), and a reference bispecific fusion protein targeting PD-L1 and OX40 (SEQ ID NOs: 43 and 44) were added to the wells and incubated for 1 h at room temperature, followed by a wash step.
  • biotinylated huOX40- His (huOX40-His-Bio, Sino Biological) was added at a constant concentration of 1 ⁇ g/mL in PBS-0.1%T-2%BSA for 1 h.
  • a 1:5000 dilution of ExtrAvidin-HRP (Sigma- Aldrich) in PBS-0.1%T-2%BSA was added to the wells and incubated for 1 h.
  • fluorogenic HRP substrate QuantaBlu, Pierce was added to each well, and the fluorescence intensity was detected using a fluorescence microplate reader.
  • Dual binding data of fusion proteins (SEQ ID NOs: 78 and 87, 88 and 79, 89 and 79, and 82 and 79) are shown in Figure 3A and 3B, together with the fit curves resulting from a 1:1 sigmoidal binding fit, where the EC 50 value and the maximum signal were free parameters, and the slope was fixed to unity.
  • the EC 50 values are summarized in Table 6. All bispecific fusion proteins show clear binding signals, demonstrating that the fusion proteins are able to engage PD-L1 and OX40 simultaneously. As expected, molecules monospecific for one of the targets such as anti-PD-L1 or anti-OX40 antibodies and Fc fusions of OX40-specific lipocalin muteins do not show binding in the simultaneous binding assay.
  • Example 6 Flow cytometric analysis of fusion proteins binding to cells expressing human and cynomolgus OX40 and PD-L1
  • Target-specific binding of fusion proteins to human and cynomolgus PD-L1- expressing cells and human and cynomolgus OX40-expressing cells was assessed by flow cytometry.
  • Flp-ln CHO cells were stably transfected with human PD-L1 , cynomolgus PD- L1, human OX40, cynomolgus OX40 or a mock control using the Flp-ln system (Life technologies) according to the manufacturer's instructions.
  • Transfected CHO cells were maintained in Ham's F12 medium (Life technologies) supplemented with 10% Fetal Calf Serum (Biochrom) and 500 ⁇ g/mL Hygromycin B (Roth). Cells were cultured in cell culture flasks according to the manufacturer’s instructions (37°C, 5% CO 2 atmosphere).
  • Example 7 Off-target binding of the fusion proteins analyzed by ELISA
  • ELISA-based assay was employed to assess the off-target binding of fusion proteins toward 30 different targets including PD-L1 , PD-L2 and other TNF receptor family proteins (Frese et al., MAbs, 2013).
  • Targets at the concentration of 5 ⁇ g/mL in PBS were coated overnight on microtiter plates at 4°C. After washing with PBS-0.05%T, the plates were blocked with PBS-0.1%T-2%BSA for 1 h at room temperature. After washing with 100 pL PBS-0.05%T five times, tested fusion proteins at 100 nM or 10 nM were added to the wells and incubated for 1 h at room temperature, followed by another wash step.
  • Bound antibodies under study were detected by incubation with 1:5000 diluted F(ab')2 Fragment goat anti-human IgG F(ab')2 HRP (Jackson Laboratory) in PBS-0.1%T-2%BSA. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well and incubated for 50 min. The fluorescence intensity was detected using a fluorescence microplate reader and normalized to the signal of a control antibody (SEQ ID NOs: 37 and 38). The normalized signals of each tested molecule binding to the non-specific targets were summed up to yield the cumulated binding ratio for the antibody at a given concentration, i.e., 100 nM or 10 nM.
  • the cumulated binding ratios at 100 nM and 10 nM for each tested molecule were further summed to yield the sum of cumulated binding ratios.
  • the control antibody SEQ ID NOs: 37 and 38
  • the fluorescence intensity resulting from binding to each of the non-specific targets and blank wells was normalized
  • Table 7 Higher cumulated binding ratio correlates to stronger off-target binding.
  • fusion proteins of SEQ ID NOs: 78 and 87, 88 and 79, 89 and 79, and 82 and 79 show no or negligible off-target binding (sum of cumulative binding rations ⁇ 150).
  • Example 8 Off-target binding of the fusion proteins analyzed by flow cytometry
  • Flow cytometry studies were employed in order to assess the off-target or non-specific binding of the fusion proteins to human endothelial cells (HUVEC) that are OX40 negative but PD-L1 positive.
  • HUVEC at a confluency of 90% were detached using a trypsin solution (Promocell) following the vendor guidelines and counted.
  • 2.5 x10 4 HUVEC per well were added to each well of a 384 well plate for FACS (V bottom). Cells were washed with PBS and live-death marker (Invitrogen) was added in a 1/1000 dilution to each well.
  • the tested fusion proteins (SEQ ID NOs: 78 and 87, 88 and 79, 89 and 79, 82 and 79, and 97 and 98), Fc fusions of OX40-specific lipocalin muteins (SEQ ID NOs: 39, 40, and 42), lipocalin scaffold (SEQ ID NO: 2) or a human lgG4 control antibody (SEQ ID NOs: 21 and 22) at concentrations ranging from 200 nM to 3.125 nM were added to the cells, with or without a 30 min pre-icubation with 2000 nM anti-PD-L1 antibody (SEQ ID NOs: 78 and 79). A 30-minute incubation at 4°C followed and two washing steps.
  • the mixtures of fusion proteins and the tracer were added to the plates and incubated for 20 min at room temperature followed by five washing steps with 100 pL PBS-0.05%T. Subsequently, a 1 :5000 dilution of ExtrAvidin-HRP (Sigma-Aldrich) was added to the wells and incubated for 1h. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Pierce) was added to each well, and the fluorescence intensity was detected using a fluorescence microplate reader.
  • ExtrAvidin-HRP Sigma-Aldrich
  • Example 10 Competition of the fusion proteins with PD-L1 in binding to PD-1 determined using ELISA
  • Fusion proteins at different concentrations were mixed with 15 nM of recombinant huPD-L1-Fc (R&D systems) as a tracer and incubated for 1 h at room temperature.
  • the mixtures of fusion proteins and the tracer were added to the plates and incubated for 20 min at room temperature followed by five washing steps with 100 pL PBS-0.05%T.
  • a 1:5000 dilution of goat anti-human IgG-Fc HRP Jackson Laboratory
  • fluorogenic HRP substrate QuantaBlu, Pierce
  • Table 8 Competition of fusion proteins with PD-L1 for binding to PD-1
  • Example 11 Assessment of T cell activation using a PD-1/PD-L1 blockade bioassay
  • PD-1-NFAT-luc Jurkat T cells a Jurkat cell line engineered to express PD-1 and the luc gene (firefly luciferase gene) driven by an NFAT response element (NFAT-RE)
  • PD-L1 aAPC/CHO-K1 cells CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate cognate TCRs in an antigen-independent manner.
  • PD-L1 aAPC/CHO-K1 cells were grown in Ham's F12 medium supplemented with 10% FCS and plated at 8.00 x 10 3 cells per well and allowed to adhere overnight at 37°C in a humidified 5% CO 2 atmosphere. On the next day, the culture media was discarded.
  • PD-1-NFAT-luc Jurkat T cells 1.00 x 10 4 PD-1-NFAT-luc Jurkat T cells were added to each well, followed by the addition of various concentrations, typically ranging from 0.005 nM to 20 nM, of tested fusion proteins (SEQ ID NOs: 82 and 79, 78 and 87, 88 and 79, 89 and 79, 93 and 90, and 91 and 94), the building block anti-PD-L1 antibody (SEQ ID NOs: 78 and 79) or a reference bispecific fusion protein targeting PD-L1 and OX40 (SEQ ID NOs: 43 and 44). Plates were covered with a gas permeable seal and incubated at 37°C in a humidified 5% CO 2 atmosphere.
  • Bio-GioTM Reagent was added to each well and the bioluminescent signal was quantified using a luminometer.
  • Four-parameter logistic curve analysis was performed with GraphPad Prism® to calculate EC 50 values which are summarized in Table 9. The assay was performed in triplicates.
  • Table 9 Assessment of T cell activation using a PD-1/PD-L1 blockade bioassay
  • Example 12 Assessment of T cell activation using human peripheral blood mononuclear cells (PBMCs)
  • a T cell assay was employed to assess the ability of the selected fusion proteins to co-stimulate T cell responses as well as to prevent co-inhibition mediated by PD- L1 binding to PD-1.
  • fusion proteins at different concentrations were added to staphylococcal enterotoxin B (SEB) stimulated human peripheral blood mononuclear cells (PBMCs) and incubated for 3 days at 37°C. IL-2 secretion levels were measured in the supernatants.
  • SEB staphylococcal enterotoxin B
  • PBMCs peripheral blood mononuclear cells
  • PBMCs from healthy volunteer donors were isolated from buffy coats by centrifugation through a polysucrose density gradient (Biocoll, 1.077 g/mL, Biochrom), following Biochrom 's protocols.
  • the purified PBMCs were resuspended in a buffer consisting of 90% FCS and 10% DMSO, immediately frozen down and stored in liquid nitrogen until further use.
  • PBMCs were thawed and rested in culture media (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1% Penicillin-Streptomycin (Life Technologies) for 16 h at 37°C in a humidified 5% CO 2 atmosphere.
  • IL-2 levels in the supernatant were assessed using the human IL-2 DuoSet kit (R&D Systems) as described in the following procedures.
  • IL-2 Capture Antibody in PBS. Subsequently, wells were washed 5 times with 80 pl PBS supplemented with 0.05% Tween (PBS-T). After 1 h blocking in PBS-0.05%T containing 1% casein (w/w), assay supernatants and a concentration series of IL-2 standard diluted in culture medium was transferred to respective wells and incubated overnight at 4°C. The next day, a mixture of 100 ng/mL goat anti-hlL-2-Bio detection antibody (R&D Systems) and 1 ⁇ g/mL Sulfotag-labelled streptavidin (Mesoscale Discovery) in PBS-T containing 0.5% casein were added and incubated at room temperature for 1 h. After washing, 25 ⁇ L reading buffer (Mesoscale Discovery) was added to each well, and the resulting electrochemiluminescence (ECL) signal was detected by a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.
  • Bispecific fusion proteins of SEQ ID NOs: 82 and 79, SEQ ID NOs: 78 and 87, SEQ ID NOs: 88 and 79, SEQ ID NOs: 89 and 79, and SEQ ID NOs: 43 and 44 and the anti-PD-L1 antibody SEQ ID NOs: 78 and 79 are capable of inducing T cell activation, which is demonstrated by increased IL-2 secretion levels compared to isotype control (hlgG4, Sigma) (Figure 9A and B). All tested fusion proteins induce a stronger T cell activation compared to the anti-PD-L1 antibody used to build the fusion proteins.
  • a T cell assay was employed to assess the ability of the fusion proteins to co- stimulate T cell activation in the presence of tumor cells. Fusion proteins were applied at different concentrations to anti-CD3 stimulated T cells, in the presence of PD-L1 expressing human breast carcinoma cells MDA-MB-231. IL-2 secretion levels were measured in the supernatants.
  • PBMC from healthy volunteer donors were isolated from buffy coats as described in Example 12. T lymphocytes were further purified from PBMC by magnetic cell sorting using a Pan T cell purification Kit (Miltenyi Biotec GmbH) following the manufacturer's instructions. Purified Pan T cells were resuspended in a buffer consisting of 90% FCS and 10% DMSO, immediately frozen down and stored in liquid nitrogen until further use.
  • T cells were thawed and rested in culture media (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1% Penicillin-Streptomycin (Life Technologies) for 16 h at 37°C in a humidified 5% CO 2 atmosphere.
  • culture media RPMI 1640, Life Technologies
  • Penicillin-Streptomycin Life Technologies
  • FIG. 10 Exemplary data are shown in Figure 10. Co-culturing of Pan T cells with MDA-MB-231 cells in presence of the fusion proteins with the lipocalin mutein fused to the C- terminus of the PD-L1 -specific antibody (SEQ ID NOs: 82 and 79, SEQ ID NOs: 78 and 87, SEQ ID NOs: 88 and 79, SEQ ID NOs: 89 and 79) led to a clear increase in IL-2 secretion compared to hlgG4 isotype control.
  • SEQ ID NOs: 82 and 79 SEQ ID NOs: 78 and 87, SEQ ID NOs: 88 and 79, SEQ ID NOs: 89 and 79
  • FIG. 10A shows that bispecific fusion proteins built with lipocalin muteins of high affinity to OX40 (picomolar range) led to the secretion of higher levels of IL-2 than bispecific fusion proteins built with lower affinity lipocalin muteins (double digit nanomolar range).
  • FIG 10B shows that the bispecific fusion protein of SEQ ID NOs: 82 and 79 induced strong T cell activation as measured by the secretion of IL-2 while neither the building blocks of this bispecific fusion protein alone or in combination nor the cocktail of anti-PD-L1 and anti-OX40 antibodies induced T cell activation.
  • the data show that the bispecific format of targeting PD-L1 and OX40 is superior to a cocktail of two separate molecules targeting OX40 and PD-L1 in presence of PD-L1 expressing target cells.
  • the bispecific fusion protein of SEQ ID NOs: 82 and 79 induced a stronger T cell activation than the reference bispecific fusion protein (SEQ ID NOs: 43 and 44).
  • Example 14 Assessment of PD-L1 dependent T cell activation induced by the fusion proteins
  • the PD-L1 target dependent T cell co-stimulation by the fusion proteins was further analyzed using a T cell activation assay. Fusion proteins were applied at different concentrations to anti-CD3 stimulated T cells, co-cultured with human PD-L1 transfected or mock transfected Flp-ln-CHO cells. IL-2 secretion levels were measured in the supernatants.
  • PBMC from healthy volunteer donors were isolated from buffy coats as described in Example 12. T lymphocytes were further purified and stored as described in Example 13. [00324] For the assay, T cells were thawed and rested in culture media (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1% Penicillin-Streptomycin (Life Technologies) overnight at 37°C in a humidified 5% CO 2 atmosphere.
  • FIG. 11 Exemplary data are shown in Figure 11. Co-culturing of Pan T cells with CHO cells transfected with human PD-L1 in presence of the bispecifc fusion proteins (SEQ ID NOs: 82 and 79, SEQ ID NOs: 78 and 87, SEQ ID NOs: 88 and 79, SEQ ID NOs: 89 and 79) led to strong dose-dependent IL-2 secretion compared to hlgG4 isotype. IL-2 levels secreted upon incubation with the lipocalin mutein bispecific fusion proteins are comparable to the IL-2 levels secreted in presence of the reference bispecific molecule.
  • the bispecifc fusion proteins SEQ ID NOs: 82 and 79, SEQ ID NOs: 78 and 87, SEQ ID NOs: 88 and 79, SEQ ID NOs: 89 and 79
  • Example 15 Mixed lymphocyte reaction (MLR) assessment with CD4 + T cells
  • a mixed lymphocyte reaction (MLR) assay was utilized to assess the ability of an exemplary fusion protein to induce CD4 + T cell stimulation in the presence of human antigen presenting cells naturally expressing PD-L1.
  • the fusion protein and various controls (SEQ ID NOs: 80 and 81, SEQ ID NOs: 78 and 79, SEQ ID NO:42, SEQ ID NOs: 82 and 79, SEQ ID NOs: 43 and 44, SEQ ID NOs: 80 and 81 + SEQ ID NOs: 25 and 26, SEQ ID NOs: 78 and 79 + SEQ ID NO: 42) at various concentrations were tested in the presence of monocyte-derived dendritic cells (moDCs) and CD4 + T cells from mismatching healthy donors in a one-way MLR. After 6 days of culturing in the presence of tested molecules, the secretion of IL-2 and interferon -gamma (IFNg) was quantified in the supernatants.
  • moDCs monocyte-
  • PBMCs were purified from platelet apheresis blood pack using a Lymphoprep solution following the manufacturer’s instructions (StemCell).
  • Total CD4 + T lymphocytes were purified from PBMC using a Miltenyi kit and frozen in a solution of 90% FBS 10% DMSO.
  • CD14 + monocytes were purified using CD14 + beads kit (Miltenyi) and used fresh.
  • MoDCs were obtained by culturing CD14 + monocytes in RPMI1640 plus 10% FBS and Pen/Strep (LifeTech) in the presence of 50 ng/mL of IL-4 and 100 ng/mL of GMCSF (Miltenyi) for 6 days at 2x10 6 cells/mL. At day 3, 10 mL of fresh medium containing cytokines was added. Phenotype (CD14, CD1a, HLADR, PD-L1) was assessed at day 7 of differentiation by FACS.
  • Figure 12B indicates that the fusion protein (SEQ ID NOs: 82 and 79) induced a dose-dependent secretion of IFNg as compared to an isotype antibody control.
  • IFNg levels induced by the fusion protein of SEQ ID NOs: 82 and 79 were higher as compared to equimolar concentrations of the cocktail of a reference PD-L1 antibody (SEQ ID NOs: 80 and81 ) and a reference OX40 antibody (SEQ ID NOs: 25 and 26), over concentrations ranging from 0.001 to 20.5 nM.
  • Example 16 Mixed lymphocyte reaction (MLR) assessment with CD3* T cells
  • a mixed lymphocyte reaction (MLR) assay was utilized to assess the ability of an exemplary fusion protein to induce CD3 T cell stimulation in the presence of human antigen presenting cells naturally expressing PD-L1.
  • MLR mixed lymphocyte reaction
  • MoDC were obtained as in Example 15. Total CD3+ cells were obtained from PBMC using a Miltenyi kit and used fresh. 10.000 moDCs were cultured in presence of 50.000 CD4 + T cells in U bottom 96 wells in complete RPMI medium, in the presence of tested molecules for 6 days in RPMI in triplicate wells. At the end of the culture, supernatants were immediately frozen and stored for IL-2, IFNg, IL-13, Granzyme A, Granzyme B and FasL secretion. Secreted factors were quantified in supernatants using Muminex technology.
  • the fusion protein of SEQ ID NOs: 82 and 79 showed stimulation of interferon gamma (IFNg) secretion, similar to the antibody of SEQ ID NOs: 78 and 79 ( Figure 13 A). However, the fusion protein of SEQ ID Nos: 82 and 79 showed a stronger induction of T cell effector cytokines, such as IL-13 and IL-2, as compared to the antibody of SEQ ID NOs: 78 and 79 ( Figure 13 B-C).
  • IFNg interferon gamma
  • the fusion protein of SEQ ID NOs: 82 and 79 also showed stronger stimulation of Granzyme A, Granzyme B and soluble Fas ligand secretion as compared to the antibody of SEQ ID NOs: 78 and 79 ( Figure 13 D-F) indicating that the tested construct stimulated secretion of cytotoxic factors from human primary T cells.
  • Example 17 Pharmacokinetics of fusion proteins in mice
  • test articles were administered as a bolus using a volume of 5 mL/kg.
  • Plasma samples from the mice were obtained at the timepoints of 5 min, 24 h, 168 h and 336 h.
  • Sufficient whole blood - taken under isoflurane anesthesia - was collected to obtain at least 30 ⁇ L Li- Heparin plasma per animal and time.
  • Drug levels were detected using a Sandwich ECL assay detecting the full bispecific construct via the target PD-L1 and the lipocalin mutein scaffold or via the target OX40 and the human IgG.
  • the anti-PD-L1 antibody used as reference was detected via the target PD-L1 and anti-human IgG.
  • the data were fited using a two-compartmental model using Phoenix WinNonlin software (Certara).
  • Figure 14 shows plots of the plasma concentration over time for the fusion proteins SEQ ID NOs: 78 and 79, SEQ ID NOs: 88 and 79, SEQ ID NOs: 89 and 79, SEQ ID NOs: 82 and 79, SEQ ID NOs: 93 and 90, SEQ ID NOs: 91 and 94, ploted together with the values obtained for the building block PD-L1 antibody (SEQ ID NOs: 78 and 79) as a reference.
  • the pharmacokinetic behavior of the bispecific fusion proteins looked similar to that of the anti-PD-L1 antibody.
  • the reference bispecific (SEQ ID NOs: 43 and 44) showed a similar profile to the other fusion proteins until 168 h followed by a drop in exposure probably due to the formation of anti-drug antibodies.
  • the terminal half-lives are summarized in Table 10.
  • Table 10 Terminal half-lives in mice determined using a non-compartmental analysis. Terminal half-life of the reference bispecific is probably influenced by anti-drug antibodies.
  • Example 18 Pharmacokinetics of fusion proteins in cynomolgus monkeys
  • Presence of anti-drug antibody was determined by a Sandwich ELISA, coating the drug itself (5 ⁇ g/mL) onto a microtiter plate, incubating it with 1 :100 diluted monkey plasma samples in PBS-0.1%T-2%BSA and detecting via HRP labeled cyno antibodies (Jackson) that bound to the coated drug using an anti-cyno IgG antibody. Signals higher than 2-fold over background (pre-dose plasma samples of each animal) were defined as anti-drug antibody positive (Table 11).
  • Figure 15 shows semi-logarithmic plots of the cynomolgus monkeys plasma concentration over time for the fusion proteins SEQ ID NOs: 82 and 79, SEQ ID NOs: 78 and 87, and SEQ ID NOs: 88 and 79 and PD-L1 antibody SEQ ID NOs: 78 and 79, Anti-drug antibody responses (indicated as dotted lines in Figure 15) were detected for all fusion protein as well as for PD-L1 antibody starting 4 to 11 days after administration.
  • Cynomolgus monkey’s drug plasma concentration with excluded anti-drug antibody positive time points were fitted using a non-compartmental analysis (NCA) model using Phoenix WinNonlin software (Certara) and NCA parameter are presented in (Table 11). Determination of total drug exposure (AUCINFobs), clearance (Cl obs ) and volume of distribution (Vz obs ) depends for most fusion proteins (except for SEQ ID NOs: 78 and 87) and PD-L1 antibody to a significant percentage on extrapolation (AUC_%Extrap_obs) from the last ADA negative timepoint to infinity.
  • NCA data imply that the fusion of the OX40-binding anticalin moiety to PD-L1 antibody SEQ ID NOs: 78 and 79 only moderately influences half live (t 1/2 ) and clearance (Cl obs ) for SEQ ID NOs: 82 and 79 and SEQ ID NOs: 88 and 79 (approximately 70% similarity to building block antibody).
  • T m s melting temperatures
  • Table 12 T m and onset melting temperature as determined by nanoDSC. The major thermal transitions are in marked as bold.
  • Example 20 Stability assessment of fusion proteins
  • exemplary fusion proteins SEQ ID NOs: 78 and 87, SEQ ID NOs: 88 and 79, SEQ ID NOs: 89 and 79, and SEQ ID NOs: 82 and 79
  • Monomeric fusion proteins were subsequently determined using analytical size exclusion by applying 2 pg of sample onto a Zenix C SEC-300, 4.6x150mm (Sepax) column (with a precolumn) at a flow rate of 0.35 mL/min and 150mM sodium phosphate pH 7,0 as running buffer.
  • Example 21 Evaluation of pl and charge of fusion proteins
  • Example 22 Assessment of activity in a Treg cell suppression assay
  • Treg suppression in vitro assay using human primary CD4 T cells (herein defined as Tresponder, Tresp) and primary in vitro induced Treg (iTreg), was used to assess the ability of fusion proteins to release the suppression that iTreg exert on Tresp. Fusion proteins, not in solution but bound to a plastic plate to provide crosslinking, were applied at fixed concentration to a co-culture of iTreg and Tresp. Proliferation of Tresp cells and expression of the surface activation marker CD25 were measured on Tresp cells in the co- culture.
  • PBMC from healthy volunteer donors were isolated from buffy coats.
  • Naive T cells were isolated from fresh healthy donor PBMC from buffy coats by negative selection using immunomagnetic beads. A fraction of naive T cells was frozen as a source of Tresp.
  • naive T cells were stimulated with anti-CD3/anti-CD28 Dynabeads in the presence of IL-2 and TGF- ⁇ 1 for six days to generate iTreg cells.
  • naive Tresp were thawed and labelled with CFSE.
  • CellTraceTM Violet labelled iTreg and anti-CD3/anti-CD28 stimulated Tresp were co-cultured at different ratios for a further four days.
  • Cells were cultured in 96-well round-bottom plates, pre-coated with a fixed dose (200 nM) of fusion protein (SEQ ID NOs: 82 and 79), an Fc fusion of an OX40-specific lipocalin mutein (SEQ ID NO: 42), an anti-PD-L1 antibody (SEQ ID NOs: 78 and 79) or an hlgG4 isotype control antibody (SEQ ID NOs: 21 and 22), respectively. Plates were covered with a gas permeable seal and incubated at 37°C in a humidified 5% CO 2 atmosphere for 4 days.
  • a statistically significant difference was detected between the fusion protein (SEQ ID NOs: 82 and 79) vs the hlgG4 isotype control at the iTreg vs Tresp ratios of 1:1 , 1 :2, 1:4, 1 :8 and 1:16.
  • Treatment with a fixed dose of the Fc fusion of the OX40-specific lipocalin mutein (SEQ ID NO: 42), crosslinked to the plastic plate, led to a significant decrease in suppression activity of iTreg on Tresp, measured as percentage of suppression compared to control (stimulated Tresp alone), using N 6 independent PBMC donors.
  • the treatment with a fixed dose of the anti-PD-L1 antibody (SEQ ID NOs: 78 and 79) did not lead to any significant difference in suppression activity of Treg, measured as Tresp cell proliferation, compared to the negative control hlgG4 isotype control.
  • Figure 18B displays that treatment with the fusion protein (SEQ ID NOs: 82 and 79) led to increased CD25 expression, a marker of T cell activation, on Tresp. This increase was statistically significantly different vs the hlgG4 isotype control treatment at the iTreg vs Tresp ratios of 1:4, 1 :8, 1 :16 and 1:32. *p ⁇ 0.5, **p ⁇ 0.01, ***p ⁇ 0.001 , ****p ⁇ 0.0001 statistical analyses were performed using a repeated-measures two-way ANOVA with Dunnet’s multiple comparisons test comparing the fusion protein (SEQ ID NOs: 82 and 79) to hlgG4 isotype.
  • the treatment with a fixed dose of the anti-PD-L1 antibody did not lead to any significant difference in suppression activity of Treg, measured as Tresp cell proliferation, compared to the negative control hlgG4 isotype.
  • the fusion protein (SEQ ID NOs: 82 and 79) and the Fc fusion of the OX40- specific lipocalin mutein showed stronger ability to overcome the suppression of Treg cells, as compared to the anti-PD-L1 antibody ( Figure 18 A, B) indicating that the tested constructs dampen the suppression of Treg cells on Tresp cells.
  • Example 23 Evaluation of anti-tumor effects in an MC38 h-OX40 knock- in mouse model
  • a fusion protein comprising the OX40-specific lipocalin mutein of SEQ ID NO: 36 and the mouse cross- reactive anti-PD-L1 antibody of SEQ ID NOs: 80 and 81 (SEQ ID NOs: 101 and 102), the anti-PD-L1 antibody (SEQ ID NOs: 80 and 81) or PBS were intraperitoneally injected into the mice on a triweekly basis for 7 injections.
  • An anti-OX40 antibody (SEQ ID NOs: 25 and 26) was intravenously injected into the mice on a triweekly basis for 7 injections.
  • the fusion protein (SEQ ID NOs: 101 and 102) slows down tumor growth to a significantly higher degree than the anti-PD-L1 antibody, as can be seen in below Table 14 showing the tumor growth inhibition rate 12 days after the first dose.
  • treatment with the fusion protein resulted in a significant decrease of the volume tumor compared to the anti-PD-L1 antibody (SEQ ID NOs: 80 and 81) and the anti-OX40 antibody (SEQ ID NOs: 25 and 26).
  • TGI Tumor growth inhibition
  • Example 24 Evaluation of anti-KLH IgG and IgM production in a KLH model
  • mice Human OX40 was knocked into C57BL/6 mice to produce humanized knock- in mice (Biocytogen). Based on weight, mice were randomized (15 mice per group). KLH (ThermoFischer) emulsified in TiterMax Gold Adjuvant (Sigma-Aldrich) was subcutaneously injected at day 0 and day 7.
  • a fusion protein comprising the OX40-specific lipocalin mutein of SEQ ID NO: 36 and the mouse cross-reactive anti-PD-L1 antibody of SEQ ID NOs: 80 and 81 (SEQ ID NOs: 101 and 102) at 50 mg/kg, the anti-PD-L1 antibody (SEQ ID NOs: 80 and 81) at 38.5 mg/kg (equimolar dose of the fusion protein) or PBS were intraperitoneally injected into the mice on a triweekly basis for a total of 7 injections. Blood sampling was performed at day minus 4 before KLH injection, day 7 and day 14 post KLH injection, and plasma was collected. Anti-KLH IgG and IgM quantification was performed with the ELISA assay IgG and IgM kits from Life Diagnostics according to the manufacturer’s recommendation.
  • Embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.
  • the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation.
  • the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.
  • ARASANZ Hlock GATO-CANAS, Mlitis ZUAZO, M., IBANEZ-VEA, Mlitis BRECKPOT, K., KOCHAN, G. & ESCORS, D. 2017. PD1 signal transduction pathways in T cells. Oncotarget, 8, 51936-51945.
  • CD4 T cell cytokine differentiation the B cell activation molecule, 0X40 ligand, instructs CD4 T cells to express interleukin 4 and upregulates expression of the chemokine receptor, Blr-1. J Exp Med, 188, 297-304.
  • ROGERS P. R complicating SONG, J., GRAMAGLIA, I., KILLEEN, N. & CROFT, M. 2001.
  • 0X40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells. Immunity, 15, 445-55.
  • MORRIS, E. S., MACDONALD, K. P., KUNS, R. D minister MORRIS, H. M compact BANOVIC, T., DON, A. L., ROWE, V., WILSON, Y. A., RAFFELT, N. C., ENGWERDA, C. R., BURMAN, A. C Manual MARKEY, K. A., GODFREY, D. I., SMYTH, M. J. & HILL, G. R. 2009. Induction of natural killer T cell-dependent alloreactivity by administration of granulocyte colony-stimulating factor after bone marrow transplantation. Nat Med, 15, 436-41.
  • VETTO J. T., LUM, S., MORRIS, A., SICOTTE, M., DAVIS, J., LEMON, M. & WEINBERG, A. 1997.
  • T-cell activation marker OX-40 Presence of the T-cell activation marker OX-40 on tumor infiltrating lymphocytes and draining lymph node cells from patients with melanoma and head and neck cancers. Am J Surg, 174, 258-65.
  • HIGGINS L M exclusively MCDONALD, S. A., WHITTLE, N., CROCKETT, N., SHIELDS, J. G. & MACDONALD, T. T. 1999. Regulation of T cell activation in vitro and in vivo by targeting the 0X40-0X40 ligand interaction: amelioration of ongoing inflammatory bowel disease with an OX40-lgG fusion protein, but not with an 0X40 ligand-IgG fusion protein. J Immunol, 162, 486-93.
  • PAKALA S. V.
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  • Blockade of CD134 (OX40)-CD134L interaction ameliorates lethal acute graft-versus-host disease in a murine model of allogeneic bone marrow transplantation. Blood, 95, 2434-9.
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  • GLAESNER W., VICK, A. M., MILLICAN, R., ELLIS, B., TSCHANG, S. H., TIAN, Y., BOKVIST, K., BRENNER,

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Abstract

La divulgation concerne des protéines de fusion spécifiques à la fois à OX40 et à PD-L1, lesquelles protéines de fusion peuvent être utilisées pour co-stimuler l'activation des lymphocytes d'une manière dépendant de la cible PD-L1. De telles protéines de fusion peuvent être utilisées dans de nombreuses applications pharmaceutiques, par exemple, en tant qu'agents anticancéreux et/ou modulateurs immunitaires. La présente divulgation concerne également des procédés de fabrication des protéines de fusion selon l'invention ainsi que des compositions comprenant lesdites protéines de fusion. La présente divulgation concerne en outre des molécules d'acide nucléique codant pour de telles protéines de fusion. En outre, la demande divulgue des utilisations thérapeutiques et/ou diagnostiques de ces protéines de fusion.
PCT/EP2022/082368 2021-11-19 2022-11-18 Nouvelle protéine de fusion spécifique à ox40 et pd-l1 Ceased WO2023089079A1 (fr)

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