A novel fusion protein specific for CD137 and CD228

Abstract

The present disclosure provides antibodies or antigen-binding domains thereof specific for CD228, and fusion proteins specific for both CD137 and CD228, which can be used to costimulate lymphocyte activation in a CD228 target-dependent manner. Such antibodies, antigen-binding domains, or fusion proteins can be used for a number of pharmaceutical applications, such as anti-cancer and / or immunomodulatory agents. The present disclosure also relates to methods of making the antibodies, antigen-binding domains, or fusion proteins described herein, and compositions comprising such antibodies, antigen-binding domains, or fusion proteins. The present disclosure further relates to nucleic acid molecules encoding such antibodies, antigen-binding domains, or fusion proteins. Furthermore, the present application discloses therapeutic and / or diagnostic uses of such antibodies, antigen-binding domains, or fusion proteins.

Description

Related Applications

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 408,634, filed September 21, 2022, U.S. Provisional Patent Application No. 63 / 413,174, filed October 4, 2022, and U.S. Provisional Patent Application No. 63 / 496,463, filed April 17, 2023, each of which is incorporated by reference in its entirety into this specification for all purposes. [Background technology]

[0002] II. Introduction and Overview Cluster of differentiation 228 or CD228 (also known as melanotransferrin, MELTF, p97, and MFI2) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein belonging to the transferrin family of iron-binding proteins, and was first described as an oncofetal protein highly expressed in malignant melanoma cells (Rose et al., Proc Natl Acad Sci USA, 1986).

[0003] CD228 is expressed in a variety of cancers, including melanoma, mesothelioma, thyroid cancer, lung cancer, liver cancer, pancreatic cancer, head and neck cancer, gastric cancer, colorectal cancer, urothelial cancer, breast cancer, and cervical cancer. Melanoma, also known as malignant melanoma, is a type of cancer that develops from melanocytes, pigment-containing cells. It is the most dangerous type of skin cancer. In 2015, 3.1 million people had active disease, and 59,800 died from melanoma. While surgery can be effective for early-stage melanoma, it may not be a treatment option for disease that has metastasized to distant organs. Metastatic melanoma often spreads to lymph nodes in the area before spreading to other sites. Surgical removal of lymph nodes has attempted to improve survival, but has been associated with numerous complications and no benefit to overall survival. Immunotherapy, chemotherapy, and radiation therapy have all been used but are often incurable, especially for late-stage melanoma. Distant metastasis generally renders the cancer incurable. The five-year survival rate for stage IV disease is 15-20%.

[0004] CD137 (also known as 4-1BB and TNFRSF9) is a costimulatory immune receptor and a member of the tumor necrosis factor receptor (TNFR) superfamily. It is primarily expressed on activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells, but is also found on resting monocytes and dendritic cells (Li and Liu, Clin Pharmacol, 2013) or endothelial cells (Snell et al., Immunol Rev, 2011). CD137 plays an important role in regulating immune responses and is therefore a target for cancer immunotherapy. CD137 ligand (CD137L), the only known natural ligand for CD137, is constitutively expressed on several types of antigen-presenting cells, such as activated B cells, monocytes, and splenic dendritic cells, and can be directed to T lymphocytes.

[0005] CD137L is a trimeric protein that exists as a membrane-bound and soluble variant. However, the ability of soluble CD137L to activate CD137, for example, on CD137-expressing lymphocytes, is limited, and high concentrations are required to induce the effect (Wyzgol et al., J Immunol, 2009). The natural method of CD137 activation is through engagement of CD137-positive cells with CD137L-positive cells. CD137 activation is then induced by CD137L-mediated clustering on the target cell, leading to signaling via TRAF1, 2, and 3 (Yao et al., Nat Rev Drug Discov, 2013; Snell et al., Immunol Rev, 2011), and subsequent downstream effects in CD137-positive T cells. For T cells activated by recognition of their cognate targets, the effects induced by CD137 costimulation are further activation, enhanced survival and proliferation, production of inflammatory cytokines, and enhanced killing capacity. Summary of the Invention

[0006]

[0006] The present disclosure provides, inter alia, novel antibodies and their antigen-binding domains that bind to CD228, along with novel approaches for simultaneously engaging CD137 and CD228 via one or more fusion proteins having binding specificity for CD137 and binding specificity for CD228.

[0007] III. Definition The following list defines terms, phrases, and abbreviations used throughout this specification. All terms listed and defined herein are intended to encompass all grammatical forms.

[0008]

[0008] As used herein, unless otherwise specified, "CD137" refers to human CD137 (huCD137). Human CD137 refers to the full-length protein, fragments thereof, or variants thereof as defined by UniProt Q07011. CD137 is also known as 4-1BB, tumor necrosis factor receptor superfamily member 9 (TNFRSF9), and is induced by lymphocyte activation (ILA). In certain embodiments, CD137 from a non-human species, such as cynomolgus monkey CD137 or mouse CD137, is used.

[0009]

[0009] As used herein, unless otherwise specified, "CD228" refers to human CD228. Human CD228 refers to the full-length protein, its mature form, its isoform, its fragment, or its variant as defined by UniProt P08582. Human CD228 is encoded by the MELTF gene. CD228 is also known as melanotransferrin, MELTF, p97, and MFI2, and these terms may be used interchangeably herein. In certain embodiments, CD228 from a non-human species, such as cynomolgus monkey CD228 or mouse CD228, is used.

[0010] As used herein, "binding affinity" refers to the ability of a biomolecule (e.g., a polypeptide or protein) of the present disclosure (e.g., a lipocalin mutein, an antibody, an antigen-binding domain thereof, a fusion protein, or any other peptide or protein) to bind to (and form a complex with) a selected target. Binding affinity is measured by several methods known to those skilled in the art, including, but not limited to, fluorescence titration, enzyme-linked immunosorbent assay (ELISA)-based assays (including direct ELISA and competitive ELISA), calorimetric methods such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). These methods are well established in the art, and some examples of such methods are further described herein. Binding affinity is thereby determined by the dissociation constant (K D ), half-maximum effective concentration (EC 50 ), or half-maximal inhibitory concentration (IC 50 ) value is reported as K D , E.C. 50 , or IC 50 A lower value of K indicates better (higher) binding ability (affinity). Thus, the binding affinity of two biomolecules for a selected target can be measured and compared. When comparing the binding affinity of two biomolecules for a selected target, the terms "comparable to," "about the same," "substantially the same," or "substantially similar" mean that one biomolecule has a K that is identical or similar to that of another molecule, within the experimental variability of binding affinity measurements. D , E.C. 50 , or IC 50 "Comparable to," "about the same," "substantially the same," or "substantially similar" refers to a value that is within 50% deviation, more preferably within 20%, and most preferably within 10% deviation from a given reference value. Experimental variability in binding affinity measurements depends on the particular method used and is known to those skilled in the art.

[0011] As used herein, the term "substantially" can also refer to the qualitative condition of exhibiting a total degree or near total degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical phenomena.

[0012] As used herein, the terms "detect," "detection," "detectable," or "detecting" are understood at both the quantitative and qualitative levels, and combinations thereof. Thus, this includes quantitative, semi-quantitative, and qualitative measurements made on the biomolecules of the present disclosure.

[0013] As used herein, "detectable affinity" generally refers to a D , E.C. 50 , or IC 50 The binding affinity between a biomolecule and its target, as reported by a value of up to about 10 -5 This means that the value is less than or equal to M. 10 -5 K is higher than M D , E.C. 50 , or IC 50 The binding affinity reported in values ​​is generally no longer measurable by common methods such as ELISA and SPR and is therefore of little importance. Therefore, a "detectable affinity" is defined as a value of about 10 as determined by ELISA or SPR, preferably SPR. -5 K below M D It may point to a value.

[0014] As used herein, "specific for," "specific binding," "specifically binds," or "binding specificity" refers to the ability of a biomolecule to distinguish between a desired target (e.g., CD137 and CD228) and one or more reference targets (e.g., the cellular receptor for neutrophil gelatinase-associated lipocalin). It is understood that such specificity is a relative, rather than absolute, property and can be determined, for example, by SPR, Western blot, ELISA, fluorescence-activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), immunohistochemistry (IHC), and peptide scanning.

[0015] As used herein in the context of a biomolecule, such as an antibody, antigen-binding domain thereof, or fusion protein of the present disclosure, that binds to CD137 and / or CD228, the terms "specific for," "specific binding," "specifically binds," or "binding specificity" mean that the biomolecule binds to, reacts with, or is directed against CD137 and / or CD228 as described herein, but does not substantially bind to another protein. The term "another protein" includes any protein that is not CD137 or CD228 and is not closely related to or homologous to CD137 or CD228. However, CD137 or CD228 from species other than human, as well as fragments and / or variants of CD137 or CD228, are not excluded by the term "another protein." The term "does not substantially bind" means that a biomolecule of the present disclosure binds to another protein with a lower binding affinity than CD137 and / or CD228, i.e., exhibits a cross-reactivity of less than 30%, preferably less than 20%, more preferably less than 10%, and particularly preferably less than 9, 8, 7, 6, or 5%. Whether a biomolecule specifically reacts as defined above can be easily tested, in particular, by comparing the reaction of a biomolecule of the present disclosure with CD137 and / or CD228 with the reaction of said biomolecule with (another) other protein(s).

[0016] As used herein, the term "lipocalin" refers to a monomeric protein weighing approximately 18-20 kDa, comprising multiple β-strands (preferably eight β-strands designated A-H) connected in pairs at one end by multiple (preferably four) loops, thereby constituting a ligand-binding pocket, and having a cylindrical β-pleated sheet supersecondary structure region that defines the entrance to the ligand-binding pocket. Preferably, the loops constituting the ligand-binding pocket used in this disclosure are those connecting the open ends of β-strands A and B, C and D, E and F, and G and H, designated loops AB, CD, EF, and GH. The diversity of these loops in the otherwise rigid lipocalin scaffold allows for a variety of different binding modes among members of the lipocalin family, allowing for targets of various sizes, shapes, and chemical characteristics to be accommodated (e.g., as reviewed in Skerra, Biochim Biophys Acta, 2000; Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996). Lipocalin family proteins have naturally evolved to bind to a wide range of ligands, and although they share an unusually low level of overall sequence conservation (often less than 20% sequence identity), their overall folding patterns are highly conserved. The positional correspondences in various lipocalins are also well known to those skilled in the art (see, e.g., U.S. Patent No. 7,250,297). Proteins within the definition of "lipocalin," as used herein, include, but are not limited to, tear lipocalin (Tlc, Lcn1), lipocalin-2 (Lcn2) or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D (ApoD), apolipoprotein M, α1-acid glycoprotein 1, α1-acid glycoprotein 2, α1-microglobulin, complement component 8γ, retinol-binding protein (RBP), epididymal retinoic acid-binding protein, glycodelin, odorant-binding protein IIa, odorant-binding protein IIb, lipocalin-15 (Lcn15), and prostaglandin D synthase.

[0017]

[0017] As used herein, unless otherwise specified, "tear lipocalin" refers to human tear lipocalin (hTlc) and further refers to mature human tear lipocalin. When used to characterize a protein, the term "mature" means a protein essentially free of a signal peptide. "Mature hTlc" in the present disclosure refers to the mature form of human tear lipocalin that does not contain a signal peptide. Mature hTlc is described by residues 19-176 of the sequence deposited in the SWISS-PROT Data Bank under accession number P31025, and its amino acid sequence is set forth in SEQ ID NO: 1.

[0018] As used herein, "lipocalin-2" or "neutrophil gelatinase-associated lipocalin" refers to human lipocalin-2 (hLcn2) or human neutrophil gelatinase-associated lipocalin (hNGAL), and further refers to mature human lipocalin-2 or mature human neutrophil gelatinase-associated lipocalin. When used to characterize a protein, the term "mature" refers to a protein essentially free of a signal peptide. "Mature hNGAL" in the present disclosure refers to the mature form of human neutrophil gelatinase-associated lipocalin without a signal peptide. Mature hNGAL is described by residues 21-198 of the sequence deposited in the SWISS-PROT Data Bank under accession number P80188, and its amino acid sequence is set forth in SEQ ID NO:2.

[0019] As used herein, "native sequence" refers to a protein or polypeptide having a naturally occurring or wild-type sequence, regardless of its mode of preparation. Such native sequence proteins or polypeptides can be isolated from nature or produced by other means, such as recombinant or synthetic methods.

[0020]

[0020] "Native sequence lipocalin" refers to a lipocalin having the same amino acid sequence as the corresponding polypeptide from nature. Thus, a native sequence lipocalin can have the amino acid sequence of each naturally occurring (wild-type) lipocalin from any organism, particularly mammals. When used in the context of lipocalin, the term "native sequence" specifically encompasses naturally occurring truncated or secreted forms of lipocalin, naturally occurring variant forms of lipocalin, such as alternatively spliced ​​forms and naturally occurring allelic variants. The terms "native sequence lipocalin" and "wild-type lipocalin" are used interchangeably herein.

[0021] As used herein, "mutein," "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 a naturally occurring (wild-type) protein or nucleic acid. The terms also include fragments of the muteins described herein. The present disclosure expressly encompasses lipocalin muteins (also referred to as Anticalin® proteins), as described herein, which comprise eight beta strands connected in pairs at one end by four loops, thereby constituting a ligand-binding pocket, and a cylindrical beta-pleated sheet supersecondary structure region defining an entrance to the ligand-binding pocket, in which at least one amino acid located within the four loops is mutated compared to native sequence lipocalin. The lipocalin muteins of the present disclosure preferably have the function of binding to CD137 as described herein.

[0022] As used herein, the term "fragment," in reference to the lipocalin muteins of the present disclosure, refers to a protein or polypeptide derived from full-length mature hTlc or hNGAL or a lipocalin mutein that has been truncated at the N-terminus and / or C-terminus, i.e., lacking at least one amino acid at the N-terminus and / or C-terminus. Such a fragment may contain at least 10 or more, e.g., 20 or 30 or more contiguous amino acids, of the primary sequence of mature hTlc or hNGAL or the lipocalin mutein from which it is derived, and is typically detectable in an immunoassay for mature hTlc or 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- and / or C-terminal amino acids. As illustrative examples, such fragments may lack one, two, three, or four N-terminal (His-His-Leu-Leu) and / or one or two C-terminal amino acids (Ser-Asp) of mature hTlc. Fragments are understood to mean functional fragments of mature hTlc or hNGAL, or the lipocalin mutein from which they are derived, and preferably retain the binding specificity of mature hTlc / hNGAL or the lipocalin mutein from which they are derived, preferably for CD137. As illustrative examples, such functional fragments may include at least amino acids 5-153, 5-150, 9-148, 12-140, 20-135, or 26-133 corresponding to the linear polypeptide sequence of mature hTlc. As another illustrative example, such functional fragments may include at least amino acids 13-157, 15-150, 18-141, 20-134, 25-134, or 28-134 corresponding to the linear polypeptide sequence of mature hNGAL.

[0023] A "fragment," with respect to an antibody, its antigen-binding domain, or the corresponding target CD137 or CD228 of a fusion protein of the present disclosure, refers to CD137 or CD228 truncated at the N-terminus and / or C-terminus, or a protein domain of CD137 or CD228. The fragments of CD137 or CD228 described herein retain the ability of full-length CD137 or CD228 to be recognized and / or bound by an antibody, its antigen-binding domain, or a fusion protein of the present disclosure. As an illustrative example, a fragment may be the extracellular domain of CD137. For example, such an extracellular domain of CD137 may include amino acids of extracellular subdomains of CD137, such as the individual or combined amino acid sequences of domain 1 (residues 24-45 of UniProt Q07011), domain 2 (residues 46-86), domain 3 (residues 87-118), and domain 4 (residues 119-159).

[0024]

[0024] As used herein, the term "variant" refers to a derivative of a protein or polypeptide that includes mutations, for example, by substitution, deletion, insertion, and / or chemical modification of the amino acid 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 include substitutions between 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. Such variants include proteins or polypeptides in which one or more amino acids are substituted by their respective D-stereoisomers, or by amino acids other than the 20 naturally occurring amino acids, such as ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline, etc. Such variants also include proteins or polypeptides in which one or more amino acid residues are added or deleted, for example, at the N-terminus and / or C-terminus. Generally, variants have at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or at least about 98% amino acid sequence identity with a native sequence protein or polypeptide. Variants preferably retain the biological activity of the protein or polypeptide from which they are derived, e.g., biological activity of binding to the same target.

[0025]

[0025] The term "variant", as used herein with respect to CD137 or CD228, relates to a fragment having one or more, for example 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 compared to CD137 or CD228 or the native sequence of CD137 or CD228 (wild-type CD137 or CD228), such as CD137 deposited at UniProt Q07011 or CD228 deposited at UniProt P08582, respectively, as described herein. Preferably, a CD137 or CD228 variant has at least 50%, 60%, 70%, 80%, 85%, 90%, or 95% amino acid identity with wild-type CD137 or CD228, respectively. The CD137 or CD228 variants described herein retain the ability to bind to CD137- and / or CD228-specific antibodies, antigen-binding domains thereof, lipocalin muteins, or fusion proteins disclosed herein.

[0026] The term "variant," as used herein with respect to a lipocalin mutein, refers to a lipocalin mutein of the present disclosure or a fragment thereof, the sequence of which has mutations, including substitutions, deletions, insertions, and / or chemical modifications. A variant of a lipocalin mutein, as used herein, retains the biological activity of the lipocalin mutein from which it is derived, e.g., the biological activity of binding to CD137. Generally, a lipocalin mutein variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 98% amino acid sequence identity with the lipocalin mutein from which it is derived.

[0027]

[0027] As used herein with respect to an antibody or antigen-binding domain thereof, the term "variant" refers to an antibody or antigen-binding domain thereof of the present disclosure in which the sequence has mutations, including substitutions, deletions, insertions, and / or chemical modifications. A variant of an antibody or antigen-binding domain thereof described herein retains the biological activity of the antibody or antigen-binding domain thereof from which it is derived, e.g., the biological activity of binding to CD228. Generally, a variant of an antibody or antigen-binding domain thereof has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 98% amino acid sequence identity with the antibody or antigen-binding domain thereof from which it is derived.

[0028] As used herein, the term "mutagenesis" refers to the introduction of mutations into a polynucleotide or amino acid sequence. Mutations are preferably introduced under experimental conditions such that a naturally occurring amino acid at a given position in a protein or polypeptide sequence can be altered, e.g., substituted with at least one amino acid. The term "mutagenesis" also includes the (additional) modification of the length of a sequence segment by deletion or insertion of one or more amino acids. Thus, for example, it is within the scope of the present disclosure to replace one amino acid at a selected sequence position with a stretch of three amino acids, adding two amino acid residues compared to the length of the respective segment of the amino acid sequence of a naturally occurring protein or polypeptide. Such insertions or deletions can be introduced independently of each other in any of the sequence segments that can be subjected to mutagenesis in the present disclosure. In one exemplary embodiment of the present disclosure, an insertion can be introduced into the amino acid sequence segment corresponding to loop AB of a native sequence lipocalin (see International Patent Publication No. WO 2005 / 019256, incorporated herein by reference in its entirety).

[0029]

[0029] As used herein, the term "random mutagenesis" means that there are no predetermined mutations (amino acid changes) at a particular sequence position, but that at least two amino acids can be incorporated at a given sequence position with a certain probability during mutagenesis.

[0030]

[0030] As used herein, the term "sequence identity" or "identity" refers to a property of sequences that measures their similarity or relationship. As used in this disclosure, the term "sequence identity" or "identity" refers to the percentage of a pair of identical residues relative to the number of residues in the longer of the two sequences, after (homologous) alignment of a protein or polypeptide of the disclosure with the sequence in question. 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.

[0031]

[0031] As used herein, the terms "sequence homology" or "homology" have their ordinary meaning, and homologous amino acids include identical amino acids and amino acids that are considered conservative substitutions at equivalent positions in the linear amino acid sequence of a protein or polypeptide of the present disclosure (e.g., any fusion protein or lipocalin mutein of the present disclosure).

[0032] Those skilled in the art will be aware of available computer programs, such as BLAST (Altschul et al., Nucleic Acids Res, 1997), BLAST2 (Altschul et al., J Mol Biol, 1990), and Smith-Waterman (Smith and Waterman, J Mol Biol, 1981), for determining sequence homology or sequence identity using standard parameters. Percentage of sequence homology or sequence identity may be determined herein, for example, using the program BLASTP, version 2.2.5 (November 16, 2002) (Altschul et al., Nucleic Acids Res, 1997). In some embodiments, the percentage of homology is determined based on an alignment of the entire protein or polypeptide sequence, including the propeptide sequence, preferably using the wild-type protein scaffold as a reference in pairwise comparisons (matrix: BLOSUM 62; gap cost: 11.1; cutoff value: 10 -3 It is calculated as a percentage of the number of "positives" (homologous amino acids) resulting in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0033]

[0033] Specifically, to determine whether the amino acid sequence of a lipocalin (mutein) differs from that of a reference (wild-type) lipocalin with respect to a particular position in the amino acid sequence of the reference (wild-type) lipocalin, one skilled in the art can use means and methods well known in the art, for example, alignment, either manually or using a computer program such as BLAST 2.0 (which stands for Basic Local Alignment Search Tool), or ClustalW, or any other suitable program suitable for generating sequence alignments. Thus, the amino acid sequence of the reference (wild-type) lipocalin can serve as the "subject sequence" or "reference sequence," while the amino acid sequence of the lipocalin mutein serves as the "query sequence." The terms "wild-type sequence," "reference sequence," and "subject sequence" are used interchangeably herein. A preferred wild-type sequence of lipocalin is the sequence of hTLc shown in SEQ ID NO: 1 or hNGAL shown in SEQ ID NO: 2.

[0034]

[0034] A "gap" is a space in an alignment that results from the addition or deletion of amino acids. Thus, two copies of the exact same sequence will have 100% identity, but sequences that are less well conserved and have deletions, additions, or substitutions may have a lower degree of sequence identity.

[0035] As used herein, the term "position" refers to either the position of an amino acid within an amino acid sequence disclosed herein or the position of a nucleotide within a nucleic acid sequence disclosed herein. When the terms "correspond" or "corresponding" are used herein in the context of an amino acid sequence position of one or more lipocalin muteins, it is understood that the corresponding position is not determined solely by the number of preceding nucleotides or amino acids. Thus, the absolute position of a given amino acid according to the present disclosure may differ from the corresponding position due to deletions or additions of amino acids elsewhere in the (mutant or wild-type) lipocalin. Similarly, the absolute position of a given nucleotide according to the present disclosure may differ from the corresponding position due to deletions or additional nucleotides elsewhere in the 5'-untranslated region (UTR) of a mutein or wild-type lipocalin, including the promoter and / or any other regulatory sequences or gene regions (including exons and introns).

[0036]

[0036] A "corresponding position" according to the present disclosure may be a sequence position that aligns to a corresponding sequence position in a pairwise or multiple sequence alignment according to the present disclosure. Preferably, for a "corresponding position" according to the present disclosure, the nucleotide or amino acid at the absolute position may be different from the adjacent nucleotide or amino acid, but it should be understood that said adjacent nucleotide or amino acid, which may be exchanged, deleted, or added, may be included in the same "corresponding position(s)."

[0037]

[0037] Additionally, with respect to corresponding positions of a lipocalin mutein based on a reference sequence according to the present disclosure, it is preferred to understand that the nucleotide or amino acid positions of the lipocalin mutein may structurally correspond to other positions of the reference lipocalin (wild-type lipocalin) or another lipocalin mutein, even if the absolute position numbers are different, as will be understood by those skilled in the art in view of the highly conserved overall folding patterns among lipocalins.

[0038]

[0038] As used interchangeably herein, the terms "conjugate," "conjugation," "fuse," "fusion," or "linked" refer to the joining of two or more subunits to one another through all forms of covalent or non-covalent bonding, including, but not limited to, genetic fusion, chemical conjugation, bonding via a linker or cross-linking agent, and non-covalent association.

[0039] The terms "fusion polypeptide" or "fusion protein," as used herein, refer to a polypeptide or protein comprising two or more subunits. In some embodiments, the fusion proteins described herein comprise two or more subunits, at least one of which is capable of specifically binding to CD137 and an additional subunit is capable of specifically binding to CD228. Within the fusion protein, the subunits may be linked by covalent or noncovalent bonds. Preferably, the fusion protein is a translational fusion between two or more subunits. A translational fusion may be generated by genetically engineering the coding sequence of one subunit in the reading frame with the coding sequence of an additional subunit. A nucleotide sequence encoding a linker may be interposed between the subunits. However, the subunits of the fusion proteins of the present disclosure may also be linked by chemical conjugation. The subunits forming a fusion protein are typically linked to each other as follows: the C-terminus of one subunit to the N-terminus of another subunit, or the C-terminus of one subunit to the N-terminus of another subunit, or the N-terminus of one subunit to the N-terminus of another subunit, or the N-terminus of one subunit to the C-terminus of another subunit. The subunits of a fusion protein may be linked in any order and may contain two or more of any of the constituent subunits. When one or more subunits are part of a protein (complex) consisting of two or more polypeptide chains, the term "fusion protein" can refer to the protein containing the fusion sequence and all other polypeptide chains of the protein (complex). As an illustrative example, when a full-length immunoglobulin / antibody is fused to a lipocalin mutein via the heavy or light chain of the immunoglobulin / antibody, the term "fusion protein" can refer to a single polypeptide chain comprising the lipocalin mutein and the heavy or light chain of the immunoglobulin / antibody. The term "fusion protein" can refer to an entire immunoglobulin / antibody (both light and heavy chains), as well as to a lipocalin mutein fused to one or both of the heavy and / or light chains.

[0040] As used herein, the term "subunit" of the fusion proteins disclosed herein refers to a single protein or separate polypeptide chains that can form a stable folded structure by itself and define a unique function that provides a binding motif for a target. In some embodiments, a preferred subunit of the present disclosure is a lipocalin mutein. In some other embodiments, a preferred subunit of the present disclosure is an antibody, e.g., a full-length antibody, or an antigen-binding domain / fragment thereof.

[0041] A "linker," which may be comprised by the fusion proteins of the present disclosure, joins two or more subunits of the fusion proteins described herein. The linkage may 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. Thus, in preferred embodiments, the linker comprises one or more amino acids, e.g., 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, including glycine-serine (GS) linkers, glycosylated GS linkers, and proline-alanine-serine polymer (PAS) linkers, are described herein. In some preferred embodiments, a GS linker, such as the (G4S)3 linker set forth in SEQ ID NO: 13, is used to join the subunits of the fusion protein together. Other preferred linkers include chemical linkers.

[0042]

[0042] As used herein, the term "albumin" includes all mammalian albumins, such as human serum albumin or bovine serum albumin or rat serum albumin.

[0043]

[0043] As used herein, the terms "organic molecule" or "small organic molecule" refer to an organic molecule containing at least two carbon atoms, preferably no more than seven or twelve rotatable carbon bonds, having a molecular weight in the range of 100 to 2,000 daltons, preferably 100 to 1,000 daltons, and optionally containing one or two metal atoms.

[0044]

[0044] A "sample" is defined as a biological sample obtained from a subject, including, but not limited to, blood, serum, urine, feces, semen, or tissue, including tumor tissue.

[0045]

[0045] 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, but not limited to, humans, domestic and farm animals, as well as zoo, sport, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, and apes, such as cynomolgus monkeys, to name a few illustrative examples. Preferably, a "mammal" as used herein is a human.

[0046] An "effective amount" is an amount sufficient to bring about beneficial or desired results. An effective amount can be administered in one or more individual administrations or doses.

[0047] As used herein, "antibody" includes whole antibodies or any antigen-binding fragments thereof (i.e., "antigen-binding portion" or "antigen-binding domain") or single chains. The terms "antibody" and "immunoglobulin" can be and are used interchangeably herein. A whole antibody refers to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain / region (V H or HCVR) and heavy chain constant region (C H The heavy chain constant region is composed of C H1 , C H2 and C H3Each light chain consists of a light chain variable domain / region (V L or LCVR) and the light chain constant region (C L The light chain constant region consists of one domain, C L The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen (e.g., CD228). The constant region of the antibody can optionally mediate the binding of the immunoglobulin 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.

[0047]

[0048] As used herein, an "antigen-binding fragment" (also referred to as an "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., CD228). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding domain" of an antibody include: (i) V H , V L , C L and C H1 (ii) a Fab fragment consisting of two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a V H , V L , C L and C H1 Domain and C H1 Domain and C H2 (iv) Fab' fragment consisting of the region between the V H and C H1 (v) a single arm V of an antibody; Hand V L (vi) a single-chain Fv fragment consisting of a V H (vii) isolated complementarity-determining regions (CDRs) or combinations of two or more isolated CDRs that can optionally be joined by synthetic linkers; (viii) V domains connected within the same polypeptide chain using short linkers. H and V L (ix) "diabodies" including V H or V L "Domain antibody fragments" (in some cases, two or more V H domains are covalently bonded together).

[0048]

[0049] Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic, or syngenic; or modified forms thereof (e.g., humanized, chimeric, or multispecific). Antibodies can also be fully human.

[0049]

[0050] As used herein, "framework" or "FR" refers to variable domain residues other than hypervariable region (CDR) residues.

[0051] "Fragment crystallizable region" or "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is usually defined to extend from the amino acid residue at Cys226, or from Pro230, to the carboxyl terminus, as numbered according to the EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). The C-terminal lysine of the Fc region (residue 447 according to the EU index of Kabat) can be removed, for example, during antibody production or purification or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Native sequence Fc regions suitable for use in the antibodies of the disclosure include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0050]

[0052] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody.

[0053] As used herein, "isolated antibody" refers to an antibody that is substantially free from its natural environment. For example, an isolated antibody is substantially free from cellular material and other proteins from the cell or tissue source from which it is derived. "Isolated antibody" also refers to an antibody that is substantially free from other antibodies with different antigen specificities. In this case, an isolated antibody that specifically binds to CD228 is substantially free from antibodies that specifically bind to antigens other than CD228. However, an isolated antibody that specifically binds to CD228 may have cross-reactivity with other antigens, such as CD228 molecules from other species.

[0051]

[0054] As used herein, "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition, which display a single binding specificity and affinity for a particular epitope.

[0052]

[0055] As used herein, a "humanized antibody" refers to an antibody consisting of CDRs from an antibody derived from a non-human mammal and a human antibody or FR and constant regions derived from a human antibody. A humanized antibody may contain variable domains that, when analyzed as a whole, have a variable region amino acid sequence closer to that of humans than to that of other species, as assessed using the DomainGapAlign tool of the Immunogenetics Information System (IMGT) described in Ehrenmann et al. (2010). Because humanized antibodies have reduced antigenicity, they may be useful as active components of therapeutic agents. The terms "therapeutic agent" or "therapeutically active agent" as used herein refer to a therapeutically useful agent. A therapeutic agent may be any agent for the prevention, amelioration, or treatment of, or the evaluation or diagnosis of, a disease, physiological condition, or symptom.

[0053]

[0056] As used herein, a "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 also is derived from human germline immunoglobulin sequences. The human antibodies of the present 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). However, 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.

[0054]

[0057] As used herein, "antibody clone X" may also be referred to as "OMTX." For example, "antibody clone 30" may also be referred to as "OMT30." Antibody clone 8 may also be referred to as "EE03"; antibody clone 24 may also be referred to as "EB03"; antibody clone 30 may also be referred to as "OC04"; antibody clone 35 may also be referred to as "OB02"; and antibody clone 36 may also be referred to as "OE12."

[0055]

[0058] As used herein, "AAFX" refers to a fusion protein containing antibody clone X and a lipocalin mutein (lipocalin mutein I) having the amino acid sequence of SEQ ID NO: 40. For example, "AAF30" refers to a fusion protein containing antibody clone 30 and lipocalin mutein I. The designation "HC" in the name of a fusion protein indicates that the lipocalin mutein therein is conjugated to the antibody via the antibody's heavy chain, and the designation "LC" in the name of a fusion protein indicates that the lipocalin mutein therein is conjugated to the antibody via the antibody's light chain. A fusion protein name followed by "HC" or "LC" after the clone number refers to a fusion protein containing that antibody clone and lipocalin mutein I conjugated via the antibody's heavy chain or light chain, respectively. For example, "30HC" refers to a fusion protein containing antibody clone 30 and lipocalin mutein I conjugated thereto via the antibody's heavy chain.

[0056]

[0059] The antibody or fusion protein may also be designated by the sequences of its heavy and light chains (e.g., a fusion protein having the sequences of SEQ ID NOs: 80 and 76), one of which (e.g., SEQ ID NO: 80) contains the sequence of a lipocalin mutein in the case of the fusion protein.

[0057] IV. Figure Description [Brief explanation of the drawings]

[0058] [Figure 1]

[0060] FIG. 1 provides a schematic diagram showing CD137 clustering by cross-linking CD137-positive T cells with CD228-expressing tumor cells using the fusion proteins provided herein. [Figure 2A]

[0061] 2A and 2B are tables showing the CDR sequences of certain antibodies provided herein. [Figure 2B] 2A and 2B are tables showing the CDR sequences of certain antibodies provided herein. [Figure 3]

[0062] FIG. 1 shows the results of an ELISA demonstrating that anti-CD228 antibodies bind to recombinant human CD228. [Figure 4-1]

[0063] Figures 4A-4C show the binding of anti-CD228 antibodies to CD228-expressing cells as measured by fluorescence intensity, and Figure 4D is a table showing the EC50 values ​​of each antibody for each cell line. [Figure 4-2] Same as description for Figure 4-1. [Figure 5A]

[0064] 5A-5E show the results of a biolayer inferometry (BLI) assay of the binding kinetics and affinity of anti-CD228 antibodies to CD228. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 6A]

[0065] 6A-6C show the results of binding assays of anti-CD228 antibodies to CD228 and certain other transferrin family members. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 7A]

[0066] 7A to 7B and 8A to 8C show the results of binding assays of anti-CD228 antibodies to human, mouse, and cynomolgus monkey CD228. [Figure 7B] Same as description for Figure 7A. [Figure 8A] Same as description for Figure 7A. [Figure 8B] Same as description for Figure 7A. [Figure 8C] Same as description for Figure 7A. [Figure 9]

[0067] 1 is a table showing the results of a cross-competition assay between various anti-CD228 antibodies. [Figure 10]

[0068] FIG. 1 shows the results of an antibody internalization assay using a CD228+ tumor cell line. [Figure 11-1]

[0069] Figure 11A provides an overview of the design of a representative fusion protein described in the present application that is bispecific for targets CD137 and CD228. Figure 11B provides an overview of the design of a representative fusion protein described in the present application that is bispecific for targets CD137 and CD228. Figure 11C provides an overview of the design of a representative fusion protein described in the present application that is bispecific for targets CD137 and CD228. Figure 11D provides an overview of the design of a representative fusion protein described in the present application that is bispecific for targets CD137 and CD228. Figure 11E provides an overview of the design of a representative fusion protein described in the present application that is bispecific for targets CD137 and CD228. Figure 11F provides an overview of the design of a representative fusion protein described in the present application that is bispecific for targets CD137 and CD228. Figure 11G provides an overview of the design of a representative fusion protein described in the present application that is bispecific for targets CD137 and CD228. Figure 11H provides an overview of the design of a representative fusion protein described herein that is bispecific for the targets CD137 and CD228. Figure 11I provides an overview of the design of a representative fusion protein described herein that is bispecific for the targets CD137 and CD228. The representative fusion protein was generated based on a CD228-specific antibody and one or more lipocalin muteins specific for CD137. One or more lipocalin muteins were genetically fused to the C-terminus and / or N-terminus of the heavy and / or light chain of the CD228-specific antibody, as shown in Figures 11A-11I. The generated fusion protein can be bivalent for CD137 (e.g., as shown in Figures 11A-11D) or tetravalent for CD137 (e.g., as shown in Figures 11E-11H), or can have even higher valency for CD137 (e.g., as shown in Figure 11I). [Figure 11-2] Same as above. [Figure 12A]

[0070] Figures 12A-12D show the results of ELISA experiments in which the binding of representative fusion proteins to human or cynomolgus monkey CD228 (Figures 12A-12B) or CD137 (Figures 12C-12D) was determined as described in Example 11. CD228 or CD137 (with a C-terminal His- or Fc-tag) was coated onto microtiter plates, and test agents were titrated starting at a top concentration of 200 nM. Bound agents were detected with anti-human IgG Fab-HRP. Data were fit using a 4PL fit with the EC50 value and maximum signal as free parameters and a slope fixed at 1. The resulting EC50 values ​​are provided in Table 4. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 13]

[0071]

[0033] Figure 1 shows the results of an ELISA experiment that determined the ability of a representative fusion protein to simultaneously bind both targets, CD228 and CD137, as described in Example 12. Recombinant huCD228-His was coated onto a microtiter plate, and then the fusion protein and a control anti-CD228 antibody were titrated, starting at a highest concentration of 200 nM. A fixed concentration of biotinylated huCD137-His was then added and detected with ExtrAvidin-Peroxidase. Data were fit using a 4PL fit with the EC50 value and maximum signal as free parameters and a slope fixed at 1. The resulting EC50 values ​​are provided in Table 5. [Figure 14]

[0072] FIG. 10 shows further results of an ELISA experiment that determined the ability of representative fusion proteins to simultaneously bind both targets, CD228 and CD137, as described in Example 12. [Figure 15A]

[0073] Figures 15A-15B show the results of flow cytometry (FACS) evaluation of target binding of fusion proteins and respective controls using CD228-positive human SH-4 cells (Figure 15A) and Flp-In-CHO cells expressing human CD137 (Figure 15B) as described in Example 13. The geometric mean of fluorescence intensity was used to calculate EC50 values ​​using nonlinear regression (shared base, SLOPE=1). The EC50 values ​​are provided in Table 6. [Figure 15B] Same as above. [Figure 16A]

[0074] Figures 16A-16E are diagrams and tables showing the potential of representative fusion proteins to costimulate T cell activation in a CD228 target-dependent manner, as assessed by a CD137 bioassay, as described in Example 14. NFκB-luc2 / CD137 Jurkat cells, tumor cells expressing high (SH-4) (Figure 16A), intermediate (A375) (Figure 16B), or low (A549) (Figure 16C) levels of CD228, were cocultured with CD228-negative RPMI-7951 tumor cells (Figure 16D) in the presence of various concentrations of fusion protein or control. After 4 hours, luciferase assay reagent was added, and luminescence signals were measured. To calculate EC50 values, a four-parameter logistic curve analysis was performed using GraphPad Prism® (see the table in Figure 16E). [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above. [Figure 16E] Same as above. [Figure 17A]

[0075] 17A-17E show the results of an antigen recall assay in which fusion proteins costimulate innate and adaptive immune cytokines from peripheral blood mononuclear cells (PBMCs) in response to viral peptides. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 17E] Same as above. [Figure 18]

[0076] 18, 19, 20, and 21 show the results of antigen recall assays in which fusion proteins costimulate T cell and NK cell responses in response to viral peptides. [Figure 19] FIG. 1 shows the results of an antigen recall assay in which fusion proteins costimulate T cell and NK cell responses in response to viral peptides. [Figure 20] FIG. 1 shows the results of an antigen recall assay in which fusion proteins costimulate T cell and NK cell responses in response to viral peptides. [Figure 21] FIG. 1 shows the results of an antigen recall assay in which fusion proteins costimulate T cell and NK cell responses in response to viral peptides. [Figure 22A]

[0077] 22A-22E show the results of an antigen recall assay in which fusion proteins costimulate cytotoxic effector molecules and cytokines from PBMCs in response to viral peptides. [Figure 22B] Same as above. [Figure 22C] Same as above. [Figure 22D] Same as above. [Figure 22E] Same as above. [Figure 23A]

[0078] Figures 23A-23D show the ability of representative fusion proteins to costimulate T cell activation in a CD228 target-dependent manner. Various tumor cell lines expressing different CD228 levels—SH-4 (CD228-high) (Figure 23A), Calu-1 (CD228-high) (Figure 23B), SK-MEL-24 (CD228-high) (Figure 23C), and RPMI-7951 (CD228-negative) (Figure 23D)—were seeded onto anti-human CD3-coated plates. Pan T cells and various concentrations of fusion proteins or controls were added and incubated for 3 days. Secreted IL-2 levels were used as a readout and were determined by an electrochemiluminescence-based assay, as described in Example 18. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above. [Figure 24A] Similarly, Figures 24A-24B show the ability of additional representative fusion proteins to costimulate T cell activation in a CD228 target-dependent manner. [Figure 24B] Same as above. [Figure 25A]

[0079] 25A-25C show T cell cytokine production in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 26]

[0080] FIG. 1 shows the proliferation of CD8 T cells in co-culture with anti-CD3 scFv-engineered CD228-expressing tumor cell lines. [Figure 27] FIG. 27 shows tumor cell killing in co-culture. [Figure 28A]

[0081] 28A-28D show evaluation of storage stability of fusion proteins and controls in PBS, 50% human plasma (HPL), or 50% mouse plasma (MPL) after 1 week of incubation at 37° C., as described in Example 21. Stability was assessed by CD137 reporter cell assay, as described in Example 14. [Figure 28B] Same as above. [Figure 28C] Same as above. [Figure 28D] Same as above. [Figure 29]

[0082] Figure 29A provides the results of pharmacokinetic analysis of the bispecific fusion protein and parent anti-CD228 antibody in mice, as described in Example 22. Figure 29B provides the results of pharmacokinetic analysis of the bispecific fusion protein and parent anti-CD228 antibody in mice, as described in Example 22. Male CD-1 nude mice (3 per time point) were intravenously injected with the fusion protein at a dose of 10 mg / kg. Drug levels were detected using a sandwich ELISA that detects whole molecules with their targets, CD228 and biotinylated CD137. Plasma levels of the anti-CD228 antibody were determined using an ELISA with the target CD228 and an anti-human IgG Fc antibody. Absolute concentrations are shown in Figure 29A, and values ​​normalized to Cmax are shown in Figure 29B. [Figure 30]

[0083] FIG. 1 is a diagram and table showing the pharmacokinetics of fusion proteins in cynomolgus monkeys. [Figure 31A]

[0084] 31A-31D, 32A-32C, and 33A-33B show the in vivo activity of the fusion proteins in a humanized xenograft model. [Figure 31B] Same as above. [Figure 31C] Same as above. [Figure 31D] Same as above. [Figure 32A] Same as above. Same as description for Figure 31A. [Figure 32B] Same as above. Same as description for Figure 31A. [Figure 32C] Same as above. Same as description for Figure 31A. [Figure 33] Same as above. Same as description for Figure 31A. [Figure 34]

[0085] Figures 34A and 34B show the proliferation of CD8 T cells in co-culture with anti-CD3 scFv-engineered CD228-expressing tumor cell lines. [Figure 35A]

[0086] FIG. 35A shows the number of CD8 T cells in treated versus untreated wells. [Figure 35B] FIG. 35B shows the mitochondrial content of CD8 T cells expressed as mean fluorescence intensity (MFI) of MitoSpy™ green FM staining. [Figure 35C] FIG. 35C shows the percentage of CD8 T cells with depolarized mitochondria as determined by staining with MitoSpy™ orange CMTMros and MitoSpy™ green FM. [Figure 36A]

[0087] FIG. 36A shows a schematic diagram of generating functionally exhausted cytotoxic T cells. [Figure 36B] Figure 36B shows the percentage of dividing cells (left), IFN-γ levels (center), and number of surviving tumor cells (right) for T cells at different passages (P0 to P4) restimulated with anti-CD3 scFv-engineered CALU-1 cells. [Figure 37A]

[0088] FIG. 37A shows a heatmap of exhausted CD8+ T cells from FIG. 36A or exhausted CD8+ T cells from the pan-cancer tumor-infiltrating lymphocyte (TIL) atlas. [Figure 37B] Figure 37B shows the expression levels of TCF7 (TCF1) (left), HAVCR2 (TIM-3) (center), and TNFRSF9 (CD137) (right) in two groups of exhausted CD8+ T cells. [Figure 38]

[0089] Figure 1 shows the fold increase in the number of carboxyfluorescein succinimidyl ester (CFSE)-low (dividing) CD8 T cells in treated versus untreated wells. DETAILED DESCRIPTION OF THE INVENTION

[0059] V. Detailed Description of the Disclosure

[0090] As described herein, in one aspect, the present disclosure provides antibodies or antigen-binding domains thereof that bind to CD228. Such anti-CD228 antibodies can be used as antibody therapeutics themselves, conjugated to therapeutic agents to form antibody-drug conjugates, included as part of bispecific or multispecific antibodies, or included as part of fusion molecules. In certain embodiments, such fusion molecules include those having binding domains that bind to targets other than CD228. For example, as described in more detail below, in some embodiments, the fusion molecule is a fusion protein that can bind to both CD137 and CD228.

[0060]

[0091] The specific fusion proteins provided herein that target both CD137 and CD228 were designed based on the inventors' recognition of several factors. For example, with regard to CD137, it was recognized that CD137 is expressed on multiple immune cell types and that its engagement enhances innate and adaptive immunity. It was also recognized that CD137 agonism can restore the function of exhausted T cells, thereby reactivating their existing anti-tumor activity. In the case of CD228, the inventors recognized that it could be an excellent antigen target for tumor-targeting anti-CD137 bispecific molecules for several reasons. First, CD228 expression is highly restricted to tumors, thereby minimizing the risk of toxicity. Second, combining two agents with different mechanisms of action may provide broader coverage and provide opportunities for further combination therapies.

[0061]

[0092] Furthermore, the fusion proteins provided herein that target both CD137 and CD228 are based on the recognition that bivalent CD137-binding agents, such as antibodies, may not be sufficient by themselves to cluster CD137 on T cells or NK cells and lead to efficient activation, similar to the lack of activity of trivalent soluble CD137L. A second problem observed with bivalent anti-CD137 antibodies is that active doses can sometimes induce on-target liver toxicity, indicating the need for a more specific, tumor-targeted mechanism of action.

[0062]

[0093] Based in part on these various insights, the present disclosure provides, inter alia, a novel approach to simultaneously engage CD137 and CD228 using one or more fusion proteins having binding specificity for CD137 and CD228. The binding specificity for CD228 can be provided by a CD228-binding antibody or its antigen-binding domain, as provided herein. The provided fusion proteins are designed to promote CD137 clustering by crosslinking CD137-positive T cells with CD228-expressing tumor cells present in the tumor microenvironment, as shown in the exemplary Figure 1. Thus, the fusion proteins are designed to locally induce antigen-specific T cells in the tumor microenvironment, potentially reducing peripheral toxicity. This is in contrast to, for example, urelumab, a 4-1BB agonist that is active in solid tumors but induces on-target liver toxicity at active doses.

[0063]

[0094] In some aspects, the present disclosure provides antibodies or antigen-binding domains thereof that bind to CD228, and fusion proteins that bind to CD137 and CD228, as well as methods and useful applications thereof. The present disclosure also provides methods for producing the CD228-binding antibodies or antigen-binding domains thereof and fusion proteins that bind to CD137 and CD228 described herein, and compositions comprising such proteins. The CD228-binding antibodies or antigen-binding domains thereof and fusion proteins that bind to CD137 and CD228 of the present disclosure, and compositions thereof, can be used in methods for detecting CD137 and / or CD228 in a sample, binding CD137 and / or CD228 in a subject, or modulating an immune response in a subject. Such antibodies, antigen-binding domains thereof, or fusion proteins with these characteristics associated with the uses provided by the present disclosure have not previously been described.

[0064] A. Exemplary Antibodies or Antigen-Binding Domains Specific for CD228

[0095] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 (e.g., a standalone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) is provided, wherein the antibody or antigen-binding domain thereof comprises: i) a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 110, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 112, and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 116, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 118; ii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 113, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 115, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 119, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 120, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 121; iii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 130, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 131, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 132, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 136, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 137, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 138; iv) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 133, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 134, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 135, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 139, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 140, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 141; v) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 150, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 152, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 156, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 157, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 158; vi) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 153, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 154, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 155, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 159, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 160, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 161; vii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 170, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 171, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 172, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; viii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 173, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 179, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 180, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 181; ix) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 190, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 191, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 192, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 196, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 197, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 198; x) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 193, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 194, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 195, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 199, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 200, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 201; xi) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; xii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221; xiii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 230, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 231, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 232, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 236, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 237, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 238; xiv) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 233, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 234, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 235, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 239, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 240, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 241; xv) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 252, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 256, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 257, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 258; xvi) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 259, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 260, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 261; xvii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 270, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 271, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 272, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 276, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 277, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 278; or xviii) A VH comprising (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 273, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 274, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 275, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 279, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 280, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 281.

[0065]

[0096] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises: i) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; ii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221; iii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 252, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 256, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 257, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 258; or iv) A VH comprising (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 253, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 254, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 259, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 260, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 261.

[0066]

[0097] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises: (a) a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216. , (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 217, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 218; or a VH having (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 213, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 214, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 215, and a VL having (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 219, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 220, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 221.

[0067]

[0098] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises: i) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 122, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 124; ii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 142, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 144; iii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 162, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 164; iv) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 182, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 184; v) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 202, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 204; vi) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 224; vii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 242, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 244; viii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 264; or ix) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 282, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 284.

[0068]

[0099] In some embodiments, an antibody or antigen-binding domain thereof that binds CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:222, and a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99% sequence identity to SEQ ID NO:224. or a VH comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:262, and a VL comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:264.

[0069]

[0100] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 224.

[0070]

[0101] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises: i) a VH comprising the amino acid sequence of SEQ ID NO: 122, and a VL comprising the amino acid sequence of SEQ ID NO: 124; ii) a VH comprising the amino acid sequence of SEQ ID NO: 142, and a VL comprising the amino acid sequence of SEQ ID NO: 144; iii) a VH comprising the amino acid sequence of SEQ ID NO: 162, and a VL comprising the amino acid sequence of SEQ ID NO: 164; iv) a VH comprising the amino acid sequence of SEQ ID NO: 182, and a VL comprising the amino acid sequence of SEQ ID NO: 184; v) a VH comprising the amino acid sequence of SEQ ID NO: 202, and a VL comprising the amino acid sequence of SEQ ID NO: 204; vi) a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224; vii) a VH comprising the amino acid sequence of SEQ ID NO: 242, and a VL comprising the amino acid sequence of SEQ ID NO: 244; viii) a VH comprising the amino acid sequence of SEQ ID NO: 262, and a VL comprising the amino acid sequence of SEQ ID NO: 264; or ix) A VH comprising the amino acid sequence of SEQ ID NO: 282, and a VL comprising the amino acid sequence of SEQ ID NO: 284.

[0071]

[0102] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224; or a VH comprising the amino acid sequence of SEQ ID NO: 262, and a VL comprising the amino acid sequence of SEQ ID NO: 264.

[0072]

[0103] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224.

[0073]

[0104] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises: i) a heavy chain (HC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 126, and a light chain (LC) comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 128; ii) an HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 146, and an LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 148; iii) an HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 166, and an LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 168; iv) an HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 186, and an LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 188; v) an HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 206, and an LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 208; vi) an HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 226, and an LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 228; vii) an HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 246, and an LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 248; viii) an HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 266, and an LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 268; or ix) an HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 286, and an LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 288.

[0074]

[0105] In some embodiments, an antibody or antigen-binding domain thereof that binds CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises an HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:226, and at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99% sequence identity to SEQ ID NO:228. or an LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:266, and an LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:268.

[0075]

[0106] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises an HC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:226, and an LC comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:228.

[0076]

[0107] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises: i) an HC comprising the amino acid sequence of SEQ ID NO: 126, and an LC comprising the amino acid sequence of SEQ ID NO: 128; ii) an HC comprising the amino acid sequence of SEQ ID NO: 146, and an LC comprising the amino acid sequence of SEQ ID NO: 148; iii) a HC comprising the amino acid sequence of SEQ ID NO: 166, and a LC comprising the amino acid sequence of SEQ ID NO: 168; iv) a HC comprising the amino acid sequence of SEQ ID NO: 186, and a LC comprising the amino acid sequence of SEQ ID NO: 188; v) a HC comprising the amino acid sequence of SEQ ID NO: 206, and a LC comprising the amino acid sequence of SEQ ID NO: 208; vi) an HC comprising the amino acid sequence of SEQ ID NO: 226, and an LC comprising the amino acid sequence of SEQ ID NO: 228; vii) an HC comprising the amino acid sequence of SEQ ID NO: 246, and an LC comprising the amino acid sequence of SEQ ID NO: 248; viii) a HC comprising the amino acid sequence of SEQ ID NO: 266, and a LC comprising the amino acid sequence of SEQ ID NO: 268; or ix) A HC comprising the amino acid sequence of SEQ ID NO: 286, and a LC comprising the amino acid sequence of SEQ ID NO: 288.

[0077]

[0108] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises an HC comprising the amino acid sequence of SEQ ID NO: 226, and an LC comprising the amino acid sequence of SEQ ID NO: 228; or an HC comprising the amino acid sequence of SEQ ID NO: 266, and an LC comprising the amino acid sequence of SEQ ID NO: 268.

[0078]

[0109] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) comprises an HC comprising the amino acid sequence of SEQ ID NO: 226, and an LC comprising the amino acid sequence of SEQ ID NO: 228.

[0079]

[0110] In some embodiments, the antibodies that bind to CD228 provided herein (e.g., independent antibodies or antibodies contained in any one of the fusion proteins disclosed herein) are monoclonal antibodies. In some embodiments, the antibodies that bind to CD228 provided herein are humanized or chimeric antibodies. In some embodiments, the antibodies that bind to CD228 provided herein are IgG1, IgG2, IgG3, or IgG4 antibodies.

[0080]

[0111] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a standalone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) has a K D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 150 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 100 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 50 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 75 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 25 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 20 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 15 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 10 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 5 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 4 nM or less. DIn some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 3 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 2.5 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 2 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 1.5 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein bind to CD228 with a K value of 1 nM or less. D Binds to CD228 at high levels.

[0081]

[0112] In some embodiments, an antibody or antigen-binding domain thereof that binds CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) binds to cynomolgus monkey CD228. In some such embodiments, an antibody or antigen-binding domain thereof that binds CD228 provided herein comprises: i) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 170, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 171, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 172, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; ii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 173, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 179, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 180, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 181; iii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; iv) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221; v) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 252, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 256, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 257, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 258; or vi) A VH comprising (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 253, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 254, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 259, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 260, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 261.

[0082] In some such embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein comprises a set of CDRs as described above, as well as: i) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 182, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 184; ii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 224; or iii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 264. Includes.

[0083] In some such embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 182 and a VL comprising the amino acid sequence of SEQ ID NO: 184; a VH comprising the amino acid sequence of SEQ ID NO: 222 and a VL comprising the amino acid sequence of SEQ ID NO: 224; or a VH comprising the amino acid sequence of SEQ ID NO: 262 and a VL comprising the amino acid sequence of SEQ ID NO: 264.

[0084]

[0113] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein (e.g., a stand-alone antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) does not bind to mouse CD228 or binds to mouse CD228 with an affinity that is 100-fold or more reduced compared to human CD228. In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein does not bind to transferrin or lactotransferrin. In some such embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein comprises: i) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 170, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 171, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 172, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 176, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 177, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 178; ii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 173, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 179, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 180, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 181; iii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 190, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 191, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 192, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 196, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 197, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 198; iv) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 193, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 194, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 195, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 199, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 200, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 201; v) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 218; vi) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 221; vii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 230, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 231, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 232, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 236, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 237, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 238; viii) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 233, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 234, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 235, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 239, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 240, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 241; ix) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 250, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 251, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 252, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 256, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 257, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 258; x) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 259, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 260, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 261; xi) a VH comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 270, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 271, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 272, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 276, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 277, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 278; or xii) A VH comprising (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 273, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 274, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 275, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 279, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 280, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 281.

[0085] In some such embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein comprises a set of CDRs as described above, as well as: i) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 182, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 184; ii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 202, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 204; iii) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 224; iv) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 242; and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 244; or v) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 264; or vi) a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 282, and a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 284. Includes.

[0086] In some such embodiments, an antibody or antigen-binding domain thereof that binds to CD228 provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 182 and a VL comprising the amino acid sequence of SEQ ID NO: 184; a VH comprising the amino acid sequence of SEQ ID NO: 202 and a VL comprising the amino acid sequence of SEQ ID NO: 204; a VH comprising the amino acid sequence of SEQ ID NO: 222 and a VL comprising the amino acid sequence of SEQ ID NO: 224; a VH comprising the amino acid sequence of SEQ ID NO: 242 and a VL comprising the amino acid sequence of SEQ ID NO: 244; a VH comprising the amino acid sequence of SEQ ID NO: 262 and a VL comprising the amino acid sequence of SEQ ID NO: 264; or a VH comprising the amino acid sequence of SEQ ID NO: 282 and a VL comprising the amino acid sequence of SEQ ID NO: 284.

[0087]

[0114] In some embodiments, an antibody or antigen-binding domain thereof that binds to CD228 (e.g., a separate antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof comprised in any one of the fusion proteins disclosed herein) is provided, wherein the antibody or antigen-binding domain thereof competes for binding to CD228 with any one of the antibodies or antigen-binding domains thereof disclosed herein. In some such embodiments, the antibody is a monoclonal antibody. In some such embodiments, the antibody is a humanized antibody or a chimeric antibody. In some such embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some such embodiments, the antibody or antigen-binding domain thereof has a K DIn some such embodiments, the antibody or antigen-binding domain thereof binds to cynomolgus monkey CD228. In some such embodiments, the antibody or antigen-binding domain thereof does not bind to mouse CD228 or binds to mouse CD228 with an affinity that is 100-fold or more reduced compared to human CD228. In some such embodiments, the antibody or antigen-binding domain thereof does not bind to transferrin or lactotransferrin. In some such embodiments, the antibody or antigen-binding domain thereof competes for binding to CD228 with an antibody or antigen-binding domain thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 122 and a VL comprising the amino acid sequence of SEQ ID NO: 124; a VH comprising the amino acid sequence of SEQ ID NO: 142 and a VL comprising the amino acid sequence of SEQ ID NO: 144; a VH comprising the amino acid sequence of SEQ ID NO: 162 and a VL comprising the amino acid sequence of SEQ ID NO: 164; a VH comprising the amino acid sequence of SEQ ID NO: 182 and a VL comprising the amino acid sequence of SEQ ID NO: 184; a VH comprising the amino acid sequence of SEQ ID NO: 202 and a VL comprising the amino acid sequence of SEQ ID NO: 204; a VH comprising the amino acid sequence of SEQ ID NO: 222 and a VL comprising the amino acid sequence of SEQ ID NO: 224; a VH comprising the amino acid sequence of SEQ ID NO: 242 and a VL comprising the amino acid sequence of SEQ ID NO: 244; a VH comprising the amino acid sequence of SEQ ID NO: 262 and a VL comprising the amino acid sequence of SEQ ID NO: 264; or a VH comprising the amino acid sequence of SEQ ID NO: 282 and a VL comprising the amino acid sequence of SEQ ID NO: 284. In some such embodiments, the antibody or antigen-binding domain thereof competes for binding to CD228 with an antibody or antigen-binding domain thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 202 and a VL comprising the amino acid sequence of SEQ ID NO: 204; a VH comprising the amino acid sequence of SEQ ID NO: 242 and a VL comprising the amino acid sequence of SEQ ID NO: 244; or a VH comprising the amino acid sequence of SEQ ID NO: 262 and a VL comprising the amino acid sequence of SEQ ID NO: 264.In some such embodiments, the antibody or antigen-binding domain thereof competes for binding to CD228 with an antibody or antigen-binding domain thereof comprising a VH comprising the amino acid sequence of SEQ ID NO: 202 and a VL comprising the amino acid sequence of SEQ ID NO: 204; or a VH comprising the amino acid sequence of SEQ ID NO: 242 and a VL comprising the amino acid sequence of SEQ ID NO: 244. In some such embodiments, competition for binding to CD228 is measured by flow cytometry using at least one labeled antibody or antigen-binding domain thereof.

[0088]

[0115] In some embodiments, the CDR sequences disclosed herein are defined according to the Kabat numbering scheme as described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. In some embodiments, the CDR sequences disclosed herein are defined according to the IMGT method as described in Lefranc, M.-P., The Immunologist, 7, 132-136 (1999).

[0089]

[0116] The specific binding antibody to CD228 contained in the fusion protein of the present disclosure may comprise an Fc portion that allows for extended in vivo half-life of the bispecific binding molecule of the present disclosure. In some embodiments, such an Fc portion is preferably of human origin, more preferably a human Fc portion of an IgG1 or IgG4 antibody, and even more preferably an engineered human Fc portion of an IgG1 or IgG4 antibody with activating or silencing effector function. In some embodiments, silencing effector function may be preferred over activating effector function. In some embodiments, such an Fc portion is engineered to silence effector function by mutation(s) at positions 234 and / or 235, numbered according to the EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). In some embodiments, mutations at positions F234 and L235 of the provided anti-CD228 antibody may be introduced to silence effector function. In other embodiments, mutations at positions D265 and P329 of the provided anti-CD228 antibodies may be introduced to silence effector function. The numbering of both sets of these potential mutations is according to the EU index of Kabat (Shields et al., J Biol Chem, 2001). In some embodiments, the provided CD228 antibodies have an engineered IgG4 backbone with the mutations S228P, F234A, and L235A.

[0090]

[0117] Various techniques for producing antibodies and their antigen-binding domains are well known in the art and are described, for example, in Altshuler et al. (2010). Thus, for example, polyclonal antibodies can be obtained from animal blood after immunization with an antigen mixed with additives and adjuvants, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous cell line culture. Examples of such techniques are described, for example, in Harlow and Lane (1999), (1988), and include the hybridoma technique first described by Kohler and Milstein, 1975, trioma technique, human B cell hybridoma technique (see, for example, Li et al., Proc Natl Acad Sci USA, 2006; Kozbor and Roder, Immunol Today, 1983), and EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., Cancer Res, 1984). Furthermore, recombinant antibodies can be derived from monoclonal antibodies or prepared de novo using various display methods, such as phage, ribosome, mRNA, or cell display. In some embodiments, suitable systems for expressing recombinant (humanized) antibodies or fragments thereof can be selected from, for example, bacteria, yeast, insect, mammalian cell lines, or transgenic animals or plants (see, e.g., U.S. Pat. No. 6,080,560; Holliger and Hudson, Nat Biotechnol, 2005). Furthermore, techniques described for the production of single-chain antibodies (see, in particular, U.S. Pat. No. 4,946,778) can be adapted to produce single-chain antibodies specific to the targets of the present invention. Surface plasmon resonance, as employed in the BIAcore system, can be used to enhance the efficiency of phage antibodies.

[0091] B. Exemplary Fusion Proteins Specific for CD137 and CD228 of the Disclosure

[0118] In some embodiments, the fusion proteins provided contain at least two subunits, in any order: (1) a first subunit comprising a full-length antibody or antigen-binding domain thereof specific for CD228, and (2) a second subunit comprising a lipocalin mutein specific for CD137. In some embodiments, in the fusion proteins provided herein, the antibody or antigen-binding domain thereof is any one of the antibodies or antigen-binding domains thereof disclosed herein, particularly any one of the antibodies or antigen-binding domains thereof disclosed in Section IV.A, above.

[0092]

[0119] In some embodiments, the provided fusion protein may also contain at least one additional subunit, such as a third subunit. For example, the fusion protein may contain a third subunit specific to CD137. In some embodiments, the third subunit may be or include a lipocalin mutein specific to CD137. For example, two lipocalin muteins may be fused to the first antibody subunit, one at the C-terminus and one at the N-terminus of the antibody. In some embodiments, the lipocalin mutein may be fused to the heavy or light chain of the antibody.

[0093]

[0120] In some embodiments, the provided fusion proteins may include one or more additional subunits (eg, a fourth, fifth, or sixth subunit).

[0121] In some embodiments, at least one subunit may be fused at its N-terminus and / or its C-terminus to another subunit.

[0094]

[0122] In some embodiments, at least one subunit can be linked to another subunit via a linker. In some further embodiments, the linker is a peptide linker, e.g., a glycine-serine (GS) linker, e.g., an unstructured GS linker or a glycosylated GS linker, or a proline-alanine-serine polymer (PAS) linker. In some embodiments, the GS linker is a (Gly4Ser)3 linker ((G4S)3), as shown in SEQ ID NO: 13. Other exemplary linkers are shown in SEQ ID NOs: 14-23. In some embodiments, the peptide linker can have 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. For example, if the first subunit comprises an antibody, such as a full-length antibody, the second subunit may be linked via a peptide linker between the N-terminus of the second subunit and the C-terminus of the heavy chain constant region (CH) of said antibody. In some further embodiments, the third subunit may be linked via a peptide linker between the N-terminus of the third subunit and the C-terminus of the light chain constant region (CL) of said antibody.

[0095]

[0123] In some embodiments, one subunit may be linked to another subunit essentially as described in Figures 11A-11I. Generally, one subunit may be fused to another subunit at its N-terminus and / or C-terminus. For example, in some embodiments, a lipocalin mutein subunit may be fused to an antibody subunit at its N-terminus and / or C-terminus. In a further example, one lipocalin mutein may be linked, preferably via a peptide linker, to the C-terminus of an antibody heavy chain (HC), the N-terminus of the HC, the C-terminus of an antibody light chain (LC), and / or the N-terminus of the LC (Figures 11A-11D).

[0096]

[0124] In some embodiments, the lipocalin mutein subunit may be fused to the antibody fragment at its N-terminus and / or C-terminus, for example, in some embodiments, the lipocalin mutein may be linked to the C-terminus of the heavy chain constant region (CH) or the C-terminus of the light chain constant region (CL) of the antibody, preferably via a peptide linker.

[0097]

[0125] In some embodiments, when one subunit comprises an antibody, such as a full-length antibody, the antibody and the second subunit may be linked between the N-terminus of the second subunit and the C-terminus of the heavy chain constant region (CH) of the antibody.

[0098]

[0126] In some embodiments, the third subunit may be linked via the N-terminus of the third subunit and the C-terminus of the light chain constant region (CL) of the antibody.

[0127] In some embodiments, the fusion protein comprises an antibody, such as a full-length antibody specific for CD228, and two lipocalin muteins specific for CD137, each of which is fused at its N-terminus to the C-terminus of the heavy chain of the antibody via a peptide linker.

[0099]

[0128] In some embodiments, the fusion protein comprises an antibody, such as a full-length antibody specific for CD228, and two lipocalin muteins specific for CD137, each of which is fused at its N-terminus to the C-terminus of the light chain of the antibody via a peptide linker.

[0100]

[0129] In some embodiments, with respect to the fusion proteins of the present disclosure, at least one subunit may be or comprise an antibody, such as a full-length antibody, and the Fc function of the antibody's Fc region on Fc receptor-positive cells may be retained while the fusion protein simultaneously engages CD137 and CD228.

[0101]

[0130] In some embodiments, at least one subunit of the provided fusion protein may be or include an antibody, such as a full-length antibody, and the Fc function of the antibody's Fc region on Fc receptor-positive cells may be reduced or completely suppressed by protein engineering, while the fusion protein simultaneously engages CD137 and CD228. In some embodiments, this may be achieved, for example, by switching from an IgG1 backbone to an IgG4 backbone, since IgG4 is known to exhibit reduced Fc-gamma receptor interaction compared to IgG1. In some embodiments, mutations such as F234A and L235A may be introduced into the IgG4 backbone to further reduce residual binding to Fc-gamma receptors. In some embodiments, a S228P mutation may be introduced into the IgG4 backbone to minimize exchange of IgG4 half-antibodies (Silva et al., J Biol Chem, 2015). In some embodiments, F234A and L235A mutations may be introduced to reduce ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010), and / or M428L and N434S mutations, or M252Y, S254T, and T256E mutations may be introduced to extend serum half-life (Dall'Acqua et al., J Biol Chem, 2006; Zalevsky et al., Nat Biotechnol, 2010). In some embodiments, an additional N297A mutation may be present in the antibody heavy chain of the fusion protein to remove a native glycosylation motif.

[0102]

[0131] In some embodiments, the Fc portion of the antibody contained in the fusion protein of the present disclosure can contribute to maintaining serum levels of the fusion protein. For example, when the Fc portion binds to Fc receptors on endothelial cells and phagocytes, the fusion protein is internalized and recycled into the bloodstream, thereby extending its half-life in the body.

[0103]

[0132] In one aspect, the fusion protein of the present disclosure binds to CD137 with high affinity. In another aspect, the provided fusion protein binds to CD228 with high affinity. In some preferred embodiments, the provided fusion protein simultaneously binds to CD137 and CD228. In some embodiments, the simultaneous binding to CD137 and CD228 allows the provided fusion protein to exhibit durable anti-tumor or anti-infective responses.

[0104]

[0133] In some embodiments, the fusion proteins of the disclosure have a K of up to about 200 nM, up to about 180 nM, up to about 150 nM, up to about 120 nM, up to about 100 nM, up to about 70 nM, or up to about 35 nM or even lower, such as about 30 nM or less, about 25 nM or less, or about 20 nM or less. D In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD228 at a K value of an antibody specific for CD228 or a variant of said antibody, e.g., an antibody having heavy and light chains provided by SEQ ID NOs: 74 and 76 or SEQ ID NOs: 77 and 79, contained in such a fusion protein. D K values ​​equal to or lower than D The provided fusion proteins may be capable of binding to CD228 at a K value. D The value can be measured, for example, in a surface plasmon resonance (SPR) assay, such as the SPR assay essentially as described in Example 10.

[0105]

[0134] In some embodiments, the fusion proteins of the present disclosure are cross-reactive with cynomolgus monkey CD228. In some embodiments, the fusion proteins of the present disclosure have a K of up to about 200 nM, up to about 180 nM, up to about 150 nM, up to about 120 nM, up to about 100 nM, up to about 70 nM, or up to about 35 nM or even lower, such as about 30 nM or less, about 25 nM or less, or about 20 nM or less. D The provided fusion proteins may be capable of binding to cynomolgus monkey CD228 at K values. DThe value can be measured, for example, in a surface plasmon resonance (SPR) assay, such as the SPR assay essentially as described in Example 10.

[0106]

[0135] In some embodiments, the fusion proteins of the disclosure have a K of up to about 10 nM or even lower, e.g., about 7 nM or less, about 6 nM or less, about 5 nM or less, or about 4 nM or less. D In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD137 at a K value of 1000 kJ / s. In some embodiments, the fusion proteins of the present disclosure may comprise a lipocalin mutein specific for CD137 contained in the particular fusion protein, such as SEQ ID NO: 40, or a lipocalin mutein fused to the Fc region of an antibody, such as SEQ ID NO: 84. D K values ​​equal to or lower than D The provided fusion proteins may be capable of binding to CD137 at a K value. D The value can be measured, for example, in an SPR assay such as the SPR assay essentially as described in Example 10.

[0107]

[0136] In some embodiments, the fusion proteins of the disclosure have an EC of up to about 2 nM, up to about 1.5 nM, or up to about 1.2 nM or even lower, such as about 1 nM or less, about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, or about 0.5 nM or less. 50 In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD228 at a K value of an antibody specific for CD228 or a variant of said antibody, e.g., an antibody having heavy and light chains provided by SEQ ID NOs: 74 and 76 or SEQ ID NOs: 77 and 79, contained in such a fusion protein. D EC values ​​equal to or lower than 50 The EC values ​​of the fusion proteins provided may be capable of binding to CD228. 50 The value can be measured, for example, in an enzyme-linked immunosorbent assay (ELISA) assay, such as the ELISA assay essentially as described in Example 11.

[0108]

[0137] In some embodiments, the fusion proteins of the present disclosure are cross-reactive with cynomolgus monkey CD228. In some embodiments, the provided fusion proteins have an EC of up to about 2 nM, up to about 1.5, or up to about 1.2 nM or even lower, such as about 1 nM or less, about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, or about 0.5 nM or less. 50 The EC values ​​of the provided fusion proteins may be capable of binding to cynomolgus monkey CD228. 50 The value can be measured, for example, in an ELISA assay such as the ELISA assay essentially as described in Example 11.

[0109]

[0138] In some embodiments, the fusion proteins of the disclosure have an EC of up to about 6 nM or even lower, such as about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2.5 nM or less, about 2 nM or less, about 1.5 nM or less, about 1 nM or less, or about 0.8 nM or less. 50 In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD137 at a level of 1000 uM. In some embodiments, the fusion proteins of the present disclosure may comprise a lipocalin mutein specific for CD137 contained in the particular fusion protein, such as SEQ ID NO: 40, or a lipocalin mutein fused to the Fc region of an antibody, such as the EC of SEQ ID NO: 84. 50 EC values ​​equal to or lower than 50 The EC values ​​of the provided fusion proteins may be capable of binding to CD137. 50 The value can be measured, for example, in an ELISA assay, such as the ELISA assay essentially as described in Example 11.

[0110]

[0139] In some embodiments, the fusion proteins of the present disclosure are cross-reactive with cynomolgus monkey CD137. In some embodiments, the provided fusion proteins have an EC of up to about 150 nM or even lower, such as about 120 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, or about 30 nM or less. 50The EC values ​​of the provided fusion proteins may be capable of binding to cynomolgus monkey CD137. 50 The value can be measured, for example, in an ELISA assay, such as an ELISA assay essentially as described in Example 11.

[0111]

[0140] In some embodiments, the fusion proteins of the present disclosure may be capable of simultaneously binding to CD137 and CD228. In some embodiments, the provided fusion proteins have an EC of up to about 20 nM, up to about 15 nM, or up to about 10 nM or even lower, such as about 7 nM or less, about 5 nM or less, about 3 nM or less, about 2.5 nM or less, about 2 nM or less, or about 1.5 nM or less. 50 At this value, the antibody may be capable of simultaneously binding to CD137 and CD228. Simultaneous binding may be determined, for example, in an ELISA assay, such as the ELISA assay essentially as described in Example 12.

[0112]

[0141] In some embodiments, the fusion proteins of the disclosure have an EC of up to about 30 nM or even lower, such as about 20 nM or less, about 15 nM or less, about 10 nM or less, about 9 nM or less, about 7 nM or less, about 5 nM or less, about 3 nM or less, about 2.5 nM or less, or about 2 nM or less. 50 The EC values ​​of the provided fusion proteins may be capable of binding to CD137 expressed on cells. 50 The value can be measured, for example, in a flow cytometry analysis essentially as described in Example 13. The CD137-expressing cells can be, for example, CHO cells transfected with human CD137 or cynomolgus CD137.

[0113]

[0142] In some embodiments, the fusion proteins of the disclosure have an EC of up to about 20 nM or even lower, such as about 10 nM or less, about 8 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2.5 nM or less, about 2 nM or less, or about 1.5 nM or less. 50 The EC values ​​of the fusion proteins provided may be capable of binding to CD228 expressed on cells. 50The value can be measured, for example, in a flow cytometry analysis essentially as described in Example 13. The CD228-expressing cell can be, for example, a CD228-expressing tumor cell, such as an SH-4 cell.

[0114]

[0143] In some embodiments, the fusion proteins of the present disclosure may be capable of costimulating T cell proliferation and / or T cell responses. In some embodiments, the provided fusion proteins result in comparable or stronger T cell activation compared to that elicited by an anti-CD137 antibody, such as the reference antibodies of SEQ ID NOs: 26 and 27. Stimulated T cell responses or T cell activation can be measured, for example, in a CD137 bioassay essentially as described in Example 14 or a functional T cell activation assay essentially as described in Example 18.

[0115]

[0144] In some embodiments, the fusion proteins of the present disclosure may be capable of costimulating T cell responses in a CD228-dependent manner. In some embodiments, the provided fusion proteins may result in the local induction of IL-2 production by T cells in the vicinity of CD228-positive cells, such as CD228-transfected cells or CD228-positive tumor cells. As used herein, "in the vicinity of CD228-positive cells" refers to the proximity of T cells and CD228-positive cells due to the provided fusion proteins simultaneously binding to CD137 and CD228. CD228-dependent activation of T cells by the provided fusion proteins can be determined, for example, in a CD137 bioassay essentially as described in Example 14 or a functional T cell activation assay essentially as described in Example 18.

[0116]

[0145] In some preferred embodiments, the provided fusion proteins may be capable of costimulating T cell responses in the presence of CD228-expressing tumor cells and / or in a (CD228-positive) tumor microenvironment. In some embodiments, the provided fusion proteins have an EC of about 1.5 nM or less, about 1.2 nM or less, about 1 nM or less, about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or less, or about 0.15 nM or less in the presence of CD228-positive tumor cells. 50 T cell activation by the provided fusion proteins in the presence of CD228-expressing tumor cells and / or in a CD228-positive tumor microenvironment can be assessed, for example, in a CD137 bioassay essentially as described in Example 14.

[0117]

[0146] In some embodiments, the provided fusion proteins are unable to costimulate T cell responses in the absence of CD228. In some embodiments, the provided fusion proteins are unable to costimulate T cell responses in the absence of CD228-expressing cells. In some embodiments, the provided fusion proteins may be able to recognize the presence of CD228 and direct the activation of corresponding T cells better than the CD137 antibodies set forth in SEQ ID NOs: 26 and 27. The CD228-dependent effects of the fusion proteins can be determined, for example, in a CD137 bioassay essentially as described in Example 14 or in a functional T cell activation assay essentially as described in Example 18.

[0118]

[0147] In some embodiments, the fusion proteins of the present disclosure may be capable of inducing increased IL-2 secretion, such as in the presence of CD228-positive (tumor) cells. In some preferred embodiments, the provided fusion proteins may be capable of inducing concentration-dependent IL-2 secretion and / or may be capable of inducing enhanced IL-2 secretion at high concentrations, preferably coating concentrations. In some embodiments, the provided fusion proteins may increase IL-2 secretion with greater efficiency compared to anti-CD137 antibodies, such as the reference antibodies of SEQ ID NOs: 26 and 27. IL-2 secretion may be measured, for example, in a functional T cell activation assay essentially as described in Example 18.

[0119]

[0148] In some embodiments, provided fusion proteins are capable of stimulating the proliferation of CD4+ and / or CD8+ T cells and / or NK cells. In some embodiments, provided fusion proteins are capable of inducing NF-κB signaling. In some embodiments, provided fusion proteins are capable of inducing increased secretion of cytokines such as interferon gamma, TNF-alpha, IL-2, IL-5, IL-10, IL-13, and IP-10 (CXCL10). In some embodiments, provided fusion proteins are capable of inducing increased secretion of cytotoxic factors such as perforin, granzyme B, and granzyme A.

[0120]

[0149] In some embodiments, the provided fusion proteins have favorable stability and / or pharmacokinetic profiles. In some embodiments, the provided fusion proteins have antibody-like pharmacokinetics. In some embodiments, the provided fusion proteins have a terminal half-life (e.g., in vivo terminal half-life in mice) of about 200 hours or more, about 250 hours or more, or about 300 hours or more. In some embodiments, the provided fusion proteins have a longer terminal half-life than anti-CD228 antibodies having the amino acid sequences of SEQ ID NOs: 74 and 76 or SEQ ID NOs: 77 and 79, respectively. The pharmacokinetic profile of the provided fusion proteins can be analyzed as described in Example 22. In some embodiments, after 336 hours, max If the % of the antibody is greater than 10%, a favorable pharmacokinetic profile or antibody-like pharmacokinetics may be considered to have been achieved.

[0121]

[0150] In some embodiments, the fusion proteins of the present disclosure may be capable of inducing an increase in mitochondrial content in CD8+ T cells and / or a decrease in mitochondrial depolarization in CD8+ T cells. In some embodiments, the provided fusion proteins may induce an increase in CD8+ T cell mitochondrial content and / or a decrease in CD8+ T cell mitochondrial depolarization in the vicinity of CD228-positive cells, such as CD228-transfected cells or CD228-positive tumor cells. The induction of an increase in CD8+ T cell mitochondrial content by the provided fusion proteins can be determined, for example, by using a polarization-independent dye, such as MitoSpy™ Green FM, essentially as described in Example 26. The induction of a decrease in CD8+ T cell mitochondrial depolarization by the provided fusion proteins can be determined, for example, by using a polarization-dependent dye, such as MitoSpy™ Orange CMTM Ros, essentially as described in Example 26.

[0122]

[0151] In some embodiments, the fusion proteins of the present disclosure may be capable of stimulating exhausted CD8+ T cell proliferation and / or may be capable of stimulating exhausted CD8+ T cell proliferation in synergy with an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the provided fusion proteins may be capable of stimulating exhausted CD8+ T cell proliferation in the vicinity of CD228-positive cells, such as CD228-transfected cells or CD228-positive tumor cells, and / or may be capable of stimulating exhausted CD8+ T cell proliferation in synergy with an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, exhausted CD8+ T cells have a reduced ability to divide, secrete cytokines (e.g., IFN-γ), and / or kill tumor cells compared to non-exhausted CD8+ T cells. In some embodiments, the fusion proteins alone may be capable of stimulating exhausted CD8+ T cell proliferation. In some embodiments, the fusion protein may be capable of stimulating exhausted CD8+ T cell proliferation in synergy with an anti-PD-1 antibody or anti-PD-L1 antibody; in such embodiments, the ability of the fusion protein to stimulate exhausted CD8+ T cell proliferation may be enhanced, whereas an anti-PD-1 antibody or anti-PD-L1 antibody alone may have no or limited ability to stimulate exhausted CD8+ T cell proliferation. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody is nivolumab, pembrolizumab, cemiplimab, dostallimab, atezolizumab, avelumab, or durvalumab. Cell proliferation may be determined using flow cytometry using a dye such as CFSE, essentially as described in Example 28.

[0123]

[0152] In some embodiments, the fusion protein provided comprises the amino acid sequence set forth in any one of SEQ ID NOs: 75, 76, and 78-83.

[0153] In some embodiments, the provided fusion proteins comprise 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 sequence set forth in any one of SEQ ID NOs: 75, 76, and 78-83.

[0124]

[0154] In some embodiments, the fusion proteins provided comprise 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 sequence set forth in SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, SEQ ID NOs: 78 and 83, SEQ ID NOs: 96 and 97, SEQ ID NOs: 98 and 99, SEQ ID NOs: 100 and 101, SEQ ID NOs: 102 and 103, SEQ ID NOs: 104 and 105, or SEQ ID NOs: 106 and 107.

[0125]

[0155] In some embodiments, provided fusion proteins comprise the amino acid sequences set forth in SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, SEQ ID NOs: 78 and 83, SEQ ID NOs: 96 and 97, SEQ ID NOs: 98 and 99, SEQ ID NOs: 100 and 101, SEQ ID NOs: 102 and 103, SEQ ID NOs: 104 and 105, or SEQ ID NOs: 106 and 107. In one embodiment, provided fusion proteins comprise the amino acid sequences set forth in SEQ ID NOs: 80 and 76. In one embodiment, provided fusion proteins comprise the amino acid sequences set forth in SEQ ID NOs: 82 and 79. In one embodiment, provided fusion proteins comprise the amino acid sequences set forth in SEQ ID NOs: 75 and 81. In one embodiment, provided fusion proteins comprise the amino acid sequences set forth in SEQ ID NOs: 78 and 83. In one embodiment, provided fusion proteins comprise the amino acid sequences set forth in SEQ ID NOs: 96 and 97. In one embodiment, provided fusion proteins comprise the amino acid sequences set forth in SEQ ID NOs: 98 and 99. In one embodiment, provided fusion proteins comprise the amino acid sequences set forth in SEQ ID NOs: 100 and 101. In one embodiment, a provided fusion protein comprises the amino acid sequence set forth in SEQ ID NOs: 102 and 103. In one embodiment, a provided fusion protein comprises the amino acid sequence set forth in SEQ ID NOs: 104 and 105. In one embodiment, a provided fusion protein comprises the amino acid sequence set forth in SEQ ID NOs: 106 and 107.

[0126] C. Exemplary Lipocalin Muteins of the Present Disclosure

[0156] Lipocalins are proteinaceous binding molecules that have naturally evolved to bind to ligands. Lipocalins are present in many organisms, including vertebrates, insects, plants, and bacteria. Members of the lipocalin protein family (Pervaiz and Brew, FASEB J, 1987) are typically small, secreted proteins with a single polypeptide chain. They are characterized by a range of molecular recognition properties: binding to a variety of primarily hydrophobic small molecules (e.g., retinoids, fatty acids, cholesterol, prostaglandins, biliverdin, pheromones, tastants, and odorants), binding to specific cell surface receptors, and forming macromolecular complexes. While lipocalins have previously been classified primarily as transport proteins, it is now clear that they perform a variety of physiological functions. These include roles in retinol transport, olfaction, pheromone signaling, and prostaglandin synthesis. Lipocalins are also involved in regulating immune responses and mediating cellular homeostasis (reviewed in, eg, Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996).

[0127]

[0157] The overall level of sequence conservation in lipocalins is unusually low, often with sequence identity below 20%. In stark contrast, the overall folding pattern is highly conserved. The core of the lipocalin structure consists of a single eight-stranded antiparallel β-sheet that closes together to form a continuously hydrogen-bonded β-barrel. This β-barrel defines a central cavity. One end of the barrel is sterically blocked by an N-terminal peptide segment across its base and three peptide loops connecting the β-strands. The other end of the β-barrel is open to solvent and encompasses a target-binding site formed by four flexible peptide loops (AB, CD, EF, and GH). It is the diversity of 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 properties (e.g., reviewed in Skerra, Biochim Biophys Acta, 2000; Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996).

[0128]

[0158] The lipocalin mutein according to the present disclosure can be a mutein of any lipocalin. Examples of suitable lipocalins (sometimes referred to as "reference lipocalin," "wild-type lipocalin," "reference protein scaffold," or simply "scaffold") for which the mutein can be used include, but are not limited to, tear lipocalin (lipocalin-1, Tlc, or von Ebner's gland protein), retinol-binding protein, neutrophil lipocalin-type prostaglandin D-synthase, β-lactoglobulin, bilin-binding protein (BBP), apolipoprotein D (APOD), neutrophil gelatinase-associated lipocalin (NGAL), α2-microglobulin-related protein (A2m), 24p3 / uterocalin (24p3), von Ebner's gland protein 1 (VEGP 1), von Ebner's gland protein 2 (VEGP 2), and major allergen Can f 1 (ALL-1). In a related embodiment, the lipocalin mutein is derived from the lipocalin group consisting of human tear lipocalin (hTlc), human neutrophil gelatinase-associated lipocalin (hNGAL), human apolipoprotein D (hAPOD), and bilin-binding protein of Pieris brassicae.

[0129]

[0159] The amino acid sequence of a lipocalin mutein according to the present disclosure may have a high degree of sequence identity with the reference (or wild-type) lipocalin from which it is derived, such as hTlc or hNGAL, when compared with sequence identity with another lipocalin (see also above). In this general context, the amino acid sequence of a lipocalin mutein according to the present disclosure is at least substantially similar to the amino acid sequence of the corresponding reference (wild-type) lipocalin, provided that gaps (as defined herein) resulting from the addition or deletion of amino acids may exist in the alignment. The sequence of each lipocalin mutein according to the present disclosure is substantially similar to the sequence of the corresponding reference (wild-type) lipocalin, and in some embodiments has 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) with the sequence of the corresponding lipocalin. In this regard, it will be understood that the lipocalin muteins of the present disclosure may contain the substitutions described herein that enable the lipocalin mutein to bind to CD137.

[0130]

[0160] Typically, lipocalin muteins contain one or more mutated amino acid residues relative to the amino acid sequence of a wild-type or reference lipocalin, such as hTlc and hNGAL, in the four loops at the open end that constitute and define the entrance to the ligand-binding pocket (see above). As explained above, these regions are essential for determining the binding specificity of the lipocalin mutein to a desired target. In some embodiments, lipocalin muteins of the present disclosure may also contain mutated amino acid residue regions outside the four loops. In some embodiments, lipocalin muteins of the present disclosure may contain one or more mutated amino acid residues in one or more of the three peptide loops (designated BC, DE, and FG) that connect the β-strands at the closed end of the lipocalin. In some embodiments, a mutein derived from tear lipocalin, NGAL, or a homolog thereof may have one, two, three, four, or more mutated amino acid residues in the N-terminal region and / or at any sequence position in the three peptide loops BC, DE, and FG located at the end of the β-barrel structure opposite the native lipocalin-binding pocket. In some embodiments, a mutein derived from tear lipocalin, NGAL, or a homolog thereof may not have a mutated amino acid residue in the peptide loop DE located at the end of the β-barrel structure compared to the wild-type sequence of tear lipocalin, NGAL, or a homolog thereof.

[0131]

[0161] In some embodiments, a lipocalin mutein according to the present disclosure can contain one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or even more, mutant amino acid residues compared to the amino acid sequence of a corresponding reference (wild-type) lipocalin, provided that such lipocalin muteins are capable of binding to CD 137. In some embodiments, a lipocalin mutein of the present disclosure contains at least two, including 2, 3, 4, 5, or even more, mutant amino acid residues in which the native amino acid residues of the corresponding reference (wild-type) lipocalin are substituted with arginine residues.

[0132]

[0162] Any type and number of mutations, including substitutions, deletions, and insertions, are contemplated, so long as the provided lipocalin mutein retains the ability to bind to CD137 and / or has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more identity to the amino acid sequence of a reference (wild-type) lipocalin, e.g., mature hTlc or mature hNGAL.

[0133]

[0163] In some embodiments, the substitution is a conservative substitution. In some embodiments, the substitution is a non-conservative substitution or one or more of the following exemplary substitutions:

[0164] Specifically, to determine whether the amino acid sequence of a lipocalin (mutein) differs from that of a reference (wild-type) lipocalin at a particular position in the amino acid sequence of the reference (wild-type) lipocalin, those skilled in the art can use means and methods well known in the art, for example, alignment, either manually or using a computer program such as BLAST2.0 (short for Basic Local Alignment Search Tool) or ClustalW or any other suitable program suitable for generating sequence alignments. Thus, the amino acid sequence of the reference (wild-type) lipocalin serves as the "subject sequence" or "reference sequence," and the amino acid sequence of the lipocalin mutein serves as the "query sequence" (see also above).

[0134]

[0165] Conservative substitutions are generally the following substitutions listed according to the amino acid being mutated, with each substitution followed by one or more substitutions that are considered conservative: Ala → Ser, Thr, or Val; Arg → Lys, Gln, Asn, or His; Asn → Gln, Glu, Asp, or His; Asp → Glu, Gln, Asn, or His; Gln → Asn, Asp, Glu, or His; Glu → Asp, Asn, Gln, or His; His → Arg, Lys, Asn, Gln, Asp, or Glu; Ile → Thr, Leu, Met, Phe, Val, Trp, Tyr, Ala, or Pro; Leu → Thr, Ile, V Lys → Arg, His, Gln, or Asn; Met → Thr, Leu, Tyr, Ile, Phe, Val, Ala, Pro, or Trp; Phe → Thr, Met, Leu, Tyr, Ile, Pro, Trp, Val, or Ala; Ser → Thr, Ala, or Val; Thr → Ser, Ala, Val, Ile, Met, Val, Phe, Pro, or Leu; Trp → Tyr, Phe, Met, Ile, or Leu; Tyr → Trp, Phe, Ile, Leu, or Met; Val → Thr, Ile, Leu, Met, Phe, Ala, Ser, or Pro. Other substitutions are permissible and can be determined empirically or in accordance with other known conservative or non-conservative substitutions. As a further direction, the following groups each contain amino acids that can typically be considered to define conservative substitutions for each other: i) alanine (Ala), serine (Ser), threonine (Thr), valine (Val); ii) aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), asparagine (Asn), histidine (His); iii) arginine (Arg), lysine (Lys), glutamine (Gln), asparagine (Asn), histidine (His); iv) isoleucine (Ile), leucine (Leu), methionine (Met), valine (Val), alanine (Ala), phenylalanine (Phe), threonine (Thr), proline (Pro); v) Isoleucine (Ile), Leucine (Leu), Methionine (Met), Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp).

[0135]

[0166] If such conservative substitutions result in altered biological activity, more substantial changes, such as those described further below with respect to amino acid classification, can be introduced and the products screened for the desired properties. Examples of such more substantial changes are: Ala → Leu or Phe; Arg → Glu; Asn → Ile, Val, or Trp; Asp → Met; Cys → Pro; Gln → Phe; Glu → Arg; His → Gly; Ile → Lys, Glu, or Gln; Leu → Lys or Ser; Lys → Tyr; Met → Glu; Phe → Glu, Gln, or Asp; Trp → Cys; Tyr → Glu or Asp; Val → Lys, Arg, His.

[0136]

[0167] In some embodiments, substantial alteration of the physical and biological properties of a lipocalin (mutein) is achieved by selecting substitutions that differ significantly in (a) the structure of the polypeptide backbone in the region of the substitution, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the effect of maintaining the bulk of the side chains.

[0137]

[0168] Naturally occurring residues are divided into groups based on common side chain properties: (1) hydrophobic: methionine, alanine, valine, leucine, isoleucine; (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. In some embodiments, substitutions may involve exchanging a member of one of these classes for a member of another class.

[0138]

[0169] Cysteine ​​residues not involved in maintaining the proper conformation of each lipocalin may also 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 a lipocalin to improve its stability.

[0139] D. Exemplary CD137-Specific Lipocalin Muteins of the Present Disclosure

[0170] As mentioned above, lipocalins are polypeptides defined by their supersecondary structure, i.e., a cylindrical β-pleated sheet supersecondary structure region comprising eight β-strands connected in pairs at one end by four loops, thereby defining a binding pocket. The present disclosure is not limited to the lipocalin muteins specifically disclosed herein. In this regard, the present disclosure relates to a lipocalin mutein having a cylindrical β-pleated sheet supersecondary structure region comprising eight β-strands connected in pairs at one end by four loops, thereby defining a binding pocket, wherein at least one amino acid in each of at least three of the four loops has been mutated, and the lipocalin is effective in binding to CD137 with detectable affinity.

[0140]

[0171] In some embodiments, the lipocalin muteins disclosed herein may be or include muteins of mature human tear lipocalin (hTlc). Muteins of mature hTlc may be referred to herein as "hTlc muteins." In some other embodiments, the lipocalin muteins disclosed herein are muteins of mature human neutrophil gelatinase-associated lipocalin (hNGAL). Muteins of mature hNGAL may be referred to herein as "hNGAL muteins."

[0141]

[0172] In one embodiment, the present disclosure includes any number of lipocalin muteins derived from a reference (wild-type) lipocalin, preferably derived from mature hTlc or mature hNGAL, that bind to CD137 with detectable affinity. In a related embodiment, the present disclosure includes various lipocalin muteins that can activate downstream signaling pathways of CD137 by binding to CD137. In this sense, CD137 can be considered a non-natural target of a reference (wild-type) lipocalin, preferably hTlc or hNGAL, where "non-natural target" refers to a substance that does not bind to the reference (wild-type) lipocalin under physiological conditions. By engineering a reference (wild-type) lipocalin with one or more mutations at certain sequence positions, the present inventors have demonstrated that high affinity and high specificity for the non-natural target CD137 are possible. In some embodiments, random mutagenesis can occur at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more nucleotide triplets encoding certain sequence positions of wild-type lipocalin by substitution at these positions with a subset of nucleotide triplets in order to generate lipocalin muteins capable of binding to CD137.

[0142]

[0173] In some embodiments, the lipocalin muteins of the present disclosure may have mutated amino acid residue(s), including substitutions, deletions, and insertions, at one or more sequence positions corresponding to the sequence positions of the linear polypeptide sequence of a reference lipocalin, preferably hTlc or hNGAL. In some embodiments, the number of mutated amino acid residues in the lipocalin muteins of the present disclosure compared to the amino acid sequence of the reference lipocalin, preferably hTlc or hNGAL, is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, for example, 25, 30, 35, 40, 45, or 50, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 being preferred, and 9, 10, or 11 being even more preferred. However, it is preferred that the lipocalin muteins of the present disclosure are still capable of binding to CD137.

[0143]

[0174] In some embodiments, a lipocalin mutein of the present disclosure may lack one, two, three, four, or more amino acids at its N-terminus and / or one, two, or more amino acids at its C-terminus compared to the respective reference (wild-type) lipocalin; e.g., SEQ ID NOS: 32-38. In some embodiments, the present disclosure encompasses hTlc muteins as defined above, in which the first one, two, three, or four N-terminal amino acid residues (His-His-Leu-Leu; positions 1-4) of the sequence of mature hTlc and / or the last one or two C-terminal amino acid residues (Ser-Asp; positions 157-158) of the linear polypeptide sequence of mature hTlc are deleted (e.g., SEQ ID NOS: 32-38). In some embodiments, the present disclosure encompasses hNGAL muteins as defined above, in which the amino acid residues (Lys-Asp-Pro; positions 46-48) of the linear polypeptide sequence of mature hNGAL are deleted (SEQ ID NO: 43). Additionally, lipocalin muteins of the present disclosure may comprise the wild-type (native) amino acid sequence of a reference (wild-type) lipocalin, preferably hTlc or hNGAL, outside of the mutated amino acid sequence positions.

[0144]

[0175] In some embodiments, one or more mutated amino acid residues incorporated into the lipocalin muteins of the present disclosure do not substantially interfere with or disrupt the binding activity to the designated target and folding of the mutein. Such mutations, including substitutions, deletions, and insertions, can be achieved at the DNA level using established standard methods (Sambrook and Russell, 2001, Molecular Cloning: A Laboratory Manual). In some embodiments, (a) mutated amino acid residue(s) at one or more sequence positions corresponding to the linear polypeptide sequence of a reference (wild-type) lipocalin, preferably hTlc or hNGAL, are introduced by random mutagenesis by substituting a subset of nucleotide triplets for the nucleotide triplet(s) encoding the corresponding sequence positions of the reference lipocalin.

[0145]

[0176] In some embodiments, the provided lipocalin muteins that bind to CD137 with detectable affinity may contain at least one amino acid substitution of a natural cysteine ​​residue with another amino acid, such as a serine residue. In some embodiments, the lipocalin muteins that bind to CD137 with detectable affinity may contain one or more non-natural cysteine ​​residues that replace one or more amino acids of a reference (wild-type) lipocalin, preferably hTlc or hNGAL. In some embodiments, the lipocalin muteins of the present disclosure contain at least two amino acid substitutions of natural amino acids with cysteine ​​residues, thereby forming one or more cysteine ​​bridges. In some embodiments, the cysteine ​​bridges may connect at least two loop regions. The definitions of these regions are used herein according to Skerra, Biochim Biophys Acta (2000), Flower (1996), and Breustedt et al. (2005).

[0146]

[0177] Generally, a lipocalin mutein of the present disclosure may have at least about 70% amino acid sequence identity, including at least about 80%, for example at least about 85%, with the amino acid sequence of mature hTlc (SEQ ID NO: 1) or mature hNGAL (SEQ ID NO: 2).

[0147]

[0178] In some embodiments, the present disclosure provides CD137-binding hTlc muteins. In this regard, the present disclosure provides CD137-binding hTlc muteins having a K of about 300 nM, 200 nM, 150 nM, 100 nM, or lower. D In some embodiments, the hTlc muteins provided have an EC of about 250 nM, 150 nM, 100 nM, 50 nM, 20 nM, or even lower. 50 In some other embodiments, the CD137-binding hTlc mutein may be cross-reactive with cynomolgus monkey CD137 (cyCD137).

[0148]

[0179] In some embodiments, the hTlc muteins of the present disclosure can interfere with the binding of CD137L to CD137.

[0180] In some embodiments, the provided hTlc muteins may contain mutant amino acid residues at one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0149]

[0181] In some embodiments, the hTlc muteins provided may contain mutant amino acid residues at one or more positions corresponding to positions 26-34, 55-58, 60-61, 65, 104-106, and 108 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0150]

[0182] In some embodiments, the hTlc muteins provided may further comprise mutated amino acid residues at one or more positions corresponding to positions 101, 111, 114, and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1).

[0151]

[0183] In some embodiments, provided hTlc muteins can contain mutated amino acid residues at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more, corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1). In some preferred embodiments, provided hTlc muteins are capable of binding to CD137, particularly human CD137.

[0152]

[0184] In some embodiments, the provided hTlc muteins can contain mutated amino acid residues at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more, corresponding to positions 26-34, 55-58, 60-61, 65, 104-106, and 108 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1). In some preferred embodiments, the provided hTlc muteins are capable of binding to CD137, particularly human CD137.

[0153]

[0185] In some embodiments, lipocalin muteins according to the present disclosure may contain at least one amino acid substitution of a native cysteine ​​residue, for example, with a serine residue. In some embodiments, hTlc muteins according to the present disclosure contain an amino acid substitution of a native cysteine ​​residue at a position corresponding to positions 61 and / or 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1) with another amino acid, such as a serine residue. In this context, it has been stated that removing the structural disulfide bond of wild-type hTlc formed by cysteine ​​residues 61 and 153 (at the level of each naive nucleic acid library) (see Breustedt et al., J Biol Chem, 2005) can provide hTlc muteins that are not only stably folded but also capable of binding with high affinity to a given non-native target. In some embodiments, elimination of the structural disulfide bond may provide the additional advantage of allowing the generation or intentional introduction of non-native disulfide bonds into muteins of the present disclosure, thereby increasing the stability of the muteins. However, hTlc muteins that bind to CD137 and have a disulfide bridge formed between Cys61 and Cys153 are also part of the present disclosure.

[0154]

[0186] In certain embodiments, hTlc muteins of the present disclosure may contain amino acid substitutions of Cys61→Ala, Phe, Lys, Arg, Thr, Asn, Gly, Gln, Asp, Asn, Leu, Tyr, Met, Ser, Pro, or Trp, and / or Cys153→Ser or Ala at positions corresponding to positions 61 and / or 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0155]

[0187] In some embodiments, two or all three of the cysteine ​​codons at positions corresponding to positions 61, 101, and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1) are replaced with codons for another amino acid. Furthermore, in some embodiments, hTlc muteins according to the present disclosure contain an amino acid substitution of a serine or histidine residue for the native cysteine ​​residue at the position corresponding to position 101 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0156]

[0188] In some embodiments, a mutein according to the present disclosure comprises an amino acid substitution of a native amino acid with a cysteine ​​residue at a position corresponding to position 28 or 105 of the linear polypeptide sequence of mature hTc (SEQ ID NO:1). Further, in some embodiments, a mutein according to the present disclosure comprises an amino acid substitution of a native arginine residue with a proline residue at a position corresponding to position 111 of the linear polypeptide sequence of mature hTc (SEQ ID NO:1). Further, in some embodiments, a mutein according to the present disclosure comprises an amino acid substitution of a native lysine residue with a tryptophan residue or glutamic acid at a position corresponding to position 114 of the linear polypeptide sequence of mature hTc (SEQ ID NO:1).

[0157]

[0189] In some embodiments, the provided CD137-binding hTlc muteins may contain one or more of the following mutated amino acid residues at one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1): Ala5→Val or Thr; Arg26→Glu; Glu27→Gly; Phe28→Cys; Pro29→Arg; Glu30→Pro; Met31→Trp; Leu 33 → Ile; Glu34 → Phe; Thr42 → Ser; Gly46 → Asp; Lys52 → Glu; Leu56 → Ala; Ser58 → Asp; Arg60 → Pro; Cys61 → Ala; Lys65 → Arg or Asn; Thr71 → Ala; Val85 → Asp; Lys94 → Arg or Glu; Cys101 → Ser; Glu104 → Val; Leu105 → Cys; His106 → Asp; Lys108 → Ser; Arg111 → Pro; Lys114 → Trp; Lys121 → Glu; Ala133 → Thr; Arg148 → Ser; Ser150 → Ile; and Cys153 → Ser. In some embodiments, hTlc muteins of the disclosure contain two or more, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more, or even all, of the mutated amino acid residues at these sequence positions of mature hTlc (SEQ ID NO: 1).

[0158]

[0190] In some embodiments, the provided CD137-binding hTlc muteins may comprise one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1): (a) Arg26 → Glu; Glu27 → Gly; Phe28 → Cys; Pro29 → Arg; Glu30 → Pro; Met31 → Trp; Leu33 → Ile; Glu34 → Phe; Leu56 → Ala; Ser58 → Asp; Arg60 → Pro; Cys61 → Ala; Cys101 → Ser; Glu104 → Val; Leu105 → Cys; His106 → Asp; Lys108 → Ser; Arg111 → Pro; Lys114 → Trp; and Cys153 → Ser; (b) Ala5 → Thr; Arg26 → Glu; Glu27 → Gly; Phe28 → Cys; Pro29 → Arg; Glu30 → Pro; Met31 → Trp; Leu33 → Ile; Glu34 → Phe; Leu56 → Ala; Ser58 → Asp; Arg60 → Pro; Cys61 → Ala; Lys65 → Arg; Val85 → Asp; Cys101 → Ser; Glu104 → Val; Leu105 → Cys; His106 → Asp; Lys108 → Ser; Arg111 → Pro; Lys114 → Trp; Lys121 → Glu; Ala133 → Thr; and Cys153 → Ser; (c) Arg26 → Glu; Glu27 → Gly; Phe28 → Cys; Pro29 → Arg; Glu30 → Pro; Met31 → Trp; Leu33 → Ile; Glu34 → Phe; Leu56 → Ala; Ser58 → Asp; Arg60 → Pro; Cys61 → Ala; Lys65 → Asn; Lys94 → Arg; Cys101 → Ser; Glu104 → Val; Leu105 → Cys; His106 → Asp; Lys108 → Ser; Arg111 → Pro; Lys114 → Trp; Lys121 → Glu; Ala133 → Thr; and Cys153 → Ser; (d) Ala5 → Val; Arg26 → Glu; Glu27 → Gly; Phe28 → Cys; Pro29 → Arg; Glu30 → Pro; Met31 → Trp; Leu33 → Ile; Glu34 → Phe; Leu56 → Ala; Ser58 → Asp; Arg60 → Pro; Cys61 → Ala; Lys65 → Arg; Lys94 → Glu; Cys101 → Ser; Glu104 → Val; Leu105 → Cys; His106 → Asp; Lys108 → Ser; Arg111 → Pro; Lys114 → Trp; Lys121 → Glu; Ala133 → Thr; and Cys153 → Ser; (e) Arg26 → Glu; Glu27 → Gly; Phe28 → Cys; Pro29 → Arg; Glu30 → Pro; Met31 → Trp; Leu33 → Ile; Glu34 → Phe; Thr42 → Ser; Leu56 → Ala; Ser58 → Asp; Arg60 → Pro; Cys61 → Ala; Cys101 → Ser; Glu104 → Val; Leu105 → Cys; His106 → Asp; Lys108 → Ser; Arg111 → Pro; Lys114 → Trp; Ser150 → Ile; and Cys153 → Ser; (f) Arg26 → Glu; Glu27 → Gly; Phe28 → Cys; Pro29 → Arg; Glu30 → Pro; Met31 → Trp; Leu33 → Ile; Glu34 → Phe; Lys52 → Glu; Leu56 → Ala; Ser58 → Asp; Arg60 → Pro; Cys61 → Ala; Thr71 → Ala; Cys101 → Ser; Glu104 → Val; Leu105 → Cys; His106 → Asp; Lys108 → Ser; Arg111 → Pro; Lys114 → Trp; Ala133 → Thr; Arg148 → Ser; Ser150 → Ile; and Cys153 → Ser; and (g) Ala5 → Thr; Arg26 → Glu; Glu27 → Gly; Phe28 → Cys; Pro29 → Arg; Glu30 → Pro; Met31 → Trp; Leu33 → Ile; Glu34 → Phe; Gly46 → Asp; Leu56 → Ala; Ser58 → Asp; Arg60 → Pro; Cys61 → Ala; Thr71 → Ala; Cys101 → Ser; Glu104 → Val; Leu105 → Cys; His106 → Asp; Lys108 → Ser; Arg111 → Pro; Lys114 → Trp; Ser150 → Ile; and Cys153 → Ser.

[0159]

[0191] In some embodiments, the remaining regions of the hTlc muteins of the present disclosure, i.e., regions that differ from positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1), may comprise the wild-type (native) amino acid sequence of the linear polypeptide sequence of mature hTlc outside of the mutant amino acid sequence positions.

[0160]

[0192] In some embodiments, hTlc muteins of the present disclosure have at least 70% sequence identity or homology to the sequence of mature hTlc (SEQ ID NO: 1). As an illustrative example, the mutein of SEQ ID NO: 32 has approximately 84% amino acid sequence identity or homology to the amino acid sequence of mature hTlc.

[0161]

[0193] In some embodiments, an hTlc mutein of the present disclosure comprises an amino acid sequence set forth in any one of SEQ ID NOs: 32-38, or a fragment or variant thereof.

[0194] In some embodiments, an hTlc mutein of the present disclosure has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to an amino acid sequence selected from SEQ ID NOs: 32-38.

[0162]

[0195] The present disclosure also includes structural homologs of hTlc muteins having an amino acid sequence selected from SEQ ID NOs: 32-38, which structural homologs have greater than about 60%, preferably greater than 65%, 70%, 75%, 80%, 85%, 90%, 92%, and most preferably greater than 95% amino acid sequence homology or sequence identity with said hTlc muteins.

[0163]

[0196] In some embodiments, the present disclosure provides CD137-binding hNGAL muteins. In this regard, the present disclosure provides CD137-binding hNGAL muteins having a K of about 800 nM, 700 nM, 200 nM, 140 nM, 100 nM or lower, preferably about 70 nM, 50 nM, 30 nM, 10 nM, 5 nM, 2 nM or lower. D In some embodiments, the hNGAL muteins provided have an EC of about 1000 nM, 500 nM, 100 nM, 80 nM, 50 nM, 25 nM, 18 nM, 15 nM, 10 nM, 5 nM, or lower. 50 It is capable of binding to CD137 at low levels.

[0164]

[0197] In some embodiments, provided CD137-binding hNGAL muteins may be cross-reactive with cynomolgus monkey CD137. In some embodiments, provided hNGAL muteins have a K of about 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, or even lower. D In some embodiments, the hNGAL muteins provided are capable of binding to cynomolgus monkey CD137 with an affinity measured by EC 50 It is capable of binding to cynomolgus monkey CD137 at low antibody levels.

[0165]

[0198] In some embodiments, the hNGAL muteins of the present disclosure may interfere with or compete with the binding of CD137L to CD137. In some other embodiments, the hNGAL muteins of the present disclosure may be capable of binding to CD137 in the presence of CD137L and / or may be capable of binding to the CD137 / CD137L complex.

[0166]

[0199] In some embodiments, the hNGAL muteins provided contain one or more mutated amino acid residues at positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0167]

[0200] In some embodiments, the hNGAL muteins provided may contain mutated amino acid residues at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the hNGAL muteins provided are capable of binding to CD137, particularly human CD137.

[0168]

[0201] In some embodiments, the provided hNGAL muteins may contain mutant amino acid residues at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 87, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the provided hNGAL muteins are capable of binding to CD137, particularly human CD137.

[0169]

[0202] In some embodiments, the provided hNGAL muteins may contain mutant amino acid residues at one or more positions corresponding to positions 36, 87, and 96 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), as well as at one or more positions corresponding to positions 28, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 94, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0170]

[0203] In some other embodiments, the provided hNGAL muteins may contain mutant amino acid residues at one or more positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0171]

[0204] In other embodiments, the hNGAL muteins provided may contain mutant amino acid residues at one or more positions corresponding to positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), and at one or more positions corresponding to positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 87, 92, 98, 101, and 122 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0172]

[0205] In some embodiments, lipocalin muteins according to the present disclosure may contain at least one amino acid substitution of a native cysteine ​​residue, for example, with a serine residue. In some embodiments, hNGAL muteins according to the present disclosure may contain an amino acid substitution of a native cysteine ​​residue at a position corresponding to positions 76 and / or 175 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) with another amino acid, such as a serine residue. In this context, it has been stated that removing the structural disulfide bond of wild-type hNGAL formed by cysteine ​​residues 76 and 175 (at the level of each naive nucleic acid library) (see Breustedt et al., J Biol Chem, 2005) can provide hNGAL muteins that are not only stable in folding but also capable of binding with high affinity to a given non-native target. In some embodiments, elimination of the structural disulfide bond may provide the additional advantage of allowing the generation or intentional introduction of a non-native disulfide bond into a mutein according to the present disclosure, thereby increasing the stability of the mutein. However, hNGAL muteins that bind to CD137 and form a disulfide bridge between Cys76 and Cys175 are also part of the present disclosure.

[0173]

[0206] In some embodiments, the provided CD137-binding hNGAL muteins may contain one or more of the following mutated amino acid residues at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gln28→His; Leu36→Gln; Ala40→Ile; Ile41→Arg or Lys; Gln49→Val, Ile, His, Ser, or Asn; Tyr52 → Met; Asn65 → Asp; Ser68 → Met, Ala or Gly; Leu70 → Ala, Lys, Ser or Thr; Arg72 → Asp; Lys73 → Asp; Asp77 → Met, Arg, Thr or Asn; Trp79 → Ala or Asp; Arg81 → Met, Trp or Ser; Phe83 → Leu; Cys87 → Ser; Leu94 → Phe; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu and Lys134 → Tyr. In some embodiments, hNGAL muteins of the present disclosure include two or more, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even more, e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all, mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 2).

[0174]

[0207] In some embodiments, the provided CD137-binding hNGAL muteins may contain one or more of the following mutated amino acid residues at one or more positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gln20→Arg; Asn25→Tyr or Asp; Gln28→His; Val33→Ile; Leu36→Met; Ala40→Asn; Ile41→Leu; Glu44→Val or Asp; Gln49 →His; Tyr52 → Ser or Gly; Lys59 → Asn; Ser68 → Asp; Leu70 → Met; Phe71 → Leu; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln or His; Tyr78 → His; Trp79 → Ile; Ile80 → Asn; Arg81 → Trp or Gln; Thr82 → Pro; Cys87 → Ser; Phe92 → Leu or Ser; Asn96 → Phe; Lys98 → Arg; Tyr100 → Asp; Pro101 → Leu; Leu103 → His or Pro; Phe122 → Tyr; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly.

[0175]

[0208] In some embodiments, the provided CD137-binding hNGAL muteins may contain one or more of the following mutated amino acid residues at one or more positions corresponding to positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Leu36→Met; Ala40→Asn; Ile41→ Leu; Gln49 → His; Tyr52 → Ser or Gly; Ser68 → Asp; Leu70 → Met; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln or His; Trp79 → Ile; Arg81 → Trp or Gln; Asn96 → Phe; Tyr100 → Asp; Leu103 → His or Pro; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly. In some embodiments, the provided CD137-binding hNGAL muteins may contain one or more of the following mutated amino acid residues at one or more positions corresponding to positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 92, 98, 101, and 122 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gln20→Arg; Asn25→Tyr or Asp; Val33→Ile; Glu44→Val or Asp; Lys59→Asn; Phe71→Leu; Tyr78→His; Ile80→Asn; Thr82→Pro; Phe92→Leu or Ser; Lys98→Arg; Pro101→Leu; and Phe122→Tyr.

[0176]

[0209] In some embodiments, the provided CD137-binding hNGAL muteins may comprise one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): (a) Gln28 → His; Leu36 → Gln; Ala40 → Ile; Ile41 → Lys; Gln49 → Asn; Tyr52 → Met; Ser68 → Gly; Leu70 → Thr; Arg72 → Asp; Lys73 → Asp; Asp77 → Thr; Trp79 → Ala; Arg81 → Ser; Cys87 → Ser; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu; and Lys134 → Tyr; (b) Gln28 → His; Leu36 → Gln; Ala40 → Ile; Ile41 → Arg; Gln49 → Ile; Tyr52 → Met; Asn65 → Asp; Ser68 → Met; Leu70 → Lys; Arg72 → Asp; Lys73 → Asp; Asp77 → Met; Trp79 → Asp; Arg81 → Trp; Cys87 → Ser; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu; and Lys134 → Tyr; (c) Gln28 → His; Leu36 → Gln; Ala40 → Ile; Ile41 → Arg; Gln49 → Asn; Tyr52 → Met; Asn65 → Asp; Ser68 → Ala; Leu70 → Ala; Arg72 → Asp; Lys73 → Asp; Asp77 → Thr; Trp79 → Asp; Arg81 → Trp; Cys87 → Ser; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu; and Lys134 → Tyr; (d) Gln28 → His; Leu36 → Gln; Ala40 → Ile; Ile41 → Lys; Gln49 → Asn; Tyr52 → Met; Asn65 → Asp; Ser68 → Ala; Leu70 → Ala; Arg72 → Asp; Lys73 → Asp; Asp77 → Thr; Trp79 → Asp; Arg81 → Trp; Cys87 → Ser; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu; and Lys134 → Tyr; (e) Gln28 → His; Leu36 → Gln; Ala40 → Ile; Ile41 → Lys; Gln49 → Ser; Tyr52 → Met; Asn65 → Asp; Ser68 → Gly; Leu70 → Ser; Arg72 → Asp; Lys73 → Asp; Asp77 → Thr; Trp79 → Ala; Arg81 → Met; Cys87 → Ser; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu; and Lys134 → Tyr; (f) Gln28 → His; Leu36 → Gln; Ala40 → Ile; Ile41 → Lys; Gln49 → Val; Tyr52 → Met; Asn65 → Asp; Ser68 → Gly; Leu70 → Thr; Arg72 → Asp; Lys73 → Asp; Asp77 → Arg; Trp79 → Asp; Arg81 → Ser; Cys87 → Ser; Leu94 → Phe; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu; and Lys134 → Tyr; (g) Gln28 → His; Leu36 → Gln; Ala40 → Ile; Ile41 → Arg; Gln49 → His; Tyr52 → Met; Asn65 → Asp; Ser68 → Gly; Leu70 → Thr; Arg72 → Asp; Lys73 → Asp; Asp77 → Thr; Trp79 → Ala; Arg81 → Ser; Cys87 → Ser; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu; and Lys134 → Tyr; (h) Gln28 → His; Leu36 → Gln; Ala40 → Ile; Ile41 → Lys; Gln49 → Asn; Tyr52 → Met; Asn65 → Asp; Ser68 → Gly; Leu70 → Thr; Arg72 → Asp; Lys73 → Asp; Asp77 → Thr; Trp79 → Ala; Arg81 → Ser; Phe83 → Leu; Cys87 → Ser; Leu94 → Phe; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu; and Lys134 → Tyr; or (i) Gln28 → His; Leu36 → Gln; Ala40 → Ile; Ile41 → Arg; Gln49 → Ser; Tyr52 → Met; Asn65 → Asp; Ser68 → Ala; Leu70 → Thr; Arg72 → Asp; Lys73 → Asp; Asp77 → Asn; Trp79 → Ala; Arg81 → Ser; Cys87 → Ser; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu; and Lys134 → Tyr.

[0177]

[0210] In some further embodiments, in the residual regions, i.e., regions different from positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), the hNGAL muteins of the present disclosure may comprise the wild-type (naturally occurring) amino acid sequence of mature hNGAL outside of the mutated amino acid sequence positions.

[0178]

[0211] In some other embodiments, the provided CD137-binding hNGAL muteins may comprise one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): (a) Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Gln49 → His; Tyr52 → Ser; Ser68 → Asp; Leu70 → Met; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln; Trp79 → Ile; Arg81 → Trp; Asn96 → Phe; Tyr100 → Asp; Leu103 → His; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly; (b) Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Gln49 → His; Tyr52 → Ser; Ser68 → Asp; Leu70 → Met; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln; Trp79 → Ile; Arg81 → Trp; Phe92 → Leu; Asn96 → Phe; Lys98 → Arg; Tyr100 → Asp; Pro101 → Leu; Leu103 → His; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly; (c) Asn25 → Tyr; Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Gln49 → His; Tyr52 → Gly; Ser68 → Asp; Leu70 → Met; Phe71 → Leu; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln; Trp79 → Ile; Arg81 → Gln; Phe92 → Ser; Asn96 → Phe; Tyr100 → Asp; Leu103 → His; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly; (d) Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Gln49 → His; Tyr52 → Gly; Ser68 → Asp; Leu70 → Met; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln; Tyr78 → His; Trp79 → Ile; Arg81 → Trp; Phe92 → Leu; Asn96 → Phe; Tyr100 → Asp; Leu103 → His; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly; (e) Asn25 → Asp; Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Gln49 → His; Tyr52 → Gly; Ser68 → Asp; Leu70 → Met; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln; Trp79 → Ile; Arg81 → Trp; Phe92 → Leu; Asn96 → Phe; Tyr100 → Asp; Leu103 → His; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly; (f) Val33 → Ile; Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Gln49 → His; Tyr52 → Gly; Ser68 → Asp; Leu70 → Met; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln; Trp79 → Ile; Arg81 → Trp; Phe92 → Leu; Asn96 → Phe; Tyr100 → Asp; Leu103 → His; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly; (g) Gln20 → Arg; Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Glu44 → Val; Gln49 → His; Tyr52 → Gly; Ser68 → Asp; Leu70 → Met; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln; Trp79 → Ile; Arg81 → Trp; Phe92 → Leu; Asn96 → Phe; Tyr100 → Asp; Leu103 → His; Phe122 → Tyr; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly; (h) Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Gln49 → His; Tyr52 → Ser; Ser68 → Asp; Leu70 → Met; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln; Trp79 → Ile; Ile80 → Asn; Arg81 → Trp; Thr82 → Pro; Asn96 → Phe; Tyr100 → Asp; Pro101 → Leu; Leu103 → Pro; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly; (i) Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Gln49 → His; Tyr52 → Gly; Lys59 → Asn; Ser68 → Asp; Leu70 → Met; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln; Trp79 → Ile; Arg81 → Trp; Phe92 → Leu; Asn96 → Phe; Tyr100 → Asp; Leu103 → His; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly; and (j) Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Glu44 → Asp; Gln49 → His; Tyr52 → Ser; Ser68 → Asp; Leu70 → Met; Phe71 → Leu; Arg72 → Leu; Lys73 → Asp; Asp77 → His; Trp79 → Ile; Arg81 → Trp; Phe92 → Leu; Asn96 → Phe; Tyr100 → Asp; Leu103 → His; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly.

[0179]

[0212] In some embodiments, the remaining regions of the hNGAL muteins of the present disclosure, i.e., regions different from positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), may comprise the wild-type (naturally occurring) amino acid sequence of mature hNGAL outside of the mutant amino acid sequence positions.

[0180]

[0213] In some embodiments, hNGAL muteins of the present disclosure have at least 70% sequence identity or homology to the sequence of mature hNGAL (SEQ ID NO: 2). As an illustrative example, the mutein of SEQ ID NO: 40 has approximately 87% amino acid sequence identity or homology to the amino acid sequence of mature hNGAL.

[0181]

[0214] In some embodiments, an hNGAL mutein of the present disclosure comprises an amino acid sequence set forth in any one of SEQ ID NOs: 39-57, or a fragment or variant thereof.

[0182]

[0215] In some embodiments, the hNGAL muteins of the present disclosure have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to an amino acid sequence selected from SEQ ID NOs: 39-57.

[0183]

[0216] The present disclosure also includes structural homologs of hNGAL muteins having an amino acid sequence selected from SEQ ID NOs: 39-57, which structural homologs have greater than about 60%, preferably greater than 65%, 70%, 75%, 80%, 85%, 90%, 92%, and most preferably greater than 95% amino acid sequence homology or sequence identity with said hNGAL muteins.

[0184]

[0217] In some embodiments, the present disclosure provides a K D The present invention provides lipocalin muteins that bind to CD137 with an affinity measured by:

[0185]

[0218] Suitable lipocalin muteins specific for CD137 are also described in WO2016 / 177762, which is incorporated herein by reference in its entirety.

[0219] In some embodiments, the lipocalin muteins of the present disclosure may contain heterologous amino acid sequences, such as a Strep II tag (sequence number 12) or a cleavage site sequence for certain restriction enzymes, at their N- or C-terminus, preferably the C-terminus, without affecting the biological activity of the lipocalin mutein (binding to its target, e.g., CD137).

[0186]

[0220] In some embodiments, further modifications of a lipocalin mutein may be introduced to modulate certain properties of the mutein, for example, to improve folding stability, serum stability, protein tolerance or water solubility, or to reduce aggregation tendency, or to introduce new properties into the mutein. In some embodiments, the modification(s) may result in modulation of two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) properties of a provided mutein.

[0187]

[0221] For example, one or more amino acid sequence positions of a lipocalin mutein can be mutated to introduce new reactive groups for conjugation with other compounds such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides, or proteins, or for the formation of non-naturally occurring disulfide bonds. Conjugated compounds, such as PEG and HES, can sometimes increase the serum half-life of the corresponding lipocalin mutein.

[0188]

[0222] In some embodiments, the reactive group of a lipocalin mutein may be naturally present in the amino acid sequence, such as a cysteine ​​residue naturally present in the amino acid sequence. In some other embodiments, such a reactive group may be introduced via mutagenesis. When a reactive group is introduced by mutagenesis, one possibility is to mutate an amino acid at an appropriate position with a cysteine ​​residue. Exemplary possibilities for such mutations to introduce a cysteine ​​residue into the amino acid sequence of an hTlc mutein include the substitution of Thr40 → Cys, Glu73 → Cys, Arg90 → Cys, Asp95 → Cys, and Glu131 → Cys in the wild-type sequence of hTlc (SEQ ID NO: 1). Exemplary possibilities for such mutations to introduce cysteine ​​residues into the amino acid sequence of an hNGAL mutein include introducing cysteine ​​residues at one or more of the sequence positions corresponding to sequence positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146, or 158 of the wild-type sequence of hNGAL (SEQ ID NO: 2). The resulting thiol moieties can be used to PEGylate or HESylate the mutein, for example, to increase the serum half-life of the respective lipocalin mutein.

[0189]

[0223] In some embodiments, artificial amino acids can be introduced into the amino acid sequence of lipocalin mutein to provide suitable amino acid side chains as new reactive groups for conjugating one of the above-mentioned compounds to lipocalin mutein.Generally, such artificial amino acids are designed to be highly reactive, so that they can be easily conjugated to desired compounds.Such artificial amino acids can be introduced by mutagenesis, for example, using artificial tRNA such as para-acetyl-phenylalanine.

[0190]

[0224] In some embodiments, the lipocalin muteins of the present disclosure are fused at their N- or C-terminus to a protein, protein domain, or peptide, such as an antibody, a signal sequence, and / or an affinity tag. In some other embodiments, the lipocalin muteins of the present disclosure are conjugated at their N- or C-terminus to a partner that is a protein, protein domain, or peptide, such as an antibody, a signal sequence, and / or an affinity tag.

[0191]

[0225] Affinity tags such as Strep-tag or Strep-tag II (Schmidt et al., J Mol Biol, 1996), c-myc tag, FLAG tag, His tag, or HA tag, or proteins such as glutathione-S-transferase, allow for easy detection and / or purification of recombinant proteins, and are examples of suitable fusion partners. Proteins with chromogenic or fluorescent properties, such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP), are also suitable fusion partners for the lipocalin muteins of the present disclosure. Generally, the lipocalin muteins of the present disclosure can be labeled with any suitable chemical or enzyme that directly or indirectly produces a detectable compound or signal through a chemical, physical, optical, or enzymatic reaction. For example, fluorescent or radioactive labels can be conjugated to the lipocalin muteins to generate fluorescence or X-rays as detectable signals. Alkaline phosphatase, horseradish peroxidase, and β-galactosidase are examples of enzyme labels (and optical labels at the same time) that catalyze the formation of a colored reaction product. In general, all labels commonly used for antibodies (except those used only for the sugar moiety of the Fc portion of immunoglobulins) can also be used for conjugation to the lipocalin muteins of the present disclosure.

[0192]

[0226] In some embodiments, the lipocalin muteins of the present disclosure may be fused or conjugated to a moiety that extends the serum half-life of the mutein (see, in this regard, International Patent Publication No. WO 2006 / 056464, where such a strategy is described with reference to a mutein of human neutrophil gelatinase-associated lipocalin (hNGAL) that has binding affinity for CTLA-4). Serum half-life extending moieties include PEG molecules, HES molecules, fatty acid molecules such as palmitic acid (Vajo and Duckworth, Pharmacol Rev, 2000), the Fc portion of an immunoglobulin, the C portion of an immunoglobulin, to name a few. H 3 domains, immunoglobulin C H The antibody may be a 4 domain, an albumin binding peptide, an albumin binding protein, or transferrin.

[0193]

[0227] In some embodiments, when PEG is used as the conjugation partner, the PEG molecule can be substituted, unsubstituted, linear, or branched. It can also be an activated polyethylene derivative. Examples of suitable compounds are those described in International Patent Publication No. WO 1999 / 64016, U.S. Patent No. 6,177,074, or U.S. Patent No. 6,403,564 in connection with interferons, or for other proteins such as PEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA), or PEG-superoxide dismutase (Fuertges and Abuchowski, Journal of Controlled Release, 1990). The molecular weight of such polymers, such as polyethylene glycol, can range from about 300 to about 70,000 daltons, including, for example, polyethylene glycols with molecular weights of about 10,000, about 20,000, about 30,000, or about 40,000 daltons. Additionally, carbohydrate oligomers and polymers, such as HES, can be conjugated to the muteins of the present disclosure for serum half-life extension, as described, for example, in US Pat. Nos. 6,500,930 or 6,620,413.

[0194]

[0228] In some embodiments, when the Fc portion of an immunoglobulin is used to extend the serum half-life of a lipocalin mutein of the present disclosure, SynFusion™ technology, commercially available from Syntonix Pharmaceuticals, Inc. (MA, USA), may be used. Use of this Fc fusion technology allows for the creation of longer acting biopharmaceuticals, which may consist of two copies of the mutein linked to the Fc region of an antibody to improve, for example, pharmacokinetics, solubility, and production efficiency.

[0195]

[0229] Examples of albumin-binding peptides that can be used to extend the serum half-life of lipocalin muteins are those having the consensus sequence Cys-Xaa1-Xaa2-Xaa3-Xaa4-Cys, where Xaa1 is Asp, Asn, Ser, Thr, or Trp; Xaa2 is Asn, Gln, His, Ile, Leu, or Lys; Xaa3 is Ala, Asp, Phe, Trp, or Tyr; and Xaa4 is Asp, Gly, Leu, Phe, Ser, or Thr as described in U.S. Patent Application Publication No. 2003 / 0069395 or Dennis et al. (2002). The albumin-binding protein fused or conjugated to lipocalin mutein to extend serum half-life can be bacterial albumin-binding protein, antibody, antibody fragment including domain antibody (see, for example, U.S. Patent No. 6,696,245), or lipocalin mutein with albumin binding activity. An example of bacterial albumin-binding protein is streptococcal protein G (Konig and Skerra, J Immunol Methods, 1998).

[0196]

[0230] In some embodiments, when the albumin-binding protein is an antibody fragment, it can be a domain antibody.Domain antibodies (dAbs) are engineered to precisely control biophysical properties and in vivo half-life, resulting in optimal safety and efficacy product profiles.Domain antibodies are commercially available, for example, from Domantis Ltd. (Cambridge, UK and MA, USA).

[0197]

[0231] In some embodiments, albumin itself (Osborn et al., J Pharmacol Exp Ther, 2002), or a biologically active fragment of albumin, can be used as a partner for the lipocalin muteins of the present disclosure to extend serum half-life. The term "albumin" includes all mammalian albumins, such as human serum albumin, bovine serum albumin, or rat albumin. Albumin or a fragment thereof can be recombinantly produced as described in U.S. Pat. No. 5,728,553 or European Patent Publication Nos. EP0330451 and EP0361991. Thus, recombinant human albumin (e.g., Recombumin® from Novozymes Delta Ltd., Nottingham, UK) can be conjugated or fused to the lipocalin muteins of the present disclosure.

[0198]

[0232] In some embodiments, when transferrin is used as a partner to extend the serum half-life of a lipocalin mutein of the present disclosure, the mutein can be genetically fused to the N-terminus, C-terminus, or both, of non-glycosylated transferrin. Non-glycosylated transferrin has a half-life of 14 to 17 days, and the transferrin fusion protein similarly extends its half-life. Transferrin carriers also provide high bioavailability, biodistribution, and circulation stability. This technology is commercially available from BioRexis (BioRexis Pharmaceutical Corporation, PA, USA). Recombinant human transferrin (DeltaFerrin™), used as a protein stabilizer / half-life extension partner, is also commercially available from Novozymes Delta Ltd. (Nottingham, UK).

[0199]

[0233] Yet another option for extending the half-life of the lipocalin muteins of the present disclosure is to fuse a long, unstructured, flexible glycine-rich sequence (e.g., a polyglycine having approximately 20-80 consecutive glycine residues) to the N- or C-terminus of the mutein. This approach, disclosed, for example, in International Patent Publication No. WO 2007 / 038619, is also known as "rPEG" (recombinant PEG).

[0200] E. Exemplary uses and applications of antibodies or antigen-binding domains thereof specific for CD228, and fusion proteins specific for CD137 and CD228.

[0234] In some embodiments, the fusion protein of the present disclosure can produce a synergistic effect by dual targeting CD137 and CD228. In some embodiments, the fusion protein of the present disclosure can produce a local anti-tumor effect by dual targeting CD137 and CD228. Therefore, the fusion protein of the present disclosure has many potential applications in medicine.

[0201]

[0235] In some embodiments, the present disclosure encompasses the use of one or more fusion proteins disclosed herein or one or more compositions comprising such fusion proteins to simultaneously bind to CD137 and CD228.

[0202]

[0236] The present disclosure also involves the use of one or more fusion proteins described for complex formation with CD137 and / or CD228.

[0237] Thus, in one embodiment of the present disclosure, the provided fusion proteins can be used for detecting CD137 and / or CD228. Such use may include contacting one or more of the fusion proteins with a sample suspected of containing CD137 and / or CD228 under appropriate conditions, thereby allowing the formation of a complex between the fusion protein and CD137 and / or CD228, and detecting the complex by an appropriate signal. The detectable signal can be caused by the label described above or by a change in physical properties due to binding, i.e., complex formation itself. One example is surface plasmon resonance, the value of which changes upon binding of one of the binding partners immobilized on a surface such as a gold foil.

[0203]

[0238] The fusion proteins of the present disclosure may be used for the isolation of CD137 and / or CD228. Such use may comprise contacting one or more of the fusion proteins with a sample suspected of containing CD137 and / or CD228 under appropriate conditions, thereby allowing the formation of a complex between the fusion protein and CD137 and / or CD228, and isolating the complex from the sample.

[0204]

[0239] In some aspects, the present disclosure provides diagnostic and / or analytical kits comprising one or more antibodies, antigen-binding domains thereof, or fusion proteins according to the present disclosure.

[0205]

[0240] In addition to diagnostic uses, in yet another aspect, the present disclosure contemplates pharmaceutical compositions comprising one or more antibodies, antigen-binding domains thereof, or fusion proteins of the present disclosure and a pharmaceutically acceptable excipient.

[0206]

[0241] Furthermore, in some embodiments, the provided antibodies, antigen-binding domains thereof, or fusion proteins can be used in therapy, for example, as anti-tumor and / or anti-infective agents and / or immunomodulators. In some embodiments, the provided antibodies, antigen-binding domains thereof, or fusion proteins can be used in the manufacture of medicaments, such as medicaments for the treatment of cancer, including CD228-positive cancers. In some embodiments, the disclosed antibodies, antigen-binding domains thereof, or fusion proteins can be used in methods for the prevention, amelioration, or treatment of human diseases, such as cancer, including CD228-positive cancers. In some embodiments, the disclosed fusion proteins can be used together with anti-PD-1 or anti-PD-L1 antibodies in methods for the prevention, amelioration, or treatment of cancer, including CD228-positive cancers. In some embodiments, the anti-PD-1 or anti-PD-L1 antibody is nivolumab, pembrolizumab, cemiplimab, dostalizumab, atezolizumab, avelumab, or durvalumab.

[0010] Accordingly, there is also provided a method for preventing, ameliorating, or treating a human disease, such as cancer, including CD228-positive cancer, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more antibodies, antigen-binding domains thereof, or fusion proteins of the present disclosure, or one or more compositions comprising such antibodies, antigen-binding domains thereof, or fusion proteins.

[0011] Accordingly, there is also provided a method for preventing, ameliorating, or treating a cancer, including CD228-positive cancer, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more fusion proteins of the present disclosure, or one or more compositions comprising such fusion proteins, and further comprising administering to the subject a therapeutically effective amount of an anti-PD-1 antibody or anti-PD-L1 antibody, or one or more compositions comprising such antibodies. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody is nivolumab, pembrolizumab, cemiplimab, dostalizumab, atezolizumab, avelumab, or durvalumab. In some embodiments, the cancer is a CD228-positive cancer.

[0207]

[0242] Examples of cancers that can be treated using the antibodies, antigen-binding domains thereof, or fusion proteins of the present disclosure include lung cancer, e.g., non-small cell lung cancer (NSCLC), melanoma, e.g., cutaneous melanoma or intraocular melanoma, pancreatic neoplasia or cancer, mesothelioma, colorectal neoplasia or cancer (CRC), thyroid cancer, breast cancer, cholangiocarcinoma, esophageal cancer, and head and neck cancer. In some embodiments, the cancer includes metastatic cancer.

[0208]

[0243] In some embodiments, the fusion proteins of the present disclosure can simultaneously target CD228-expressing tumor cells and activate lymphocytes of the host immune system adjacent to such tumor cells. In some embodiments, the fusion proteins of the present disclosure can increase targeted anti-tumor T cell activity, enhance anti-tumor immunity, and / or have a direct inhibitory effect on tumor growth, thereby producing synergistic anti-tumor results. In some embodiments, the fusion proteins of the present disclosure can activate immune responses in the tumor microenvironment. In some embodiments, the fusion proteins of the present disclosure can reduce the side effects of effector lymphocytes on healthy cells, i.e., off-target toxicity, for example, by locally inhibiting oncogene activity and / or inducing lymphocyte activation.

[0209]

[0244] In some embodiments, the present disclosure encompasses the use of a fusion protein of the present disclosure or a composition comprising a provided fusion protein to induce localized lymphocyte responses in the vicinity of CD228-positive tumor cells. Accordingly, in some embodiments, the present disclosure provides a method for inducing a localized lymphocyte response in the vicinity of CD228-positive tumor cells, comprising applying one or more fusion proteins of the present disclosure or one or more compositions comprising such fusion proteins. By "localized," we mean that upon simultaneous binding to T cells via CD137 and engagement with CD228-positive tumor cells, the T cells produce cytokines, particularly IL-2 and / or IFN-gamma, in the vicinity of the CD228-positive cells. Such cytokines reflect T cell activation, and the T cells may be able to kill CD228-positive cells directly or indirectly by attracting other killer cells, such as cytotoxic T cells and / or NK cells. The provided fusion proteins can also be used to increase the secretion of cytotoxic factors such as perforin, granzyme B, and granzyme A by T cells in the vicinity of CD228-positive tumor cells.

[0210]

[0245] In some embodiments, the present disclosure encompasses the use of a fusion protein of the present disclosure, or a composition comprising such a fusion protein, to costimulate T cells and / or activate the downstream signaling pathway of CD137. Preferably, the provided fusion proteins costimulate T cells and / or activate the downstream signaling pathway of CD137 when engaged with CD228-positive tumor cells. Accordingly, the present disclosure provides a method of inducing T lymphocyte proliferation / activity and / or activating the downstream signaling pathway of CD137, preferably comprising applying one or more fusion proteins of the present disclosure and / or one or more compositions comprising such fusion protein(s) when engaged with CD228-expressing tumor cells.

[0211]

[0246] In some embodiments, the present disclosure encompasses the use of a fusion protein of the present disclosure, or a composition comprising such a fusion protein, to induce CD137 clustering and activation in T cells and direct such T cells to CD228-expressing tumor cells.

[0212]

[0247] In some embodiments, the present disclosure encompasses the use of a fusion protein of the present disclosure, or a composition comprising such a fusion protein, to induce an increase in CD8+ T cell mitochondrial content and / or a decrease in CD8+ T cell mitochondrial depolarization in the vicinity of CD228-positive tumor cells, comprising applying one or more fusion proteins of the present disclosure and / or one or more compositions comprising such fusion proteins to tissue comprising a tumor. In some embodiments, the present disclosure encompasses the use of a fusion protein of the present disclosure, or a composition comprising such a fusion protein, to induce an increase in CD8+ T cell mitochondrial content, comprising applying one or more fusion proteins of the present disclosure and / or one or more compositions comprising such fusion proteins to tissue comprising a tumor. In some embodiments, the present disclosure encompasses the use of a fusion protein of the present disclosure, or a composition comprising such a fusion protein, to induce mitochondrial depolarization in CD8+ T cells in the vicinity of CD228-positive tumor cells, comprising applying one or more fusion proteins of the present disclosure and / or one or more compositions comprising such fusion proteins to tissue comprising a tumor.

[0213]

[0248] In some embodiments, the present disclosure encompasses the use of a fusion protein of the present disclosure, or a composition comprising such a fusion protein, to stimulate exhausted CD8+ T cell proliferation in the vicinity of CD228-positive tumor cells, the use comprising applying one or more fusion proteins of the present disclosure and / or one or more compositions comprising such fusion proteins to tissue comprising a tumor. In some embodiments, the present disclosure encompasses the use of a fusion protein of the present disclosure, or a composition comprising such a fusion protein, to stimulate exhausted CD8+ T cell proliferation in the vicinity of CD228-positive tumor cells, the use comprising applying one or more fusion proteins of the present disclosure and / or one or more compositions comprising such fusion proteins and applying an anti-PD-1 antibody or anti-PD-L1 antibody, or one or more compositions comprising such an antibody, to tissue comprising a tumor. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody is nivolumab, pembrolizumab, cemiplimab, dostalizumab, atezolizumab, avelumab, or durvalumab.

[0214] F. Exemplary provided are antibodies or antigen-binding domains thereof specific for CD228, and the production of fusion proteins specific for CD137 and CD228.

[0249] In some embodiments, the present disclosure provides nucleic acid molecules (e.g., DNA or RNA) comprising a nucleotide sequence encoding the provided antibodies, antigen-binding domains thereof, or fusion proteins. In some embodiments, the present disclosure encompasses vectors containing the provided nucleic acid molecules. In some embodiments, the present disclosure encompasses host cells containing the provided nucleic acid molecules or vectors. Because the degeneracy of the genetic code permits the substitution of certain codons with other codons that specify the same amino acid, the present disclosure is not limited to the particular nucleic acid molecules encoding the antibodies, antigen-binding domains thereof, or fusion proteins described herein, but rather encompasses all nucleic acid molecules comprising a nucleotide sequence encoding a functional antibody, antigen-binding domain, or fusion protein. In this regard, the present disclosure also relates to nucleotide sequences encoding the provided antibodies, antigen-binding domains thereof, or fusion proteins.

[0215]

[0250] Nucleic acid molecules, such as DNA, contain sequence elements containing information for transcriptional and / or translational regulation. When such sequences are "operably linked" to a nucleotide sequence encoding a protein, they are said to "permit expression of the nucleic acid molecule" or "permit expression of the nucleotide sequence." An operable linkage is one in which the regulatory sequence elements and the sequence to be expressed are connected in a manner that allows gene expression. While the exact nature of the regulatory regions required for gene expression can vary between species, these regions generally include a promoter, which in prokaryotes contains both the promoter itself, i.e., the DNA element that directs the initiation of transcription, and the DNA element that, once transcribed into RNA, signals the initiation of translation. Such promoter regions usually include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35 / -10 box and Shine-Dalgarno element in prokaryotes, and the TATA box, CAAT sequence, and 5'-capping element in eukaryotes. These regions may also contain enhancer or repressor elements, as well as translational signals and leader sequences for targeting the native protein to specific compartments of the host cell.

[0216]

[0251] In addition, the 3' non-coding sequences may contain regulatory elements involved in transcription termination, polyadenylation, etc. However, if these termination sequences do not function satisfactorily in a particular host cell, they may be substituted with signals that are functional in that cell.

[0217]

[0252] Therefore, the nucleic acid molecule of the present disclosure can be "operably linked" to one or more regulatory sequences, such as a promoter sequence, to allow the expression of the nucleic acid molecule. In some embodiments, the nucleic acid molecule of the present disclosure includes a promoter sequence and a transcription termination sequence. Suitable prokaryotic promoters include, for example, the tet promoter, the lacUV5 promoter, or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells include the SV40 promoter or the CMV promoter.

[0218]

[0253] In some embodiments, a nucleic acid molecule encoding a lipocalin mutein disclosed in the present application may be "operably linked" to another nucleic acid molecule encoding an antibody of the present disclosure to allow expression of a fusion protein as disclosed herein.

[0219]

[0254] In some embodiments, provided methods can include mutagenizing at least one nucleic acid molecule encoding mature hTlc at nucleotide triplets encoding one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of hTlc (SEQ ID NO: 1) to obtain a lipocalin mutein that is included in a provided fusion protein. In some embodiments, provided methods may include subjecting at least one nucleic acid molecule encoding mature hNGAL to mutagenesis at nucleotide triplets encoding one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of hNGAL (SEQ ID NO: 2) to obtain a lipocalin mutein that is included in a provided fusion protein. In some embodiments, provided methods may include subjecting at least one nucleic acid molecule encoding mature hNGAL to mutagenesis at nucleotide triplets encoding one or more positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of hNGAL (SEQ ID NO: 2) to obtain a lipocalin mutein that is included in a provided fusion protein.

[0220]

[0255] Additionally, with respect to hTlc or hNGAL muteins of the present disclosure that are included in fusion proteins, in some embodiments, the naturally occurring disulfide bond between Cys61 and Cys153 or between Cys76 and Cys175, respectively, can be eliminated, and thus such muteins can be produced in cellular compartments that have a reducing redox environment, for example, in the cytoplasm of Gram-negative bacteria.

[0221]

[0256] Furthermore, with respect to the hTlc or hNGAL muteins provided herein that are included in fusion proteins, the present disclosure also includes nucleic acid molecules encoding such muteins, which in some embodiments may contain one or more additional mutations outside the indicated sequence positions of experimental mutagenesis. Such mutations are often tolerated or may even be advantageous if they contribute, for example, to improved folding efficiency, serum stability, thermal stability, or ligand-binding affinity of the lipocalin mutein and / or fusion protein.

[0222]

[0257] In some embodiments, the provided nucleic acid molecules can also be part of a vector or any other type of cloning vehicle, such as a plasmid, phagemid, phage, baculovirus, cosmid, or artificial chromosome.

[0223]

[0258] In some embodiments, the provided nucleic acid molecule may be contained in a phagemid.In this context, a phagemid vector refers to a vector encoding the intergenic region of a temperate phage such as M13 or f1, or its functional portion fused to a target cDNA.For example, in some embodiments, after hyperinfecting bacterial host cells with such a provided phagemid vector and suitable helper phage (for example, M13K07, VCS-M13 or R408), intact phage particles are produced, thereby allowing the coded heterologous cDNA to be physically linked to the corresponding polypeptide displayed on the phage surface (Lowman, Annu Rev Biophys Biomol Struct, 1997; Rodi and Makowski, Curr Opin Biotechnol, 1999).

[0224]

[0259] According to various embodiments, the cloning vehicle may contain, in addition to the regulatory sequences described above and nucleic acid sequences encoding the antibodies, antigen-binding domains thereof, or fusion proteins described herein, replication and control sequences derived from a species compatible with the host cell used for expression, and a selectable marker that confers a selectable phenotype on transformed or transfected cells. Many suitable cloning vectors are known in the art and are commercially available.

[0225]

[0260] The present disclosure also relates, in some embodiments, to methods for producing the antibodies, antigen-binding domains, or fusion proteins of the present disclosure, starting with a nucleic acid encoding the antibody, antigen-binding domain, or fusion protein, or any subunit(s) thereof, using genetic engineering methods. In some embodiments, the provided methods can be performed in vivo, and the provided antibodies, antigen-binding domains, or fusion proteins can be produced, for example, in a bacterial or eukaryotic host organism and then isolated from the host organism or a culture thereof. The antibodies, antigen-binding domains, or fusion proteins of the present disclosure can also be produced in vitro, for example, using an in vitro translation system.

[0226]

[0261] For in vivo production of antibodies, antigen-binding domains thereof, or fusion proteins, nucleic acids encoding such antibodies, antigen-binding domains thereof, or fusion proteins may be introduced into suitable bacterial or eukaryotic host organisms using recombinant DNA techniques well known in the art. In some embodiments, DNA molecules encoding the antibodies, antigen-binding domains thereof, or fusion proteins described herein (e.g., SEQ ID NOS: 91-94, and the sequence pairs SEQ ID NOS: 91 and 88, 93 and 90, 87 and 92, or 89 and 94), particularly cloning vectors containing the coding sequences for such antibodies, antigen-binding domains thereof, or fusion proteins, can be transformed into host cells capable of expressing the genes. Transformation can be carried out using standard techniques. Thus, the present disclosure also covers host cells containing the nucleic acid molecules disclosed herein.

[0227]

[0262] In some embodiments, transformed host cells can be cultured under conditions suitable for expression of nucleotide sequences encoding antibodies, antigen-binding domains thereof, or fusion proteins of the present disclosure. In some embodiments, host cells are prokaryotic cells such as Escherichia coli (E. coli) or Bacillus subtilis, or eukaryotic cells 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.

[0228]

[0263] In some embodiments, when a lipocalin mutein of the present disclosure, including those contained in the fusion proteins disclosed herein, contains an intramolecular disulfide bond, it may be preferable to use an appropriate signal sequence to direct the nascent protein to a cellular compartment having an oxidative redox environment. Such an oxidative environment may be provided by the periplasm of Gram-negative bacteria such as E. coli, the extracellular environment of Gram-positive bacteria, or the lumen of the endoplasmic reticulum of eukaryotic cells, and typically promotes the formation of structural disulfide bonds.

[0229]

[0264] In some embodiments, the antibody, its antigen-binding domain, or fusion protein of the present disclosure can be produced in the cytoplasm of a host cell, preferably E. coli. In this case, the provided antibody, its antigen-binding domain, or fusion protein can be directly obtained in a soluble and folded state, or recovered in the form of inclusion bodies and then re-denatured in vitro. Another option is to use a specific host strain with an oxidative intracellular environment, which can allow disulfide bond formation in the cytoplasm (Venturi et al., J Mol Biol, 2002).

[0230]

[0265] In some embodiments, the antibodies, antigen-binding domains thereof, or fusion proteins of the present disclosure described herein need not necessarily be generated or produced, in whole or in part, by genetic engineering. Rather, such proteins can be obtained by any of a number of conventional, well-known techniques, such as straightforward organic synthesis strategies, solid-phase-assisted synthesis techniques, commercially available automated synthesizers, or in vitro transcription and translation. For example, promising antibodies, antigen-binding domains thereof, or fusion proteins or lipokine muteins contained in such fusion proteins can be identified using molecular modeling, synthesized in vitro, and investigated for binding activity to the target(s) of interest. Methods for 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).

[0231]

[0266] In some embodiments, an antibody, antigen-binding domain thereof, or fusion protein of the disclosure may be produced by in vitro transcription / translation using well-established methods known to those skilled in the art.

[0232]

[0267] In some further embodiments, the antibodies, antigen-binding domains thereof, or fusion proteins described herein may be prepared using conventional recombinant techniques alone or in combination with conventional synthetic techniques.

[0233]

[0268] Furthermore, in some embodiments, a fusion protein according to the present disclosure may be obtained by conjugating the individual subunits comprised in the fusion protein, e.g., the antibody and the mutein, to one another. Such conjugation may be achieved, for example, by any form of covalent or non-covalent linkage using conventional methods.

[0234]

[0269] Those skilled in the art will understand methods useful for preparing antibodies, their antigen-binding domains, or fusion proteins contemplated by the present disclosure, even though the protein or nucleic acid sequences are not explicitly disclosed herein. Generally, such amino acid sequence modifications include, for example, directed mutagenesis of single amino acid positions to simplify subcloning of protein genes or portions thereof by incorporating a specific restriction enzyme cleavage site. These mutations can also be incorporated to further improve the affinity of the antibody, its antigen-binding domain, or fusion protein for its target(s) (e.g., CD137 and / or CD228). Furthermore, mutations can be introduced, as needed, to modulate one or more properties of the protein, such as improving folding stability, serum stability, protein resistance, or water solubility, or reducing aggregation tendency.

[0235]

[0270] Further objects, advantages, and features of the present disclosure will become apparent to those skilled in the art upon examination of the following examples and accompanying drawings, which are not intended to be limiting. Thus, while the present disclosure has been specifically disclosed by exemplary embodiments and optional features, it should be understood that modifications and variations of the disclosure embodied therein disclosed herein may be resorted to by those skilled in the art, and such modifications and variations are considered to be within the scope of the present disclosure.

[0236] VI. Working Examples

[0271] [Example]

[0237] Example 1: Generation of anti-CD228 antibodies

[0272] A human immunoglobulin transgenic rat strain (OmniRat®; OMT, Inc.) was used to develop monoclonal antibody-expressing hybridoma cells. OmniRat® contains a chimeric human / rat IgH locus (containing 22 human VHs, with all human D and JH segments linked in their natural configuration to the rat CH locus) together with a fully human IgL locus (12 Vκs linked to Jκ-Cκs and 16 Vλs linked to Jλ-Cλs). See Osborn et al. (2013) J Immunol 190(4):1481-1490; WO2014 / 093908. In response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity IgG monoclonal antibodies.

[0238]

[0273] Transgenic rats were immunized with recombinant human CD228 protein. Subcutaneous injections included 48.2 μg of recombinant protein in complete Freund's adjuvant on day 1 and 48 μg of recombinant protein in incomplete Freund's adjuvant on days 33, 81, and 127.

[0239]

[0274] The presence of antibodies directed against human CD228 in the serum of transgenic rats was monitored by flow cytometry on days 33, 81, and 127 using RPMI-7951 cells engineered to stably express human CD228. Transgenic rats with detectable immune responses were boosted 5 and 7 days before harvesting the spleen and lymph nodes. The boosts before organ harvest included intravenous injection of 90 μg and intraperitoneal injection of 18 μg of recombinant human CD228 protein suspended in phosphate-buffered saline (PBS).

[0240]

[0275] Spleen cells and lymphocytes showing B cell lineage and specificity for fluorescently labeled recombinant human CD228 were sorted as single cells into cell lysis buffer by flow-activated cell sorting (FACS). RNA from single B cells was reverse transcribed into cDNA and amplified using primer sets against known human variable regions. Amplicons were purified for Sanger sequencing with appropriate sequencing primers, and heavy and light chain sequences were subsequently identified and annotated using IgBLAST.

[0241]

[0276] Highly productive heavy and light chain sequences of human variable regions identified from single B cell cloning and Sanger sequencing were selected for gene synthesis and cloned into expression vectors using conventional cloning techniques. The expression vectors encoded human IgG1, kappa, or lambda constant regions, which were in frame with and downstream of the human variable regions. Antibody expression constructs were verified by Sanger sequencing.

[0242]

[0277] To generate full-length antibodies, heavy and light chain expression vectors were co-transfected into ExpiCHO cells according to the manufacturer's protocol (Life Technologies). Nine days after transfection, cell culture supernatants were collected by centrifugation. The filtered culture supernatants were spiked with 10% Triton-X (final concentration 0.1%) and left overnight at 4°C on a shaking platform. The supernatants were loaded onto a 5mL HiTrap MabSelectSuRe column at 2.5mL / min using an AKTA Avant 25 chromatography system. The column was then washed with 5 column volumes (CV) of end-wash buffer (1x PBS, 0.1% Triton-X), 5CV of high-salt buffer (1x PBS, 0.5M NaCl), and 7.5CV of standard 1x PBS (pH 7.4). Protein was eluted with 1 CV of 20 mM citrate (pH 3) and immediately buffer exchanged into 1× PBS (pH 7.4) using a HiPrep Sephadex G-25 Desalting column. Fractions were determined by A280 UV watch parameters to allow optimal collection of purified protein.

[0243]

[0278] Figures 2A and 2B show the CDR sequences of antibodies as defined by Kabat (Figure 2A) and IMGT (Figure 2B). [Example]

[0244]

[0279] Example 2: Binding of anti-CD228 antibodies to recombinant human CD228

[0280] Humanized antibodies specific for human melanotransferrin (CD228) were evaluated for binding to recombinant human CD228 (R&D Systems) by ELISA. Antibodies were titrated on plates coated with 1 μg / ml recombinant human CD228, detected with HRP-conjugated goat anti-human IgG secondary reagent (Thermo), and developed with TMB substrate (Thermo). OD450 values ​​were read on a plate reader using SoftMax Pro software. OD450 values ​​from each experiment were transferred to GraphPad Prism 8 for plotting and analysis. The results are shown in Figure 3. [Example]

[0245]

[0281] Example 3: Binding of anti-CD228 antibodies to cells

[0282] Humanized antibodies specific for human CD228 were evaluated for relative binding to CD228-expressing cancer cell lines. Cells were incubated with titrations of Alexa-647-labeled humanized antibody clones, washed, and fluorescence intensity was assessed by flow cytometry on an Attune NXT flow cytometer. MFI values ​​were transferred to GraphPad Prism 8 for plotting and analysis. EC50 values ​​were determined by nonlinear regression. Numbers next to cell line names reflect surface CD228 copy numbers estimated using the QIFIKIT quantitative analysis kit (Agilent).

[0246]

[0283] The results are shown in Figures 4A-4C as raw mean fluorescence intensity (MFI) values. Figure 4D shows the EC values ​​of each antibody for each cell line. 50 Indicates the value. [Example]

[0247]

[0284] Example 4: Binding Profiles of Anti-CD228 Antibodies

[0285] The binding kinetics and affinity of human CD228 bearing a C-terminal polyhistidine tag (R&D Systems) were determined by biolayer interferometry (BLI) using the Octet® RED384 system (Sartorius). An anti-human antibody capture AHC (GE Healthcare) biosensor was used for the analysis. Anti-CD228 antibodies OMT8, OMT24, OMT30, OMT35, and OMT36 (IgG) were then applied to the chip surface at 0.5 μg / mL in HBS-EP+ buffer for 180 seconds, followed by capture with an anti-human IgG-Fc antibody. After each capture step, the biosensor was washed with HBS-EB+ blank. For affinity determination, dilutions of recombinant huCD228 (100 nM, 40 nM, 16 nM, 6.4 nM, 2.6 nM, and 1.0 nM) or a blank were prepared in HBS-EP+ buffer and applied to the biosensor. Binding assays were performed with a contact time of 300 seconds and a dissociation time of 1,200 seconds. All measurements were performed at 25°C. Fresh AHC biosensors were used for each analysis. Data were evaluated using Satorius Octet® Data Analysis Software (v12.0), and the results are shown in Figures 5A-5E. A single reference was used and the raw data were fitted using a 1:1 binding model. [Example]

[0248]

[0286] Example 5: Lack of cross-reactivity of anti-CD228 antibodies to transferrin and lactotransferrin

[0287] Humanized antibodies specific for human melanotransferrin (CD228) were evaluated by ELISA for binding to lactotransferrin and transferrin, related transferrin family members. The antibodies were titrated on plates coated with 1 μg / mL recombinant human melanotransferrin (CD228), lactotransferrin, or transferrin, and detected with HRP-conjugated goat anti-human IgG secondary reagent (Thermo) developed with TMB substrate (Thermo). OD450 values ​​were read using a plate reader equipped with SoftMax Pro software. OD450 values ​​from each experiment were imported into GraphPad Prism 8 for plotting and analysis. These results, shown in Figures 6A-6C, demonstrate no cross-reactive binding of the anti-CD228 antibody to other transferrin family members. [Example]

[0249]

[0288] Example 6: Cross-reactivity of anti-CD228 antibodies to non-human CD228

[0289] The CD228-negative human melanoma cell line RPMI-7951 was engineered to express cynomolgus monkey CD228 to test the binding of anti-CD228 antibody clones. 50,000 RPMI-7951 cells were incubated with titrations of the humanized antibody clones, washed with staining buffer to remove excess antibody, and incubated with 250 ng / mL of a fluorochrome-labeled monoclonal antibody against human IgG1 (Thermo) to detect bound antibody. Results are shown in Figure 7A as mean fluorescence intensity (MFI) determined by measurement on an Attune NXT flow cytometer (Thermo).

[0250]

[0290] The reactivity of CD228 antibody clones to recombinant his-tagged cynomolgus CD228 was tested by ELISA. Plates were coated with 1 μg / ml of recombinant his-cynomolgus CD228 in phosphate-buffered saline, blocked with 250 μL of Superblock Blocking Buffer (Thermo). The antibody was titrated across the plate, and bound antibody was detected with HRP-conjugated goat anti-human IgG (Sigma) and developed with TMB substrate (Thermo). OD450 values ​​were read using a plate reader equipped with SoftmaxPro software. The results are shown in Figure 7B.

[0251]

[0291] Humanized antibodies specific for human CD228 were evaluated for binding to recombinant cynomolgus monkey and mouse CD228 by ELISA. Antibodies were titrated on plates coated with 1 μg / mL recombinant human, cynomolgus monkey, and mouse CD228, detected with HRP-conjugated goat anti-human IgG secondary reagent (Thermo), developed with TMB substrate (Thermo), and OD450 values ​​were read using a plate reader equipped with SoftMax Pro software. OD450 values ​​for each experiment are shown in Figures 8A-8C.

[0252]

[0292] These results showed that a subset of antibody clones (OMT30, OMT35, OMT24, OMT8, OMT36, and L235) were cross-reactive with cynomolgus monkey CD228 and showed minimal cross-reactivity with mouse CD228. [Example]

[0253]

[0293] Example 7: Anti-CD228 antibody cross-competition assay

[0294] Fluorochrome-labeled humanized monoclonal antibodies specific for human CD228 were evaluated for binding to HT-1080 and SK-MEL-5 tumor cell lines in competition with unlabeled humanized antibodies to identify potential shared epitopes. HT-1080 and SK-MEL-5 cells were preincubated with 1 μg / mL of unlabeled humanized antibody in staining buffer for 30 minutes at 4°C, washed with staining buffer, and then incubated with 200 ng / mL of the A647-labeled version. Bound fluorochrome-labeled antibody was determined by flow cytometry using an Attune NXT flow cytometer. Results are shown in Figure 9 as the percent mean fluorescence intensity compared to preincubation with a control nonbinding human IgG1 (Sigma). Clones with direct epitopes or steric competition show low staining percentage values. These results indicate that antibody clones 28, 32, and 35 share closely related epitopes, while clones 8, 11, 24, 30, and 36 likely bind to unique CD228 epitopes. [Example]

[0254]

[0295] Example 8: Internalization of antibody clones in CD228+ tumor cell lines

[0296] Humanized antibodies specific for human CD228 were evaluated for relative internalization in the melanoma cell line SK-MEL-5 and lung cancer cell line Calu-1, which express CD228, respectively. Cells were incubated with 2 μg / ml anti-CD228 antibody, washed three times to remove free antibody, and incubated at 37°C in 5% CO2 for the indicated times. At each time point, cells were fixed (BD Cytofix) and stained for bound human IgG1 with a fluorochrome-conjugated anti-human IgG1 antibody (Invitrogen). At the end of the time course, cells were assessed by FACS for the mean fluorescence intensity of surface-bound antibody on an Attune NXT flow cytometer. Internalization results are shown in Figure 10 as a percentage of the MFI relative to the starting MFI at time point 0. These results demonstrate a range of internalization rates among the antibody clones, with antibody clone 35 internalizing at the fastest rate. [Example]

[0255]

[0297] Example 9: Expression and analysis of representative fusion proteins

[0298] Representative antibody-lipocalin mutein fusion proteins were generated by fusing a CD228-specific antibody and a CD137-specific lipocalin mutein, such as the lipocalin mutein of SEQ ID NO: 40, to each other via a linker, such as the unstructured (G4S)3 linker of SEQ ID NO: 13, and simultaneously engaging CD228 and CD137. Two different exemplary CD228-specific antibodies were used. The first CD228-specific antibody had a heavy chain provided by SEQ ID NO: 75 (or comprising the heavy chain variable domain of SEQ ID NO: 70, or comprising the heavy chain CDRs (HCDR1, HCDR2, HCDR3) of SEQ ID NOs: 58-60) and a light chain provided by SEQ ID NO: 76 (or comprising the light chain variable domain of SEQ ID NO: 71, or comprising the light chain CDRs (LCDR1, LCDR2, LCDR3) of SEQ ID NOs: 61-63). The second CD228-specific antibody had a heavy chain provided by SEQ ID NO: 78 (or comprising the heavy chain variable domain of SEQ ID NO: 72, or comprising the heavy chain CDRs (HCDR1, HCDR2, HCDR3) of SEQ ID NOs: 64-66) and a light chain provided by SEQ ID NO: 79 (or comprising the light chain variable domain of SEQ ID NO: 73, or comprising the light chain CDRs (LCDR1, LCDR2, LCDR3) of SEQ ID NOs: 67-69). Various formats of fusion proteins generated are shown in Figures 11A-11I, including fusion proteins that are bivalent with respect to CD137 (e.g., as shown in Figures 11A-11D), or tetravalent with respect to CD137 (e.g., as shown in Figures 11E-11H), or have even higher valency with respect to CD137 (e.g., as shown in Figure 11I). Exemplary fusion proteins of SEQ ID NOs: 80 and 76 (encoded by the nucleotide sequences of SEQ ID NOs: 91 and 88), SEQ ID NOs: 82 and 79 (encoded by the nucleotide sequences of SEQ ID NOs: 93 and 90), SEQ ID NOs: 75 and 81 (encoded by the nucleotide sequences of SEQ ID NOs: 87 and 92), and SEQ ID NOs: 78 and 83 (encoded by the nucleotide sequences of SEQ ID NOs: 89 and 94) were bivalent for CD137 with a CD137-specific lipocalin mutein fused to the C-terminus of each heavy chain (Figure 11A) or each light chain (Figure 11B).

[0256]

[0299] The CD228-specific antibody and all antibody-lipocalin mutein fusion proteins described in this example had an engineered IgG4 backbone containing the S228P mutation to minimize IgG4 half-antibody exchange in vitro and in vivo (Silva et al., J Biol Chem, 2015). Additional mutations in the IgG4 backbone, including any one or more of the mutations F234A, L235A, M428L, N434S, M252Y, S254T, and T256E, may be present in all antibodies and fusion proteins described herein. The F234A and L235A mutations may be introduced to reduce ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010). The M428L and N434S mutations, or the M252Y, S254T, and T256E mutations, can be introduced to extend serum half-life (Dall'Acqua et al., J Biol Chem, 2006; Zalevsky et al., Nat Biotechnol, 2010). All antibodies were expressed without a carboxy-terminal lysine to avoid heterogeneity.

[0257]

[0300] The present disclosure also embodies asymmetric antibody-lipocalin mutein fusion formats, for example, where one light chain of the antibody may be fused to a lipocalin mutein and the other may not.

[0301] Exemplary fusion protein constructs were generated by gene synthesis and cloned into mammalian expression vectors. They were then transiently expressed in suspension-adapted CHO-K1 cells. The concentration of fusion protein in the cell culture medium was measured by BLItz (ForteBio) using a Protein A biosensor for kinetic assays. The resulting titers are summarized in Table 1.

[0258] [Table 1]

[0259]

[0303] For each construct, 1 liter of the aforementioned cell culture medium was purified using Protein A chromatography (MabSelect SuRe, Cytiva) followed by size-exclusion chromatography (SEC) in a buffer consisting of 20 mM histidine, 60 mM NaCl, pH 6.0. The purity of the final product, i.e., monomer content, was determined by analytical size-exclusion chromatography using an Agilent AdvanceBio SEC column (300 Å, 2.7 μm, 7.8 × 300 mm) and DPBS as the running buffer at 0.8 ml / min. The yield and monomer content after the two-step purification process are summarized in Table 2.

[0260] [Table 2] [Example]

[0261]

[0305] Example 10: Binding of fusion proteins to huCD228, cyCD228 or huCD137 determined by surface plasmon resonance (SPR)

[0306] The binding kinetics and affinity of exemplary fusion proteins to huCD228-His, cyCD228-His, or huCD137-His (recombinant human CD228, cynomolgus monkey CD228, or human CD137 with a C-terminal polyhistidine tag, R&D Systems) were determined by surface plasmon resonance (SPR) using a Biacore 8K (GE Healthcare).

[0262]

[0307] Anti-human IgG Fc antibody (GE Healthcare) was immobilized on a CM5 sensor chip according to the manufacturer's instructions. The fusion proteins to be tested (SEQ ID NOs: 80 and 76, 82 and 79, 75 and 81, and 78 and 83) were then captured by the anti-human IgG-Fc antibody on the chip surface at 0.5 μg / mL in HBS-EP+ buffer at a flow rate of 10 μL / min for 180 seconds. After each capture step, the needle was washed. The hIgG1 versions of the two anti-CD228 antibodies contained in the fusion proteins (SEQ ID NOs: 74 / 76 and 77 / 79, respectively) were also tested as controls.

[0263]

[0308] To determine affinity, dilutions of recombinant huCD228-His, cyCD228-His, or huCD137-His (500 nM, 125 nM, 31.25 nM, and 7.8 nM) or a blank were prepared in HBS-EP+ buffer and applied to the prepared chip surface. Binding assays were performed with a contact time of 180 s, a dissociation time of 1200 s, and a flow rate of 30 μL / min. All measurements were performed at 25°C. Chip surface regeneration was achieved by injecting 3 M MgCl2 for 120 s. Three conditioning cycles were performed prior to protein measurement. Data were evaluated using Biacore 8K Evaluation software (v1.1.1). Double referencing was used, and raw data was fitted using a 1:1 binding model.

[0264]

[0309] k of representative fusion proteins and control antibodies on , k off , and the resulting equilibrium dissociation constant (K D ) are summarized in Table 3. All bispecific fusion proteins (SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, and SEQ ID NOs: 78 and 83) bound to huCD228 and cyCD228 with nanomolar affinities similar to their respective antibodies. All bispecific fusion proteins bound CD137 with affinities in the single-digit nanomolar range.

[0265] [Table 3] [Example]

[0266]

[0311] Example 11: Binding of fusion proteins to CD228 or CD137 in enzyme-linked immunosorbent assay (ELISA)

[0312] Enzyme-linked immunosorbent assays (ELISA) were used to determine the binding ability of exemplary fusion proteins to human and cynomolgus monkey CD228 or CD137.

[0267]

[0313] Recombinant huCD228-His or cyCD228-His (human or cynomolgus monkey CD228 with a C-terminal polyhistidine tag, R&D Systems or Sino Biologics) was coated onto microtiter plates at a concentration of 1 μg / mL in PBS overnight 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 hour at room temperature. After washing five times with 100 μL of PBS-0.05% T, various concentrations (maximum concentration: 200 nM) of exemplary fusion proteins (SEQ ID NOs: 80 and 76, 82 and 79, 75 and 81, and 78 and 83) and the hIgG1 versions of the two anti-CD228 antibodies contained in the fusion proteins (SEQ ID NOs: 74 / 76 and 77 / 79, respectively) were added to the wells and incubated at room temperature for 1 hour, followed by another washing step. The binding molecules under study were detected by incubation with anti-human IgG Fab-HRP (Jackson Laboratory) diluted 1:5000 in PBS-0.1% T-2% BSA. After an additional washing step, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well, and the fluorescence intensity was detected using a fluorescence microplate reader.

[0268]

[0314] The same ELISA setup was also used to determine the binding ability of fusion proteins to CD137, except that huCD137-His (human CD137 with a C-terminal polyhistidine tag, R&D Systems) or cyCD137-Fc (cynomolgus monkey CD137 fused to an Fc at the C-terminus) were coated onto microtiter plates instead. Test drugs were similarly titrated, and bound drugs were detected with anti-human IgG Fab-HRP (Jackson Laboratory).

[0269]

[0315] The results of an exemplary experiment are shown in EC 50 The resulting EC values ​​and maximum signals are shown in Figures 12A-12D, along with the resulting fit curves from a 4PL fit where the slope was fixed to unity and the value and maximum signal were free parameters. 50 The values ​​are provided in Table 4.

[0270]

[0316] Measured EC of fusion proteins against human CD228 and cynomolgus monkey CD228 (SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, and SEQ ID NOs: 78 and 83) 50 The values ​​were similar for each antibody. These antibodies showed curves approximately 2-fold higher than the plateau, but such a difference was not observed when detecting using an anti-human IgG Fc-HRP antibody, which may be due to the accessibility of the detection antibody (data not shown). Regarding binding to human CD137, all fusion proteins exhibited EC 50 The fusion proteins also showed cross-reactivity with cynomolgus monkey CD137, with values ​​in the upper single-digit nanomolar range. Overall, the EC values ​​of the HC fusion proteins (SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79) were 50 Values ​​are the EC of the corresponding LC fusion proteins (SEQ ID NOs: 75 and 81, SEQ ID NOs: 78 and 83). 50 was slightly better than the value.

[0271] [Table 4] [Example]

[0272]

[0318] Example 12: Simultaneous binding of fusion proteins to CD228 and CD137 in ELISA

[0319] A dual binding ELISA format was used to demonstrate simultaneous binding of exemplary fusion proteins to CD228 and CD137.

[0273]

[0320] Recombinant huCD228-His in PBS (1 μg / mL) was coated onto microtiter plates overnight at 4°C. After each incubation step, the plates were washed five times with 100 μL of PBS-0.05% T. The plates were blocked with 2% BSA (w / v) in PBS-0.1% T at room temperature for 1 hour and then washed again. Different concentrations (maximum concentration: 200 nM) of the tested fusion proteins and a control anti-CD228 antibody were added to the wells and incubated at room temperature for 1 hour, followed by a washing step. Biotinylated huCD137-His (huCD137-His-Bio, Sino Biological) was then added at a constant concentration of 1 μg / mL in PBS-0.1% T-2% BSA for 1 hour. After washing, 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. After a further washing step, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well, and the fluorescence intensity was detected using a fluorescence microplate reader.

[0274]

[0321] The dual binding data of the fusion proteins (SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, and SEQ ID NOs: 78 and 83) were analyzed by EC 50 The EC values ​​and maximum signals are free parameters, along with the resulting fit curve from a 4PL fit where the slope was fixed to unity. 50The values ​​are summarized in Table 5. In contrast to the anti-CD228 antibody, all bispecific fusion proteins showed clear binding signals, demonstrating that the fusion proteins can simultaneously engage CD228 and CD137. Additional dual binding data for fusion proteins (SEQ ID NOS: 76, 79, 80, 82, 96, 97, 100, 101, 102, and 103) are shown in Figure 14.

[0275] [Table 5] [Example]

[0276]

[0323] Example 13: Flow cytometry analysis of fusion protein binding to CD228-positive cells and cells expressing human or cynomolgus CD137

[0324] Target-specific binding of the fusion proteins to human CD228-expressing cells, and human and cynomolgus monkey CD137-expressing cells, was assessed by flow cytometry (FACS).

[0277]

[0325] Human melanoma SH-4 cells (ATCC, CRL7724) were used as huCD228-positive cells. CHO cells were stably transfected with human CD137 or cynomolgus monkey CD137 using the Flp-In system (Life Technologies) according to the manufacturer's instructions.

[0278]

[0326] SH-4 cells were maintained in DMEM (PAN Biotech) supplemented with 10% fetal calf serum (Biochrom). Transfected CHO cells were maintained in Ham's F12 medium (Life Technologies) supplemented with 10% fetal calf serum (Biochrom) and 500 μg / ml hygromycin B (Roth). Cells were cultured in cell culture flasks (37°C, 5% CO2 atmosphere) according to the manufacturer's instructions.

[0279]

[0327] For flow cytometry analysis, each cell line was incubated with the fusion proteins (SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, and SEQ ID NOs: 78 and 83), hIgG1 versions of the two anti-CD228 antibodies contained in the fusion proteins (SEQ ID NOs: 74 / 76 and 77 / 79, respectively), an IgG4 isotype control (SEQ ID NOs: 24 and 25), and a monoclonal anti-CD137 antibody (SEQ ID NOs: 26 and 27), which were detected using fluorescently labeled anti-human IgG or anti-human NGAL antibodies in FACS analysis as described below:

[0328] 5 x 10 per well 4 Cells were incubated for 1 hour in ice-cold PBS containing 5% fetal calf serum (PBS-FCS). Serial dilutions of the fusion proteins and control antibodies were added to the cells and incubated on ice for 1 hour. After washing twice with PBS, cells were incubated with a goat anti-hIgG Alexa647-conjugated antibody (Life Technologies) or a proprietary rabbit anti-NGAL Alexa488-conjugated antibody for 30 minutes on ice. Cells were then washed and analyzed using an iQue flow cytometer (Intellicyte Screener). Geometric mean values ​​of the fluorescent signals were plotted and fitted using nonlinear regression (shared base, slope = 1) in Graphpad software.

[0280]

[0329] The ability of the fusion proteins to bind human CD228 as well as human CD137 expressed on cells is shown in Figures 15A-15B. The binding affinity (EC 50) are in the single-digit nanomolar range comparable to their respective control antibodies, and the fusion proteins tested are fully cross-reactive with cynomolgus CD137, in contrast to the anti-CD137 antibodies of SEQ ID NOs: 26 and 27 (summarized in Table 6). Neither fusion protein bound to mock-transfected cells (data not shown).

[0281] [Table 6] [Example]

[0282]

[0331] Example 14: CD228-dependent T cell costimulation using a CD137 reporter cell assay

[0332] The potential of the exemplary fusion protein to induce activation of the CD137 signaling pathway in the presence of CD228 was assessed using a commercially available, bistably transfected Jurkat cell line expressing CD137 and the luc2 gene (a humanized version of firefly luciferase, where luc2 expression was driven by an NFκB-responsive element). In this bioassay, CD137 engagement leads to intracellular signaling of CD137, resulting in NFκB-mediated luminescence.

[0283]

[0333] Tumor cells expressing high (SH-4), intermediate (A375), or low (A549) levels of CD228 and CD228-negative RPMI-7951 tumor cells were cultured under standard conditions. One day before the assay, tumor cells were plated at 1.25 × 10 per well. 4 Cells were plated at 100x the number of cells and allowed to adhere overnight at 37°C in a humidified 5% CO2 atmosphere.

[0284]

[0334] The next day, 3.75 x 10 4NF-kB-Luc2 / CD137 Jurkat reporter cells were added to each well, followed by various concentrations ranging from 0.0002 nM to 10 nM of the fusion protein, a reference anti-CD137 antibody (urelumab, SEQ ID NOs: 26 and 27), or an IgG4 fusion with a CD137-specific lipocalin mutein (SEQ ID NO: 84) contained in the fusion protein. The plate was covered with a gas-permeable seal and incubated at 37°C in a humidified 5% CO2 atmosphere. After 4 hours, 30 μL of Bio-Glo™ reagent was added to each well, and the bioluminescent signal was quantified using a luminometer (PHERAstar). A four-parameter logistic curve analysis was performed using GraphPad Prism® to determine the EC 50 Values ​​(shared base, fixed slope) were calculated and are summarized in the table in Figure 16E. To demonstrate the CD228 dependence of CD137 engagement by the fusion proteins, the same experiment was performed in parallel in the presence of CD228-negative cells (RPMI-7951) or in the absence of target cells. Assays were performed in triplicate.

[0285]

[0335] The results of a representative experiment are shown in Figures 16A-16D. The data demonstrate that all fusion proteins tested induced CD137-mediated T cell activation in a dose-dependent manner in the presence of tumor cells expressing CD228. In the case of SH-4 cells (Figure 16A) and A375 cells (Figure 16B), all fusion proteins resulted in higher maximal activation than the reference anti-CD137 antibody. Overall, the fusion molecules of SEQ ID NOs: 80 and 76 (based on the CD228-specific antibody of SEQ ID NOs: 75 and 76) performed better than the other fusion molecules, i.e., exhibited lower EC 50 Figure 16D shows that activation of CD137 by the fusion protein was CD228-dependent, since activation of NF-kB-Luc2 / CD137 Jurkat cells was not detected in CD228-negative cells (RPMI-7951). In contrast, the reference anti-CD137 mAb (SEQ ID NOs: 26 and 27) demonstrated CD137-mediated T cell costimulation regardless of CD228 expression. [Example]

[0286]

[0336] Example 15: Evaluation of PBMC responses to viral peptides in the presence of tumor cells with or without high CD228 expression

[0337] Assays were performed to assess the ability of the fusion proteins to costimulate innate and adaptive immune cytokines from PBMCs in response to viral peptides in a CD228 target-dependent manner. PBMCs isolated from healthy donors were co-incubated at a 10:1 ratio with engineered CD228+ or wild-type (CD228-) RPMI-7951 tumor cell lines in RPMI containing 10% FCS and presented with viral peptides from CMV, EBV, and influenza viruses (CEF peptides). The bispecific fusion proteins and controls were titrated into the assay, and changes in IFN-γ, TNF-α, IL-5, IL-12 (p70), and CXCL10 (IP-10) were measured at the end of 4 days of stimulation at 37°C in 5% CO2. Supernatants were evaluated for T cell (IFN-γ and TNF-α) and myeloid cell (IL-12 and CXCL10) responses using Luminex® multiplex cytokine arrays. Data are presented in Figures 17A-17E as fold changes in cytokines relative to untreated control wells. Samples were pooled from triplicate test article treatments prior to cytokine measurement. These results demonstrate that, compared to antibodies, fusion proteins (e.g., AAF30(HC)) costimulate various innate and adaptive immune cytokines from PBMCs in response to viral peptides in a CD228 target- and dose-dependent manner. [Example]

[0287]

[0338] Example 16: Evaluation of PBMC cellular responses to viral peptides in the presence of CD228+ tumor cells

[0339] To assess the activity of the bispecific fusion proteins in antigen recall assays, cryopreserved PBMCs (Bloodworks Northwest) from healthy donors were thawed in prewarmed RPMI 10% FCS, washed, and labeled with 1.5 mL of 10 nM CFSE in PBS 3% FCS at room temperature. To quench the labeling reaction, cells were washed twice with 12 mL of RPMI 10% FCS. Cells were counted and cultured in RPMI-complete (10% FCS, 1x Glutamax, 1x MEM NEAA, 1x sodium pyruvate, 1x penicillin / streptomycin (Gibco™)) containing the CD228-expressing cell line RPMI-7951. CD228 (ATCC) (engineered to express human CD228) at a 10:1 ratio and plated in a non-adherent 96-well round-bottom plate (Sbio) at 1.5 × 10 5 Cells were distributed at 100 cells / well. CEF peptide was added to a final concentration of 100 ng / mL, and the fusion protein bispecific and control were added in triplicate at equimolar titrations. Assays were incubated for 5 days at 37°C with 5% CO2. Figures 18, 19, and 20 show representative examples of CD8+ T cells, NK cells, and CD8+ T cell / Treg ratios calculated from antigen recall assays in cocultures with CD228-engineered RPMI-7951 cell lines, respectively. Similarly, Figure 21 shows representative examples of NK cell and CD8+ T cell / Treg ratios calculated from antigen recall assays. These results demonstrate that, compared with antibodies, fusion proteins (e.g., AAF30(HC)) costimulate the proliferation / division of CD8+ T cells and NK cells in response to viral peptides in a CD228 target- and dose-dependent manner. [Example]

[0288]

[0340] Example 17: Evaluation of PBMC cytokine responses to viral peptides in the presence of CD228+ tumor cells

[0341] To assess the activity of the bispecific fusion proteins in antigen recall assays, cryopreserved PBMCs (Bloodworks Northwest) from healthy donors were thawed in prewarmed RPMI 10% FCS, washed, and labeled with 1.5 mL of 10 nM CFSE in PBS 3% FCS at room temperature. To quench the labeling reaction, cells were washed twice with 12 mL of RPMI 10% FCS. Cells were counted and cultured in RPMI-complete (10% FCS, 1x Glutamax, 1x MEM NEAA, 1x sodium pyruvate, 1x penicillin / streptomycin (Gibco™)) containing the CD228-expressing cell line RPMI-7951. CD228 (ATCC) (engineered to express human CD228), CALU-1 cells (ATCC), or H3677 cells (Seagen) were mixed at a 10:1 ratio and plated in a non-adherent 96-well round-bottom plate (Sbio) at 1.5 × 10 5 Cells were distributed at 100 cells / well. CEF peptide was added to a final concentration of 100 ng / mL, and the fusion protein bispecific and control were added in triplicate in equimolar titrations. Assays were incubated at 37°C with 5% CO2 for 5 days. After assay completion, plates were spun down and supernatants were collected for cytokine assessment. Cytokines were measured using MILLIPLEX® MAP Human CD8+ T Cell Magnetic Bead Panel Premixed 17 Plex and read on a Luminex® MAGPIX® system. Figures 22A-22E show representative mean fold changes in cytokines across three CD228-expressing cell lines. Raw Luminex® data were exported and analyzed in Microsoft Excel. These data highlight the consistent changes in cytotoxic effector molecules and various cytokines upon antigen recall in the presence of the bispecific fusion protein. [Example]

[0289]

[0342] Example 18: Evaluation of T cell activation in the presence of tumor cells with or without high CD228 expression

[0343] To evaluate the ability of the fusion protein to costimulate T cell activation in a CD228 target-dependent manner, an additional T cell assay was used. Different concentrations of the fusion protein were applied to anti-CD3 stimulated T cells in the presence of tumor cell lines with different levels of CD228 expression. The tumor cell lines tested included SH-4 (CD228-high), Calu-1 (CD228-high), SK-MEL-24 (CD228-high), and RPMI-7951 (CD228-negative). IL-2 secretion levels in the supernatant were used as a readout.

[0290]

[0344] PBMCs from healthy volunteer donors were isolated from buffy coats by centrifugation through a polysucrose density gradient (Biocoll, 1.077 g / mL, Biochrom) according to Biochrom's protocol. T lymphocytes were further purified from PBMCs by magnetic cell sorting using a Pan T cell purification kit (Miltenyi Biotec GmbH) according to the manufacturer's instructions. Purified Pan T cells were resuspended in a buffer consisting of 90% FCS and 10% DMSO and immediately frozen and stored in liquid nitrogen until further use.

[0291]

[0345] For the assay, T cells were thawed and rested for 16 h at 37°C in a humidified 5% CO atmosphere in culture medium (RPMI1640, Life Technologies) supplemented with 10% FCS and 1% penicillin-streptomycin (Life Technologies).

[0292]

[0346] The following procedure was performed in triplicate for each experimental condition: flat-bottom tissue culture plates were precoated with 0.25 μg / mL anti-human CD3 antibody for 1 hour at 37°C, then washed twice with PBS. The tumor cell lines SH-4, Calu-1, SK-MEL-24, and RPMI-7951 were treated with 30 μg / mL mitomycin C (Sigma-Aldrich) for 30 minutes to block proliferation. The mitomycin-treated tumor cells were then washed twice with PBS and plated at 2.5 × 10 cells per well. 4Cells were plated in culture medium and allowed to adhere overnight at 37°C in a humidified 5% CO2 atmosphere. Target cells were grown under standard conditions, then detached using Accutase (PAA Laboratories) and resuspended in culture medium.

[0293]

[0347] The next day, the plates were washed twice with PBS and then 1.25 × 10 cells were added per well. 4 T cells were added to tumor cells. A dilution series of bispecific fusion proteins (SEQ ID NOS: 80 and 76, 82 and 79, 75 and 81, and 78 and 83), reference anti-CD137 antibodies (SEQ ID NOS: 26 and 27), a CD137-specific lipocalin mutein fusion of IgG4 in a fusion protein (SEQ ID NOS: 84), or an isotype control (SEQ ID NOS: 24 and 25) was added to corresponding wells at concentrations ranging from 0.0002 nM to 10 nM. The plate was covered with a gas-permeable seal and incubated at 37°C in a humidified 5% CO2 atmosphere for 3 days.

[0294]

[0348] After 3 days of coculture, IL-2 levels in the supernatants were assessed using the human IL-2 DuoSet kit (R&D Systems) as described below: 384-well plates were coated with 1 μg / mL "Human IL-2 Capture Antibody" in PBS for 2 hours at room temperature. The wells were then washed five times with 80 μl of PBS supplemented with 0.05% Tween (PBS-T). After blocking for 1 hour in PBS-0.05%T containing 1% casein (w / w), assay supernatants and a concentration series of IL-2 standards diluted in culture medium were transferred to each well and incubated overnight at 4°C. The next day, a mixture of 100 ng / mL goat anti-hIL-2-Bio detection antibody (R&D Systems) and 1 μg / mL Sulfotag-labeled streptavidin (Mesoscale Discovery) in PBS-T containing 0.5% casein was added and incubated for 1 hour at room temperature. After washing, 25 μL of read buffer (Mesoscale Discovery) was added to each well, and the resulting electrochemiluminescence (ECL) signal was detected using a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.

[0295]

[0349] Representative data are shown in Figures 23A-23D. Co-culturing Pan T cells with SH-4 cells (CD228 high), Calu-1 cells (CD228 high), or SK-MEL-24 cells (CD228 high) in the presence of fusion proteins (SEQ ID NOs: 80 and 76, 82 and 79, 75 and 81, or 78 and 83) resulted in a clear, dose-dependent increase in IL-2 secretion compared to the hIgG4 isotype control (Figures 23A-23C). Overall, the increase in IL-2 secretion induced by the fusion proteins was significantly higher than that induced by the reference anti-CD137 antibody (SEQ ID NOs: 26 and 27). No increase in IL-2 secretion was observed with the fusion of IgG4 with the CD137-specific lipocalin mutein contained in the fusion protein (SEQ ID NO: 84). Furthermore, co-culture with CD228-negative RPMI-7951 cells did not increase IL-2 secretion levels with either fusion protein, but did with the reference anti-CD137 antibodies (SEQ ID NOs: 26 and 27) (Figure 23D).

[0296]

[0350] Similar assays were performed using CD228+ Calu-1 or CD228-SK-BR-3 cells with a different but overlapping set of fusion proteins, and Figures 24A-24B show IL-2 secretion in response to stimulation with the fusion proteins and urelumab. These results demonstrate that fusion proteins (e.g., AAF35(HC)), compared with antibodies, costimulate T cell activation in a CD228 target- and dose-dependent manner.

[0297]

[0351] This data indicates that the functional activity of the bispecific fusion protein, as measured by its ability to activate T cells or increase IL-2 secretion, is CD228 dependent. [Example]

[0298]

[0352] Example 19: Evaluation of T cell cytokines produced in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines

[0353] Experiments were conducted to evaluate the effect of the fusion protein on cytokine and soluble 4-1BB (sCD137) production from T cells directly stimulated with CD228-expressing tumor cells. PBMCs from healthy donors were cocultured with CD228+ CALU-1 tumor cells (10:1 in RPMI 10% FCS) engineered to express surface anti-CD3 scFv to induce direct T cell receptor engagement by tumor cells. Titrations of fusion protein or control were added in triplicate to 96-well round-bottom plates. At the end of 3 days of stimulation at 37°C and 5% CO2, supernatants were collected and the soluble cytokines IL-2, IL-13, and sCD137 were measured by Luminex® multiplex array. Samples were pooled from triplicate test article treatments before cytokine measurement. The results are shown in Figures 25A-25C and demonstrate that the fusion protein increases cytokines from direct T cell-tumor cell interaction. [Example]

[0299]

[0354] Example 20: Evaluation of CD8 T cell proliferation in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines

[0355] Experiments were performed to evaluate the effect of fusion protein bispecifics on cytotoxic T cells stimulated directly by CD228-expressing tumor cells. PBMCs from healthy donors were cocultured (10:1) with CFSE-labeled CD228+ CALU-1 tumor cells engineered to express surface anti-CD3 scFv to induce T cell receptor engagement. Titrations of bispecific fusion proteins or controls were added in triplicate to 96-well round-bottom plates. After 72 hours of incubation at 37°C in 5% CO2, plates were washed and stained with live / dead viability dye and antibodies specific for CD3, CD4, and CD8, as well as CD228. Plates were assessed for proliferation of live CD8+ T cells and CD228+ tumor cells using an Attune™ NxT flow cytometer. Figure 26 shows the percentage of CD8+ T cells containing diluted CFSE compared to untreated wells. Figure 27 shows the percent of viable tumor cells remaining compared to untreated wells, reflecting tumor cell killing. These results demonstrate that in the presence of CD228, fusion proteins (e.g., AAF30(HC)) dose-dependently costimulate CD8+ T cell proliferation and tumor cell killing compared to antibodies. [Example]

[0300]

[0356] Example 21: Evaluation of storage stability of fusion proteins

[0357] To assess storage stability, exemplary fusion proteins (SEQ ID NOS: 80 and 76, 82 and 79, 75 and 81, or 78 and 83) were diluted to a concentration of 1 mg / ml in PBS or 0.5 mg / ml in 50% human plasma (HPL) or 50% mouse plasma (MPL), respectively. Aliquots of these samples were incubated at 37°C for 1 week or stored at -20°C. The activity of the fusion proteins was then determined by the CD137 reporter cell assay described in Example 14 using CD228-expressing SH-4 cells as target cells. Fusion proteins diluted with PBS, 50% HPL, or 50% MPL immediately before the assay served as the reference, and fusion proteins always stored at -20°C served as the untreated reference. Anti-CD137 antibodies (SEQ ID NOS: 26 and 27) and IgG4 isotype antibodies (SEQ ID NOS: 24 and 25) were used as additional controls. Representative results are shown in Figures 28A-28D.

[0301]

[0358] Fusion molecules of SEQ ID NOs: 80 and 76 and SEQ ID NOs: 75 and 81 (both based on the CD228-specific antibodies of SEQ ID NOs: 75 and 76) showed excellent stability under all tested conditions (Figures 28A and 28C, respectively). Fusion molecules of SEQ ID NOs: 82 and 79 and SEQ ID NOs: 78 and 83 (both based on the CD228-specific antibodies of SEQ ID NOs: 78 and 79) showed excellent stability in PBS and 50% human plasma, but showed approximately 3-fold higher EC 50 There was a slight decrease in stability as indicated by the values ​​and approximately 30% lower plateau (Figures 28B and 28D, respectively). [Example]

[0302]

[0359] Example 22: Pharmacokinetics of fusion proteins in mice

[0360] Pharmacokinetic analysis of representative fusion proteins (SEQ ID NOs: 80 and 76, 82 and 79, 75 and 81, or 78 and 83) was performed in mice. Approximately 5-week-old male CD-1 NUDE mice (3 mice per time point; Charles River Laboratories, Research Models and Services, Germany GmbH) were injected with the fusion protein at a dose of 10 mg / kg via the tail vein. The test article was administered as a bolus using a volume of 5 mL / kg. Plasma samples from the mice were obtained at 5 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, 4 days, 7 days, 14 days, 21 days, and 28 days. Sufficient whole blood was collected under isoflurane anesthesia to obtain at least 50 μL of Li-heparin plasma per animal and every hour.

[0303]

[0361] Fusion protein levels were detected using a sandwich ELISA, which detects the complete bispecific construct via targets CD228 and CD137 in a free PK assay. To this end, huCD228-His (human CD228 with a C-terminal polyhistidine tag) was dissolved in PBS (1 μg / mL) and coated onto microtiter plates overnight at 4°C. After each incubation step, the plates were washed five times with 80 μL of PBS supplemented with 0.05% (v / v) Tween 20. The plates were blocked with PBS / BSA / Tween (PBS containing 2% BSA (w / v) and 0.1% (v / v) Tween 20) for 1 h at room temperature and then washed. Plasma samples were diluted to a plasma concentration of 20% in PBS / BSA / Tween / standard CD-1 mouse plasma, added to the wells, and incubated for 1 h at room temperature. Another washing step was then performed. Bound drugs were detected under study conditions after 1 hour of incubation with a mixture of biotinylated human CD137 and streptavidin SULFO-TAG (Mesoscale Discovery) (1 μg / mL each), each diluted in PBS containing 2% BSA (w / v) and 0.1% (v / v) Tween 20. After an additional washing step, 35 μL of reading buffer was added to each well, and the electrochemiluminescence (ECL) signal of each well was read using a Mesoscale Discovery reader. The level of parent anti-CD228 antibody was detected in a similar manner using target CD228 and anti-human IgG Fc antibodies (GE Healthcare). Data were transferred to Excel for analysis and quantification. A calibration curve with standard protein dilutions was generated for back-calculation of plasma concentrations. Noncompartmental analysis was performed using WinNonLin software.

[0304]

[0362] 29A-29B show plasma concentrations over time (29A: mean concentrations; 29B: C) for fusion proteins SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, or SEQ ID NOs: 78 and 83, plotted alongside the values ​​obtained for the parent anti-CD228 antibodies (SEQ ID NOs: 74 and 76, SEQ ID NOs: 77 and 79, respectively) as a reference. max A plot of α (normalized to α) is shown. The corresponding terminal half-lives are summarized in Table 7.

[0305]

[0363] This data demonstrates that the fusion protein has a long, antibody-like terminal half-life in mice. Because the assay employed to determine the plasma concentration of the fusion protein requires retention of binding activity to both CD228 and CD137, the results also demonstrate that the bispecific molecule remains intact over a 28-day time course.

[0306] [Table 7] [Example]

[0307]

[0365] Example 23: Pharmacokinetics of fusion proteins in cynomolgus monkeys

[0366] The heavy chain fusion protein bispecifics 30HC and 35HC (SEQ ID NOs: 80 and 76, and SEQ ID NOs: 82 and 79, respectively) were compared for differences in pharmacokinetics in cynomolgus monkeys. Animals received a single dose of the fusion bispecific at a dose of 1 mg / kg or 6 mg / kg. As shown in Figure 30, the bispecific fusion proteins exhibited similar pharmacokinetic profiles, with slight differences after day 14, which may be due to anti-drug antibody responses. [Example]

[0308]

[0367] Example 24: Evaluation of in vivo activity of fusion proteins in a humanized xenograft model

[0368] To evaluate the activity of the bispecific fusion protein in vivo, a humanized xenograft model was performed using the CD228+ melanoma cell lines CALU-1 (ATCC) and H3677 (SGEN, Japan). Tumor cells were implanted into immunodeficient NSG mice in 25% Matrigel® (Corning), and tumors grew to an average volume of 100 mm. 3 The tumor growth curves were monitored until a 100% T cell count was reached, at which point peripheral blood mononuclear cells (PBMCs) from healthy donors were adoptively transferred via tail vein injection to serve as a source of human T cells. Mice were then administered equimolar amounts of the bispecific fusion protein (10 mg / kg), antibody (non-bispecific antibody, anti-PD-1, and / or anti-4-1BB), or control (8 mg / kg) every 5 days, and tumor growth was monitored. The growth curve for Calu-1 over the study is shown in Figure 31A, and the final tumor volume at the end of the study on day 80 is shown in Figure 31B. The growth curve for H3677 over the study is shown in Figure 31C, and the final tumor volume at the end of the study on day 22 is shown in Figure 31D. Statistics are based on Tukey's analysis. These results demonstrate that fusion proteins (e.g., AAF30(HC)) compared with antibodies reduce the growth of CD228+ tumor cells in vivo.

[0309]

[0369] To evaluate intratumoral PD effects from animals treated with the bispecific fusion in a humanized CD228+ Calu-1 xenograft model, tumors were harvested, processed into single-cell suspensions, and tumor and immune cell populations were stained and analyzed by flow cytometry. Figure 32A shows the ratio of CD8+ T cells to tumor cells, determined by dividing the number of CD8+ T cells by the number of CD228+ non-immune cells. Figure 32B shows the change in the CD8+ / CD4+ T cell ratio. Figure 32C shows the percentage of actively degranulating CD8+ T cells, as determined by surface expression of CD107a, at the end of the study. The dashed lines in the figures indicate the CD8+ / CD4+ T cell ratio (Figure 32B) or the percentage of CD107a-expressing CD8+ T cells (Figure 32C) in resting PBMC (i.e., non-tumor model) samples from the same donor. These data demonstrate that treatment with the bispecific fusion protein enhanced the expansion and activation of cytotoxic CD8+ T cells compared to controls. Statistics are from Tukey's analysis.

[0310]

[0370] To evaluate intratumoral PD efficacy from animals treated with the bispecific fusion in separate humanized CD228+ Calu-1 xenograft models, tumors were harvested, processed into single-cell suspensions, and staining and flow cytometry of tumor and immune cell populations were performed. Figure 33A shows the ratio of CD8+ T cells to CD4+ T cells. Figure 33B shows the change in intracellular TCF1 expression. These data demonstrate that treatment with the bispecific fusion protein targeting 4-1BB promoted the expansion and differentiation of cytotoxic CD8+ T cells, transforming them into cells with stem cell-like properties that exhibited greater antitumor potential, as determined by the expression of the transcription factor TCF1. Statistics provided are the results of Tukey's analysis. [Example]

[0311]

[0371] Example 25: Evaluation of CD8 T cell proliferation in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines

[0372] Similar to Example 20, an experiment was performed to evaluate the effect of fusion protein bispecifics on cytotoxic T cells receiving T cell receptor stimulation directly from CD228-expressing tumor cells. PBMCs from healthy donors were cocultured with CFSE-labeled CD228+ A2058 and SK-MEL-5 melanoma cells (10:1) engineered to express anti-CD3 scFv on their surface to induce T cell receptor engagement. Titrations of bispecific fusion proteins or controls were added in triplicate to 96-well round-bottom plates. After 72 hours of incubation at 37°C under 5% CO2, plates were washed and stained with antibodies specific for CD3, CD4, and CD8. Plates were assessed for viable CD8 T cell proliferation using an Attune™ NxT flow cytometer. Figures 34A and 34B show the percentage of divided CD8 T cells relative to untreated wells. These results show that in the presence of CD228, fusion proteins (e.g., AAF30(HC)) costimulate CD8+ T cell proliferation in a dose-dependent manner compared to antibodies, control fusion proteins, and urelumab. [Example]

[0312]

[0373] Example 26: Evaluation of CD8 T cell proliferation and mitochondrial function in co-culture with anti-CD3 scFv-engineered CD228-expressing tumor cell lines

[0374] Similar to Example 20, an experiment was performed to evaluate the effect of fusion protein bispecifics on cytotoxic T cells receiving T cell receptor stimulation directly from CD228-expressing tumor cells. PBMCs from healthy donors were cocultured with CD228+ CALU-1 lung cancer cells (2.5:1) engineered to express surface anti-CD3 scFv to induce T cell receptor engagement. Titrations of bispecific fusion proteins or controls were added in quadruplicate to 96-well round-bottom plates. After 6 days of incubation at 37°C under 5% CO2, plates were washed and stained with antibodies specific for CD2, CD4, and CD8. Plates were assessed for viable CD8 T cell expansion using an Attune™ NxT flow cytometer. Figure 35A shows the number of CD8 T cells in treated versus untreated wells. To measure mitochondrial content in CD8 T cells, cells were stained with 75 nM MitoSpy™ Green FM for 20 minutes at 37°C and 5% CO2. Figure 35B shows the mitochondrial content of CD8+ T cells, expressed as mean fluorescence intensity (MFI) of MitoSpy™ Green FM staining. Figure 35C shows the percentage of CD8+ T cells with depolarized mitochondria, as determined by staining with the polarization-dependent dyes MitoSpy™ Orange CMT Cross (25 nM, 20 min at 37°C in 5% CO2) and MitoSpy™ Green FM (75 nM, 20 min at 37°C in 5% CO2). These results demonstrate that in the presence of CD228, the fusion protein (e.g., AAF30(HC)) dose-dependently costimulates CD8+ T cell proliferation and measurably improves the metabolic fitness of activated cytotoxic T cells compared to a non-bispecific CD228-binding antibody or urelumab (20H4.9). [Example]

[0313]

[0375] Example 27: Preparation and characterization of a T cell exhaustion model

[0376] To generate a model of functionally exhausted cytotoxic T cells receiving direct T cell receptor stimulation from CD228-expressing tumor cells, CALU-1 lung cancer cells were engineered to express membrane-bound anti-CD3 scFv fragments. PBMCs from healthy donors were serially passaged (P0–P4) on CALU-1 cell lawns to allow the progression of T cell functional impairment. Figure 36A shows a schematic diagram of the generation of such functionally exhausted cytotoxic T cells. Cells recovered and banked from serial passages were analyzed for proliferation, interferon gamma (IFN-γ) secretion, and tumor cell killing ability. Figure 36B shows the percentage of dividing cells (left), IFN-γ levels (center), and the number of remaining viable tumor cells (right) after restimulation of T cells from different passages in coculture with anti-CD3 scFv-engineered CALU-1 cells. Passage 4 (P4) T cells showed significantly reduced ability to divide, secrete cytokines, and kill tumor cells.

[0314]

[0377] CD8+ T cells from P4 were analyzed by single-cell RNA sequencing (RNAseq) and clustered into two states using the Uniform Manifold Approximation and Projection (UMAP) method. These two states resemble CD8+ T cells: progenitor (P4 Tpex_like) and terminally exhausted (P4 Texterm_like). The average gene expression per state was scaled for each dataset and shown in Figure 37A. This is a heatmap of P4 cells or exhausted CD8+ T cells from the pan-cancer tumor-infiltrating lymphocyte (TIL) atlas, as described in Zheng et al., Pan-cancer single-cell landscape of tumor-infiltrating T cells, Science 374:6574 (2021). Furthermore, Figure 37B shows the expression levels of TCF7 (TCF1) (left), HAVCR2 (TIM-3) (center), and TNFRSF9 (CD137) (right) in Tpex_like and P4 Texterm_like cells. The similarity of Tpex_like and Texterm_like cells to true TIL CD8+ T cells was assessed by hierarchical clustering based on marker genes of exhausted T cell state, and the results demonstrated that Tpex_like and P4 Texterm_like cells have significantly overlapping gene signatures with true exhausted TILs. [Example]

[0315]

[0378] Example 28: Evaluation of functional reactivation of exhausted CD8 T cells in co-culture with anti-CD3 scFv-engineered CD228-expressing tumor cell lines

[0379] Experiments were conducted to evaluate the effect of fusion protein bispecifics on functionally exhausted cytotoxic T cells. Exhausted T cells from Example 27 were harvested, CFSE-labeled, and co-cultured again with CD228+ anti-CD3 scFv CALU-1 tumor cells (1:1). Titrations of the bispecific fusion protein, a costimulatory antibody against CD28 (clone CD28.2, BioLegend, San Diego, CA, catalog numbers 302901 / 302902), the anti-PD-1 antibody nivolumab, and the anti-4-1BB agonist antibody 20H4.9 were added in triplicate to a 96-well round-bottom plate. After 96 hours of incubation at 37°C under 5% CO2, the plate was washed and stained with antibodies specific for CD3, CD4, and CD8. The plate was evaluated for viable CD8 T cell proliferation using an Attune™ NxT flow cytometer. Figure 38 shows the fold increase in the number of CFSE-low (divided) CD8+ T cells in treated versus untreated wells (dashed line = 1). These results demonstrate that agonists of 4-1BB, fusion proteins (e.g., AAF30(HC)), and 20H4.9 dose-dependently stimulate the proliferation of functionally exhausted CD8+ T cells, whereas the anti-PD-1 antibody nivolumab and agonistic antibodies against CD28 alone do not increase proliferation. In the presence of CD228-expressing CALU-1 tumor cells, AAF30(HC) increased costimulation compared with 20H4.9, and the proliferative effects of AAF30(HC) and 20H4.9 on exhausted cytotoxic T cells were further amplified by blocking the PD-1 / PD-L1 pathway with the anti-PD-1 antibody nivolumab, demonstrating the complementarity of their mechanisms of action and supporting the clinical combination of fusion protein bispecifics with agents that block the PD-1 / PD-L1 pathway, such as anti-PD-1 or anti-PD-L1 antibodies.

[0316]

[0380] The embodiments illustratively described herein may suitably be practiced in the absence of any element(s), limitation(ies) not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" are intended to be interpreted expansively and without limitation. Furthermore, the terms and phrases employed herein are used as terms of description and not of limitation, and the use of such terms and phrases is not intended to exclude equivalents of the illustrated and described features or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Thus, while the present embodiments have been specifically disclosed in terms of preferred embodiments and optional features, it should be understood that those skilled in the art may resort to equivalents, modifications, and variations thereof, and that such equivalents, modifications, and variations are considered to be within the scope of the present invention. All patents, patent applications, textbooks, and (peer-reviewed) publications mentioned herein are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, if a definition or use of a term in a reference incorporated herein by reference contradicts or contradicts the definition of that term provided herein, the definition of that term provided herein applies, and the definition of that term in the reference does not apply. Each narrower species and subgeneric group falling within the generic disclosure also forms part of the present invention. This includes the generic description of the invention with a provisos or negative limitation removing any subject matter from the genus, regardless of whether the excluded material is specifically described herein. Furthermore, where features are described in terms of a Markush group, one skilled in the art will recognize that the disclosure is thereby also described in terms of any individual member or subgroup of members of the Markush group. Further embodiments will become apparent from the claims that follow.

[0317] VII. Non-patent literature 1. ROSE, T. M., PLOWMAN, G. D., TEPLOW, D. B., DREYER, W. J., HELLSTROM, K. E. & BROWN, J. P. 1986. Primary structure of the human melanoma-associated antigen p97 (melanotransferrin) deduced from the mRNA sequence. Proc Natl Acad Sci U S A, 83(5), 1261-5. 2. LI, S. Y. & LIU, Y. 2013. Immunotherapy of melanoma with the immune costimulatory monoclonal antibodies targeting CD137. Clin Pharmacol, 5,47-53. 3. SNELL, L. M., LIN, G. H., MCPHERSON, A. J., MORAES, T. J. & WATTS, T. H. 2011. T-cell intrinsic effects of GITR and 4-1BB during viral infection and cancer immunotherapy. Immunol Rev, 244, 197-217. 4. WYZGOL, A., MULLER, N., FICK, A., MUNKEL, S., GRIGOLEIT, G. U., PFIZENMAIER, K. & WAJANT, H. 2009. Trimer stabilization, oligomerization, and antibody-mediated cell surface immobilization improve the activity of soluble trimers of CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand. J Immunol, 183, 1851-61. 5. YAO, S., ZHU, Y. & CHEN, L. 2013. Advances in targeting cell surface signalling molecules for immune modulation. Nat Rev Drug Discov, 12, 130-46. 6. MELERO, I., BACH, N., HELLSTROM, K. E., ARUFFO, A., MITTLER, R. S. & CHEN, L. 1998. Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway. Eur J Immunol,28, 1116-21. 7. YANG, Y., YANG, S., YE, Z., JAFFAR, J., ZHOU, Y., CUTTER, E., LIEBER, A., HELLSTROM, I. & HELLSTROM, K. E. 2007. Tumor cells expressing anti-CD137 scFv induce a tumor-destructive environment. Cancer Res, 67, 2339-44. 8. ZHANG, H., KNUTSON, K. L., HELLSTROM, K. E., DISIS, M. L. & HELLSTROM, I. 2006. Antitumor efficacy of CD137 ligation is maximized by the use of a CD137 single-chain Fv-expressing whole-cell tumor vaccine compared with CD137-specific monoclonal antibody infusion. Mol Cancer Ther, 5, 149-55. 9. YE, Z., HELLSTROM, I., HAYDEN-LEDBETTER, M., DAHLIN, A., LEDBETTER, J. A. & HELLSTROM, K. E. 2002. Gene therapy for cancer using single-chain Fv fragments specific for 4-1BB. Nat Med, 8, 343-8. 10. MARTINET, O., DIVINO, C. M., ZANG, Y., GAN, Y., MANDELI, J., THUNG, S., PAN, P. Y. & CHEN, S. H. 2002. T cell activation with systemic agonistic antibody versus local 4-1BB ligand gene delivery combined with interleukin-12 eradicate liver metastases of breast cancer. Gene Ther, 9, 786-92. 11. YE, Q., SONG, D. G., POUSSIN, M., YAMAMOTO, T., BEST, A., LI, C., COUKOS, G. & POWELL, D. J., JR. 2014. CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor. Clin Cancer Res, 20, 44-55. 12. CHACON, J. A., WU, R. C., SUKHUMALCHANDRA, P., MOLLDREM, J. J., SARNAIK, A., PILON-THOMAS, S., WEBER, J., HWU, P. & RADVANYI, L. 2013. Co-stimulation through 4-1BB / CD137 improves the expansion and function of CD8(+) melanoma tumor-infiltrating lymphocytes for adoptive T-cell therapy. PLoS One, 8, e60031. 13. FISHER, T. S., KAMPERSCHROER, C., OLIPHANT, T., LOVE, V. A., LIRA, P. D., DOYONNAS, R., BERGQVIST, S., BAXI, S. M., ROHNER, A., SHEN, A. C., HUANG, C., SOKOLOWSKI, S. A. & SHARP, L. L. 2012. Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity. Cancer Immunol Immunother, 61, 1721-33. 14. SKERRA, A. 2000. Lipocalins as a scaffold. Biochim Biophys Acta, 1482, 337-50. 15. FLOWER, D. R., NORTH, A. C. & SANSOM, C. E. 2000. The lipocalin protein family: structural and sequence overview. Biochim Biophys Acta, 1482, 9-24. 16. FLOWER, D. R. 1996. The lipocalin protein family: structure and function. Biochem J, 318 ( Pt 1), 1-14. 17. ALTSCHUL, S. F., MADDEN, T. L., SCHAFFER, A. A., ZHANG, J., ZHANG, Z., MILLER, W. & LIPMAN, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 25, 3389-402. 18. ALTSCHUL, S. F., GISH, W., MILLER, W., MYERS, E. W. & LIPMAN, D. J. 1990. Basic local alignment search tool. J Mol Biol, 215, 403-10. 19. SMITH, T. F. & WATERMAN, M. S. 1981. Identification of common molecular subsequences. J Mol Biol, 147, 195-7. 20. WARD, E. S., GUSSOW, D., GRIFFITHS, A. D., JONES, P. T. & WINTER, G. 1989. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature, 341, 544-6. 21. HOLLIGER, P., PROSPERO, T. & WINTER, G. 1993. "Diabodies": small bivalent and bispecific antibody fragments. Proc Natl Acad Sci U S A, 90, 6444-8. 22. JOHNSON, G. & WU, T. T. 2000. Kabat database and its applications: 30 years after the first variability plot. Nucleic Acids Res, 28, 214-8. 23. EHRENMANN, F., KAAS, Q. & LEFRANC, M. P. 2010. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF. Nucleic Acids Res, 38, D301-7. 24. BULLIARD, Y., JOLICOEUR, R., ZHANG, J., DRANOFF, G., WILSON, N. S. & BROGDON, J. L. 2014. OX40 engagement depletes intratumoral Tregs via activating FcgammaRs, leading to antitumor efficacy. Immunol Cell Biol, 92, 475-80. 25. BULLIARD, Y., JOLICOEUR, R., WINDMAN, M., RUE, S. M., ETTENBERG, S., KNEE, D. A., WILSON, N. S., DRANOFF, G. & BROGDON, J. L. 2013. Activating Fc gamma receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies. J Exp Med, 210, 1685-93. 26. SILVA, J. P., VETTERLEIN, O., JOSE, J., PETERS, S. & KIRBY, H. 2015. The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. J Biol Chem,290, 5462-9. 27. GLAESNER, W., VICK, A. M., MILLICAN, R., ELLIS, B., TSCHANG, S. H., TIAN, Y., BOKVIST, K., BRENNER, M., KOESTER, A., PORKSEN, N., ETGEN, G. & BUMOL, T. 2010. Engineering and characterization of the long-acting glucagon-like peptide-1 analogue LY2189265, an Fc fusion protein. Diabetes Metab Res Rev, 26, 287-96. 28. DALL'ACQUA, W. F., KIENER, P. A. & WU, H. 2006. Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn). J Biol Chem, 281, 23514-24. 29. ZALEVSKY, J., CHAMBERLAIN, A. K., HORTON, H. M., KARKI, S., LEUNG, I. W., SPROULE, T. J., LAZAR, G. A., ROOPENIAN, D. C. & DESJARLAIS, J. R. 2010. Enhanced antibody half-life improves in vivo activity. Nat Biotechnol, 28, 157-9. 30. SHIELDS, R. L., NAMENUK, A. K., HONG, K., MENG, Y. G., RAE, J., BRIGGS, J., XIE, D., LAI, J., STADLEN, A., LI, B., FOX, J. A. & PRESTA, L. G. 2001. High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R. J Biol Chem, 276,6591-604. 31. ALTSHULER, E. P., SEREBRYANAYA, D. V. & KATRUKHA, A. G. 2010. Generation of recombinant antibodies and means for increasing their affinity. Biochemistry (Mosc), 75, 1584-605. 32. HARLOW, E. & LANE, D. 1999. Using antibodies : a laboratory manual, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press. 33. HARLOW, E. & LANE, D. 1988. Antibodies : a laboratory manual, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory. 34. LI, J., SAI, T., BERGER, M., CHAO, Q., DAVIDSON, D., DESHMUKH, G., DROZDOWSKI, B., EBEL, W., HARLEY, S., HENRY, M., JACOB, S., KLINE, B., LAZO, E., ROTELLA, F., ROUTHIER, E., RUDOLPH, K., SAGE, J., SIMON, P., YAO, J., ZHOU, Y., KAVURU, M., BONFIELD, T., THOMASSEN, M. J., SASS, P. M., NICOLAIDES, N. C. & GRASSO, L. 2006. Human antibodies for immunotherapy development generated via a human B cell hybridoma technology. Proc Natl Acad Sci U S A, 103, 3557-62. 35. KOZBOR, D. & RODER, J. C. 1983. The production of monoclonal antibodies from human lymphocytes. Immunol Today, 4, 72-9. 36. COLE, S. P., CAMPLING, B. G., LOUWMAN, I. H., KOZBOR, D. & RODER, J. C. 1984. A strategy for the production of human monoclonal antibodies reactive with lung tumor cell lines. Cancer Res, 44, 2750-3. 37. HOLLIGER, P. & HUDSON, P. J. 2005. Engineered antibody fragments and the rise of single domains. Nat Biotechnol, 23, 1126-36. 38. PERVAIZ, S. & BREW, K. 1987. Homology and structure-function correlations between alpha 1-acid glycoprotein and serum retinol-binding protein and its relatives. FASEB J, 1, 209-14. 39. SAMBROOK, J. & RUSSELL, D. W. 2001. Molecular cloning : a laboratory manual, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press. 40. FLOWER, D. R. 2000. Beyond the superfamily: the lipocalin receptors. Biochim Biophys Acta, 1482, 327-36. 41. BREUSTEDT, D. A., KORNDORFER, I. P., REDL, B. & SKERRA, A. 2005. The 1.8-A crystal structure of human tear lipocalin reveals an extended branched cavity with capacity for multiple ligands. J Biol Chem, 280, 484-93. 42. SCHMIDT, T. G., KOEPKE, J., FRANK, R. & SKERRA, A. 1996. Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. J Mol Biol, 255, 753-66. 43. VAJO, Z. & DUCKWORTH, W. C. 2000. Genetically engineered insulin analogs: diabetes in the new millenium. Pharmacol Rev, 52, 1-9. 44. FUERTGES, F. & ABUCHOWSKI, A. 1990. The clinical efficacy of poly(ethylene glycol)-modified proteins. Journal of Controlled Release, 11, 139-148. 45. DENNIS, M. S., ZHANG, M., MENG, Y. G., KADKHODAYAN, M., KIRCHHOFER, D., COMBS, D. & DAMICO, L. A. 2002. Albumin binding as a general strategy for improving the pharmacokinetics of proteins. J Biol Chem, 277, 35035-43. 46. KONIG, T. & SKERRA, A. 1998. Use of an albumin-binding domain for the selective immobilisation of recombinant capture antibody fragments on ELISA plates. J Immunol Methods, 218, 73-83. 47. OSBORN, B. L., OLSEN, H. S., NARDELLI, B., MURRAY, J. H., ZHOU, J. X., GARCIA, A., MOODY, G., ZARITSKAYA, L. S. & SUNG, C. 2002. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys. J Pharmacol Exp Ther, 303, 540-8. 48. LOWMAN, H. B. 1997. Bacteriophage display and discovery of peptide leads for drug development. Annu Rev Biophys Biomol Struct, 26, 401-24. 49. RODI, D. J. & MAKOWSKI, L. 1999. Phage-display technology--finding a needle in a vast molecular haystack. Curr Opin Biotechnol, 10, 87-93. 50. VENTURI, M., SEIFERT, C. & HUNTE, C. 2002. High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm. J Mol Biol, 315, 1-8. 51. BRUCKDORFER, T., MARDER, O. & ALBERICIO, F. 2004. From production of peptides in milligram amounts for research to multi-tons quantities for drugs of the future. Curr Pharm Biotechnol, 5, 29-43. VIII. Allocation list

[0381]

[0318]

Table 8-1

[0319]

Table 8-2

[0320]

Table 8-3

[0321]

Table 8-4

[0322]

Table 8-5

[0323]

Table 8-6

[0324]

Table 8-7

[0325]

Table 8-8

[0326]

Table 8-9

[0327]

Table 8-10

[0328]

Table 8-11

[0329]

Table 8-12

[0330]

Table 8-13

[0331]

Table 8-14

[0332]

Table 8-15

[0333]

Table 8-16

[0334]

Table 8-17

[0335]

Table 8-18

[0336]

Table 8-19

[0337]

Table 8-20

[0338]

Table 8-21

[0339]

Table 8-22

[0340]

Table 8-23

[0341]

Table 8-24

[0342]

Table 8-25

[0343]

Table 8-26

[0344]

Table 8-27

[0345]

Table 8-28

[0346]

Table 8-29

[0347]

Table 8-30

[0348]

Table 8-31

[0349]

Table 8-32

[0350]

Table 8-33

[0351]

Table 8-34

[0352]

Table 8-35

[0353]

Table 8-36

Claims

1. A fusion protein capable of binding to both CD137 and CD228, comprising at least two subunits in any order, wherein the first subunit comprises an antibody or its antigen-binding domain specific to CD228, and the second subunit comprises a lipocalin mutein specific to CD137.

2. The fusion protein of claim 1, further comprising a third subunit, wherein the third subunit comprises a lipocalin mutein specific for CD137.

3. K below 150 nM Ｄ The fusion protein of claim 1 or 2, which is capable of binding to CD228 at a value of 1:

1.

4. K<7 nM Ｄ A fusion protein according to any one of claims 1 to 3, which is capable of binding to CD137 at a value of 0.1 to 0.

25.

5. The above K Ｄ 5. The fusion protein of claim 3 or 4, wherein the value is determined by a surface plasmon resonance (SPR) assay.

6. EC ≤ 1.5 nM ５０ 6. The fusion protein of claim 1, wherein the fusion protein is capable of binding to CD228 at a value of 0.1 to 0.

5.

7. EC ≤ 4 nM ５０ 7. The fusion protein of claim 1, wherein the fusion protein is capable of binding to CD137 at a value of 0.1 to 0.

25.

8. The EC ５０ 8. The fusion protein of claim 6 or 7, wherein the value is determined by an enzyme-linked immunosorbent assay (ELISA) assay.

9. A fusion protein described in any one of claims 1 to 8, which binds to cynomolgus monkey CD228.

10. A fusion protein described in any one of claims 1 to 9, which binds to cynomolgus monkey CD137.

11. EC<10 nM as measured in an ELISA assay ５０ 11. The fusion protein of claim 1, which is capable of simultaneously binding to CD137 and CD228 at a value of 0.1 to 0.

1.

12. EC ≤ 30 nM as measured by flow cytometry analysis ５０ 12. The fusion protein of claim 1, wherein the fusion protein is capable of binding to CD137 expressed on a cell at a specific level.

13. EC50 of 10 nM or less as measured by flow cytometry analysis ５０ 13. The fusion protein of claim 1, wherein the fusion protein is capable of binding to CD228 expressed on a cell at a specific level.

14. A fusion protein according to any one of claims 1 to 13, which is capable of binding to CD228-expressing tumor cells.

15. 15. The fusion protein of any one of claims 1 to 14, which is capable of stimulating T cell proliferation and / or T cell responses.

16. 16. The fusion protein of any one of claims 1 to 15, which is capable of stimulating CD4+ and / or CD8+ T cell proliferation.

17. 17. The fusion protein of any one of claims 1 to 16, which is capable of stimulating exhausted CD8+ T cell proliferation and / or is capable of stimulating exhausted CD8+ T cell proliferation in synergy with an anti-PD-1 antibody or an anti-PD-L1 antibody.

18. 18. The fusion protein of any one of claims 1 to 17, which is capable of inducing an increase in CD8+ T cell mitochondrial content and / or a decrease in CD8+ T cell mitochondrial depolarization.

19. The fusion protein of any one of claims 1 to 18, which is capable of inducing increased secretion of IL-2 and / or IFN-gamma.

20. 20. The fusion protein of claim 1, which is capable of inducing increased secretion of a cytotoxic factor.

21. 21. A fusion protein according to any one of claims 1 to 20, capable of costimulating a T cell response in a CD228-dependent manner.

22. 22. The fusion protein of any one of claims 1 to 21, which is capable of costimulating a T cell response in the tumor microenvironment.

23. 23. The fusion protein of any one of claims 1 to 22, which does not costimulate a T cell response in the absence of CD228.

24. 24. The fusion protein of any one of claims 1 to 23, having an antibody-like pharmacokinetic profile.

25. 25. The fusion protein of any one of claims 1 to 24, wherein the lipocalin mutein comprises one or more mutated amino acid residues at positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1).

26. The amino acid sequence of the lipocalin mutein may contain the following mutated amino acid residues at one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1): Ala5→Val or Thr; Arg26→Glu; Glu27→Gly; Phe28→Cys; Pro29→Arg; Glu30→Pro; Met31→Trp; Leu33→Ile; Glu34 →Phe; Thr42 → Ser; Gly46 → Asp; Lys52 → Glu; Leu56 → Ala; Ser58 → Asp; Arg60 → Pro; Cys61 → Ala; Lys65 → Arg or Asn; Thr71 → Ala; Val85 → Asp; Lys94 → Arg or Glu; Cys101 → Ser; Glu104 → Val; Leu105 → Cys; His106 → Asp; Lys108 → Ser; Arg111 → Pro; Lys114 → Trp; Lys121 → Glu; Ala133 → Thr; Arg148 → Ser; Ser150 → Ile; and Cys153 → Ser 26. The fusion protein of claim 25, comprising one or more of:

27. The amino acid sequence of the lipocalin mutein comprises the following set of variant amino acid sequences compared to the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1): (a) Arg26→Glu; Glu27→Gly; Phe28→Cys; Pro29→Arg; Glu30→Pro; Met31→Trp; Leu33→Ile; Glu34→Phe; Leu56→Ala; Ser58→Asp; Arg60→Pro; Cys61→Ala; Cys101→Ser; Glu104→Val; Leu105→Cys; His106→Asp; Lys108→Ser; Arg111→Pro; Lys114→Trp; and Cys153→Ser; (b) Ala5→Thr; Arg26→Glu; Glu27→Gly; Phe28→Cys; Pro29→Arg; Glu30→Pro; Met3 1→Trp;Leu33→Ile;Glu34→Phe;Leu56→Ala;Ser58→Asp;Arg60→Pro;Cys61→Ala; Lys65 → Arg; Val85 → Asp; Cys101 → Ser; Glu104 → Val; Leu105 → Cys; His106 → Asp; Lys108 → Ser; Arg111 → Pro; Lys114 → Trp; Lys121 → Glu; Ala133 → Thr; and Cys153 → Ser; (c) Arg26→Glu; Glu27→Gly; Phe28→Cys; Pro29→Arg; Glu30→Pro; Met31→Trp; Leu33→Ile; Glu34→Phe; Leu56→Ala; Ser58→Asp; Arg60→Pro; Cys61→Ala; Lys65→Asn; Lys94→Arg; Cys101→Ser; Glu104→Val; Leu105→Cys; His106→Asp; Lys108→Ser; Arg111→Pro; Lys114→Trp; Lys121→Glu; Ala133→Thr; and Cys153→Ser; (d) Ala5→Val; Arg26→Glu; Glu27→Gly; Phe28→Cys; Pro29→Arg; Glu30→Pro; Met3 1→Trp;Leu33→Ile;Glu34→Phe;Leu56→Ala;Ser58→Asp;Arg60→Pro;Cys61→Ala; Lys65 → Arg; Lys94 → Glu; Cys101 → Ser; Glu104 → Val; Leu105 → Cys; His106 → Asp; Lys108 → Ser; Arg111 → Pro; Lys114 → Trp; Lys121 → Glu; Ala133 → Thr; and Cys153 → Ser; (e) Arg26→Glu; Glu27→Gly; Phe28→Cys; Pro29→Arg; Glu30→Pro; Met31→Trp; Leu33→Ile; Glu34→Phe; Thr42→Ser; Leu56→Ala; Ser58→Asp; Arg60→Pro; Cys61→Ala; Cys101→Ser; Glu104→Val; Leu105→Cys; His106→Asp; Lys108→Ser; Arg111→Pro; Lys114→Trp; Ser150→Ile; and Cys153→Ser; (f) Arg26→Glu; Glu27→Gly; Phe28→Cys; Pro29→Arg; Glu30→Pro; Met31→Trp; Leu3 3→Ile; Glu34→Phe; Lys52→Glu; Leu56→Ala; Ser58→Asp; Arg60→Pro; Cys61→Ala; Th r71→Ala; Cys101→Ser; Glu104→Val; Leu105→Cys; His106→Asp; Lys108→Ser; Arg111→Pro; Lys114→Trp; Ala133→Thr; Arg148→Ser; Ser150→Ile; and Cys153→Ser; and (g) Ala5→Thr; Arg26→Glu; Glu27→Gly; Phe28→Cys; Pro29→Arg; Glu30→Pro; Met31→Trp; Leu33→Ile; Glu34→Phe; Gly46→Asp; Leu56→Ala; Ser58→Asp; Arg60→Pro; Cys61→Ala; Thr71→Ala; Cys101→Ser; Glu104→Val; Leu105→Cys; His106→Asp; Lys108→Ser; Arg111→Pro; Lys114→Trp; Ser150→Ile; and Cys153→Ser 27. The fusion protein of claim 25 or 26, comprising one of:

28. 28. The fusion protein of any one of claims 1 to 27, wherein the amino acid sequence of the lipocalin mutein has at least 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 32 to 38.

29. 29. The fusion protein of any one of claims 1 to 28, wherein the amino acid sequence of the lipocalin mutein comprises an amino acid sequence selected from SEQ ID NOs: 32 to 38, or a fragment or variant thereof.

30. 25. The fusion protein of any one of claims 1 to 24, wherein the lipocalin mutein comprises one or more mutated amino acid residues at positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature human neutrophil gelatinase-binding lipocalin (hNGAL) (SEQ ID NO: 2).

31. The amino acid sequence of the lipocalin mutein contains the following mutated amino acid residues at positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature human neutrophil gelatinase-binding lipocalin (hNGAL) (SEQ ID NO: 2): Gln28→His; Leu36→Gln; Ala40→Ile; Ile41→Arg or Lys; Gln49→Val, Ile, His, Ser, or Asn; Tyr52→Me t; Asn65 → Asp; Ser68 → Met, Ala, or Gly; Leu70 → Ala, Lys, Ser, or Thr; Arg72 → Asp; Lys73 → Asp; Asp77 → Met, Arg, Thr, or Asn; Trp79 → Ala or Asp; Arg81 → Met, Trp, or Ser; Phe83 → Leu; Cys87 → Ser; Leu94 → Phe; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu; and Lys134 → Tyr 31. The fusion protein of claim 30, comprising one or more of:

32. 25. The fusion protein of any one of claims 1 to 24, wherein the lipocalin mutein comprises one or more mutated amino acid residues at positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 of the linear polypeptide sequence of mature human neutrophil gelatinase-binding lipocalin (hNGAL) (SEQ ID NO: 2).

33. The amino acid sequence of the lipocalin mutein contains the following mutated amino acid residues at positions corresponding to 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gln20→Arg; Asn25→Tyr or Asp; Gln28→His; Val33→Ile; Leu36→Met; Ala40→Asn; Ile41→Leu; Glu44→Val or Asp; Gln49→His; Tyr52→Ser or G Ly; Lys59 → Asn; Ser68 → Asp; Leu70 → Met; Phe71 → Leu; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln or His; Tyr78 → His; Trp79 → Ile; Ile80 → Asn; Arg81 → Trp or Gln; Thr82 → Pro; Cys 87 → Ser; Phe92 → Leu or Ser; Asn96 → Phe; Lys98 → Arg; Tyr100 → Asp; Pro101 → Leu; Leu103 → His or Pro; Phe122 → Tyr; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly 33. The fusion protein of claim 32, comprising one or more of:

34. The amino acid sequence of the lipocalin mutein, as compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), comprises the following set of mutated amino acid residues: (a) Gln28→His; Leu36→Gln; Ala40→Ile; Ile41→Lys; Gln49→Asn; Tyr52→Met; Ser68→Gly; Leu70→Thr; Arg72→Asp; Lys73→Asp; Asp77→Thr; Trp79→Ala; Arg81→Ser; Cys87→Ser; Asn96→Lys; Tyr100→Phe; Leu103→His; Tyr106→Ser; Lys125→Phe; Ser127→Phe; Tyr132→Glu; and Lys134→Tyr; (b) Gln28→His; Leu36→Gln; Ala40→Ile; Ile41→Arg; Gln49→Ile; Tyr52→Met; Asn65→Asp; Ser68→Met; Leu70→Lys; Arg72→Asp; Lys73→Asp; Asp77→Met; Trp79→Asp; Arg81→Trp; Cys87→Ser; Asn96→Lys; Tyr100→Phe; Leu103→His; Tyr106→Ser; Lys125→Phe; Ser127→Phe; Tyr132→Glu; and Lys134→Tyr; (c) Gln28→His; Leu36→Gln; Ala40→Ile; Ile41→Arg; Gln49→Asn; Tyr52→Met; Asn65→Asp; Ser68→Ala; Leu70→Ala; Arg72→Asp; Lys73→Asp; Asp77→Thr; Trp79→Asp; Arg81→Trp; Cys87→Ser; Asn96→Lys; Tyr100→Phe; Leu103→His; Tyr106→Ser; Lys125→Phe; Ser127→Phe; Tyr132→Glu; and Lys134→Tyr; (d) Gln28→His; Leu36→Gln; Ala40→Ile; Ile41→Lys; Gln49→Asn; Tyr52→Met; Asn65→Asp; Ser68→Ala; Leu70→Ala; Arg72→Asp; Lys73→Asp; Asp77→Thr; Trp79→Asp; Arg81→Trp; Cys87→Ser; Asn96→Lys; Tyr100→Phe; Leu103→His; Tyr106→Ser; Lys125→Phe; Ser127→Phe; Tyr132→Glu; and Lys134→Tyr; (e) Gln28→His; Leu36→Gln; Ala40→Ile; Ile41→Lys; Gln49→Ser; Tyr52→Met; Asn65→Asp; Ser68→Gly; Leu70→Ser; Arg72→Asp; Lys73→Asp; Asp77→Thr; Trp79→Ala; Arg81→Met; Cys87→Ser; Asn96→Lys; Tyr100→Phe; Leu103→His; Tyr106→Ser; Lys125→Phe; Ser127→Phe; Tyr132→Glu; and Lys134→Tyr; (f) Gln28→His; Leu36→Gln; Ala40→Ile; Ile41→Lys; Gln49→Val; Tyr52→Met; Asn65→Asp; Ser68→Gly; Leu70→Thr; Arg72→Asp; Lys73→Asp; Asp77→Arg; Trp79→Asp; Arg81→Ser; Cys87→Ser; Leu94→Phe; Asn96→Lys; Tyr100→Phe; Leu103→His; Tyr106→Ser; Lys125→Phe; Ser127→Phe; Tyr132→Glu; and Lys134→Tyr; (g) Gln28→His; Leu36→Gln; Ala40→Ile; Ile41→Arg; Gln49→His; Tyr52→Met; Asn65→Asp; Ser68→Gly; Leu70→Thr; Arg72→Asp; Lys73→Asp; Asp77→Thr; Trp79→Ala; Arg81→Ser; Cys87→Ser; Asn96→Lys; Tyr100→Phe; Leu103→His; Tyr106→Ser; Lys125→Phe; Ser127→Phe; Tyr132→Glu; and Lys134→Tyr; (h) Gln28→His; Leu36→Gln; Ala40→Ile; Ile41→Lys; Gln49→Asn; Tyr52→Met; As n65→Asp; Ser68→Gly; Leu70→Thr; Arg72→Asp; Lys73→Asp; Asp77→Thr; Trp79→Al a; Arg81 → Ser; Phe83 → Leu; Cys87 → Ser; Leu94 → Phe; Asn96 → Lys; Tyr100 → Phe; Leu103 → His; Tyr106 → Ser; Lys125 → Phe; Ser127 → Phe; Tyr132 → Glu; and Lys134 → Tyr; (i) Gln28→His; Leu36→Gln; Ala40→Ile; Ile41→Arg; Gln49→Ser; Tyr52→Met; Asn65→Asp; Ser68→Ala; Leu70→Thr; Arg72→Asp; Lys73→Asp; Asp77→Asn; Trp79→Ala; Arg81→Ser; Cys87→Ser; Asn96→Lys; Tyr100→Phe; Leu103→His; Tyr106→Ser; Lys125→Phe; Ser127→Phe; Tyr132→Glu; and Lys134→Tyr; (j) Leu36→Met; Ala40→Asn; Ile41→Leu; Gln49→His; Tyr52→Ser; Ser68→Asp; Leu70→Met; Arg72→Leu; Lys73→Asp; Asp77→Gln; Trp79→Ile; Arg81→Trp; Asn96→Phe; Tyr100→Asp; Leu103→His; Lys125→Ser; Ser127→Ile; Tyr132→Trp; and Lys134→Gly; (k) Leu36→Met; Ala40→Asn; Ile41→Leu; Gln49→His; Tyr52→Ser; Ser68→Asp; Leu70→Met; Arg72→Leu; Lys73→Asp; Asp77→Gln; Trp79→Ile; Arg81→Trp; Phe92→Leu; Asn96→Phe; Lys98→Arg; Tyr100→Asp; Pro101→Leu; Leu103→His; Lys125→Ser; Ser127→Ile; Tyr132→Trp; and Lys134→Gly; (l) Asn25→Tyr; Leu36→Met; Ala40→Asn; Ile41→Leu; Gln49→His; Tyr52→Gly; Ser68→Asp; Leu70→Met; Phe71→Leu; Arg72→Leu; Lys73→Asp; Asp77→Gln; Trp79→Ile; Arg81→Gln; Phe92→Ser; Asn96→Phe; Tyr100→Asp; Leu103→His; Lys125→Ser; Ser127→Ile; Tyr132→Trp; and Lys134→Gly; (m) Leu36→Met; Ala40→Asn; Ile41→Leu; Gln49→His; Tyr52→Gly; Ser68→Asp; Leu70→Met; Arg72→Leu; Lys73→Asp; Asp77→Gln; Tyr78→His; Trp79→Ile; Arg81→Trp; Phe92→Leu; Asn96→Phe; Tyr100→Asp; Leu103→His; Lys125→Ser; Ser127→Ile; Tyr132→Trp; and Lys134→Gly; (n) Asn25→Asp; Leu36→Met; Ala40→Asn; Ile41→Leu; Gln49→His; Tyr52→Gly; Ser68→Asp; Leu70→Met; Arg72→Leu; Lys73→Asp; Asp77→Gln; Trp79→Ile; Arg81→Trp; Phe92→Leu; Asn96→Phe; Tyr100→Asp; Leu103→His; Lys125→Ser; Ser127→Ile; Tyr132→Trp; and Lys134→Gly; (o) Val33 → Ile; Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Gln49 → His; Tyr52 → Gly; Ser68 → Asp; Leu70 → Met; Arg72 → Leu; Lys73 → Asp; Asp77 → Gln; Trp79 → Ile; Arg81 → Trp; Phe92 → Leu; Asn96 → Phe; Tyr100 → Asp; Leu103 → His; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly; (p) Gln20→Arg; Leu36→Met; Ala40→Asn; Ile41→Leu; Glu44→Val; Gln49→His; Tyr52→Gly; Ser68→Asp; Leu70→Met; Arg72→Leu; Lys73→Asp; Asp77→Gln; Trp79→Ile; Arg81→Trp; Phe92→Leu; Asn96→Phe; Tyr100→Asp; Leu103→His; Phe122→Tyr; Lys125→Ser; Ser127→Ile; Tyr132→Trp; and Lys134→Gly; (q) Leu36→Met; Ala40→Asn; Ile41→Leu; Gln49→His; Tyr52→Ser; Ser68→Asp; Leu70→Met; Arg72→Leu; Lys73→Asp; Asp77→Gln; Trp79→Ile; Ile80→Asn; Arg81→Trp; Thr82→Pro; Asn96→Phe; Tyr100→Asp; Pro101→Leu; Leu103→Pro; Lys125→Ser; Ser127→Ile; Tyr132→Trp; and Lys134→Gly; (r) Leu36→Met; Ala40→Asn; Ile41→Leu; Gln49→His; Tyr52→Gly; Lys59→Asn; Ser68→Asp; Leu70→Met; Arg72→Leu; Lys73→Asp; Asp77→Gln; Trp79→Ile; Arg81→Trp; Phe92→Leu; Asn96→Phe; Tyr100→Asp; Leu103→His; Lys125→Ser; Ser127→Ile; Tyr132→Trp; and Lys134→Gly; and (s) Leu36 → Met; Ala40 → Asn; Ile41 → Leu; Glu44 → Asp; Gln49 → His; Tyr52 → Ser; Ser68 → Asp; Leu70 → Met; Phe71 → Leu; Arg72 → Leu; Lys73 → Asp; Asp77 → His; Trp79 → Ile; Arg81 → Trp; Phe92 → Leu; Asn96 → Phe; Tyr100 → Asp; Leu103 → His; Lys125 → Ser; Ser127 → Ile; Tyr132 → Trp; and Lys134 → Gly 34. The fusion protein of any one of claims 30 to 33, comprising one of:

35. 35. The fusion protein of any one of claims 1 to 24 and 30 to 34, wherein the amino acid sequence of the lipocalin mutein has at least 85% sequence identity to an amino acid sequence selected from SEQ ID NOs: 39 to 57.

36. 36. The fusion protein of any one of claims 1 to 24 and 30 to 35, wherein the amino acid sequence of the lipocalin mutein comprises an amino acid sequence selected from SEQ ID NOs: 39 to 57, or a fragment or variant thereof.

37. 37. A fusion protein according to any one of claims 1 to 24 and 30 to 36, wherein the amino acid sequence of the lipocalin mutein comprises the amino acid sequence of SEQ ID NO:

40.

38. 38. The fusion protein of any one of claims 1 to 37, wherein one subunit is linked to another subunit via a linker.

39. 39. The fusion protein of any one of claims 1 to 38, wherein the second subunit is linked at its N-terminus to the N-terminus or C-terminus of each heavy chain constant region (CH) of the first subunit or to the N-terminus or C-terminus of each light chain constant region (CL) of the first subunit via a linker.

40. 40. The fusion protein of any one of claims 1 to 39, wherein the third subunit is linked at its N-terminus to the N-terminus or C-terminus of each heavy chain constant region (CH) of the first subunit, the N-terminus or C-terminus of each light chain constant region (CL) of the first subunit, or the C-terminus of each second subunit via a linker.

41. 41. The fusion protein of any one of claims 38 to 40, wherein the linker is an unstructured glycine-serine linker, a polyproline linker, a proline-alanine-serine polymer, or a linker selected from SEQ ID NOs: 13-23.

42. The linker is unstructured (Gly-Gly-Gly-Gly-Ser) ３ 42. A fusion protein according to any one of claims 38 to 41, wherein the linker is (SEQ ID NO: 13).

43. the antibody or the antigen-binding domain thereof, a heavy chain variable domain (VH) comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 110, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 112, and a light chain variable domain (VL) comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 116, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 118; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 113, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 114, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 115, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 119, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 120, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 121; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 130, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 131, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 132, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 136, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 137, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 138; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 133, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 134, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 135, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 139, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 140, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 141; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 150, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 151, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 152, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 156, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 157, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 158; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 153, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 154, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 155, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 159, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 160, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 161; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 170, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 171, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 172, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 176, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 177, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 178; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 173, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 174, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 175, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 179, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 180, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 181; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 190, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 191, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 192, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 196, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 197, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 198; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 193, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 194, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 195, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 199, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 200, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 201; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:210, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO:211, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:212, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:216, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:217, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:218; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:213, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO:214, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:215, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:219, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:220, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:221; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 230, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 231, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 232, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 236, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 237, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 238; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 233, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 234, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 235, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 239, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 240, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 241; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:250, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO:251, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:252, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:256, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:257, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:258; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:253, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO:254, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:255, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:259, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:260, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:261; (a) a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:270, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:271, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:272, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:276, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:277, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:278; or (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 273, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 274, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 275; and (d) a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 279, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 280, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:

281.

43. The fusion protein of any one of claims 1 to 42, comprising:

44. the antibody or the antigen-binding domain thereof, (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:210, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO:211, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:212, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:216, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:217, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:218; (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:213, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO:214, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:215, and a VL comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO:219, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:220, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:221; (a) a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:250, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:251, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:252, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:256, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:257, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:258; or (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 253, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 254, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 255; and (d) a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 259, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 260, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:

261.

44. The fusion protein of claim 43, comprising:

45. the antibody or the antigen-binding domain thereof, (a) a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:210, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:211, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:212, and a VL comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:216, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:217, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:218; or (a) a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 213, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 214, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 215; and (d) a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 219, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 220, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:

221.

45. The fusion protein of claim 43 or 44, comprising:

46. the antibody or the antigen-binding domain thereof a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 122, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 124; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 142, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 144; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 162, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 164; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 182, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 184; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 202, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 204; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 224; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 242, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 244; a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 264; or A VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 282, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

284.

44. The fusion protein of claim 43, comprising:

47. the antibody or the antigen-binding domain thereof, a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 224; or A VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

264.

47. The fusion protein of any one of claims 43, 44 and 46, comprising:

48. the antibody or the antigen-binding domain thereof, A VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 222, and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

224.

48. The fusion protein of any one of claims 43 to 47, comprising:

49. the antibody or the antigen-binding domain thereof, a VH comprising the amino acid sequence of SEQ ID NO: 122, and a VL comprising the amino acid sequence of SEQ ID NO: 124; a VH comprising the amino acid sequence of SEQ ID NO: 142, and a VL comprising the amino acid sequence of SEQ ID NO: 144; a VH comprising the amino acid sequence of SEQ ID NO: 162, and a VL comprising the amino acid sequence of SEQ ID NO: 164; a VH comprising the amino acid sequence of SEQ ID NO: 182, and a VL comprising the amino acid sequence of SEQ ID NO: 184; a VH comprising the amino acid sequence of SEQ ID NO: 202, and a VL comprising the amino acid sequence of SEQ ID NO: 204; a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224; a VH comprising the amino acid sequence of SEQ ID NO: 242, and a VL comprising the amino acid sequence of SEQ ID NO: 244; a VH comprising the amino acid sequence of SEQ ID NO: 262, and a VL comprising the amino acid sequence of SEQ ID NO: 264; or VH comprising the amino acid sequence of SEQ ID NO: 282, and VL comprising the amino acid sequence of SEQ ID NO: 284 47. The fusion protein of claim 43 or 46, comprising:

50. the antibody or the antigen-binding domain thereof, a VH comprising the amino acid sequence of SEQ ID NO: 222, and a VL comprising the amino acid sequence of SEQ ID NO: 224; or VH comprising the amino acid sequence of SEQ ID NO: 262, and VL comprising the amino acid sequence of SEQ ID NO: 264 50. The fusion protein of any one of claims 43, 44, 46, 47 and 49, comprising:

51. the antibody or the antigen-binding domain thereof, VH comprising the amino acid sequence of SEQ ID NO: 222, and VL comprising the amino acid sequence of SEQ ID NO: 224 51. The fusion protein of any one of claims 43 to 50, comprising:

52. the antibody or the antigen-binding domain thereof, a heavy chain (HC) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 126, and a light chain (LC) comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 128; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 146, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 148; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 166, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 168; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 186, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 188; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 206, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 208; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 228; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:246, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:248; an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 266, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 268; or HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 286, and LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

288.

50. The fusion protein of any one of claims 43, 46 and 49, comprising:

53. the antibody or the antigen-binding domain thereof, an HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and an LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 228; or HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 266, and LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

268.

53. The fusion protein of any one of claims 43, 44, 46, 47, 49, 50 and 52, comprising:

54. an antibody or antigen-binding domain thereof, HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

228.

54. The fusion protein of any one of claims 43 to 53, comprising:

55. the antibody or the antigen-binding domain thereof, an HC comprising the amino acid sequence of SEQ ID NO: 126, and an LC comprising the amino acid sequence of SEQ ID NO: 128; an HC comprising the amino acid sequence of SEQ ID NO: 146, and an LC comprising the amino acid sequence of SEQ ID NO: 148; an HC comprising the amino acid sequence of SEQ ID NO: 166, and an LC comprising the amino acid sequence of SEQ ID NO: 168; an HC comprising the amino acid sequence of SEQ ID NO: 186, and an LC comprising the amino acid sequence of SEQ ID NO: 188; an HC comprising the amino acid sequence of SEQ ID NO: 206, and an LC comprising the amino acid sequence of SEQ ID NO: 208; an HC comprising the amino acid sequence of SEQ ID NO: 226, and an LC comprising the amino acid sequence of SEQ ID NO: 228; an HC comprising the amino acid sequence of SEQ ID NO: 246, and an LC comprising the amino acid sequence of SEQ ID NO: 248; an HC comprising the amino acid sequence of SEQ ID NO: 266, and an LC comprising the amino acid sequence of SEQ ID NO: 268; or HC comprising the amino acid sequence of SEQ ID NO: 286, and LC comprising the amino acid sequence of SEQ ID NO: 288 53. The fusion protein of any one of claims 43, 46, 49, and 52, comprising:

56. the antibody or the antigen-binding domain thereof, an HC comprising the amino acid sequence of SEQ ID NO: 226, and an LC comprising the amino acid sequence of SEQ ID NO: 228; or HC comprising the amino acid sequence of SEQ ID NO: 266, and LC comprising the amino acid sequence of SEQ ID NO: 268 56. The fusion protein of any one of claims 43, 44, 46, 47, 49, 50, 52, 53, and 55, comprising:

57. the antibody or the antigen-binding domain thereof, HC comprising the amino acid sequence of SEQ ID NO: 226, and LC comprising the amino acid sequence of SEQ ID NO:

228.

57. The fusion protein of any one of claims 43 to 56, comprising:

58. 58. The fusion protein of any one of claims 1 to 57, wherein the antibody is a monoclonal antibody.

59. 59. The fusion protein of any one of claims 1 to 58, wherein the antibody is a humanized antibody or a chimeric antibody.

60. 60. The fusion protein of any one of claims 1 to 59, wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.

61. The antibody or the antigen-binding domain thereof has a K Ｄ 61. The fusion protein of any one of claims 1 to 60, which binds to CD228 at a value

62. 62. The fusion protein of any one of claims 1 to 61, wherein the antibody or the antigen-binding domain thereof binds to cynomolgus monkey CD228.

63. A fusion protein described in any one of claims 1 to 62, wherein the antibody or its antigen-binding domain does not bind to mouse CD228 or binds to mouse CD228 with an affinity that is 100-fold or less reduced compared to human CD228.

64. 64. The fusion protein of any one of claims 1 to 63, wherein the antibody or the antigen-binding domain thereof does not bind to transferrin or lactotransferrin.

65. 65. The fusion protein of any one of claims 1 to 64, wherein the antibody is an IgG4 antibody.

66. 66. The fusion protein of claim 65, wherein the antibody comprises one or more of the following mutations: S228P, N297A, F234A, L235A, M428L, N434S, M252Y, S254T, and T256E.

67. 67. The fusion protein of any one of claims 1 to 44, 46, 47, 49, 50, 52, 53, 55, 56, and 58 to 66, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 75, 76, and 78-83, or comprising 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 more sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 75, 76, and 78-83.

68. 68. The fusion protein of any one of claims 1 to 44, 46, 47, 49, 50, 52, 53, 55, 56, and 58 to 67, comprising 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 more sequence identity to the amino acid sequence set forth in SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, or SEQ ID NOs: 78 and 83.

69. 69. A fusion protein according to any one of claims 1 to 44, 46, 47, 49, 50, 52, 53, 55, 56, and 58 to 68, comprising the amino acid sequence set forth in SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, or SEQ ID NOs: 78 and 83.

70. 70. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1 to 69.

71. 71. The nucleic acid molecule of claim 70, operably linked to a regulatory sequence to allow expression of the nucleic acid molecule.

72. 71. A vector comprising the nucleic acid molecule of claim 69 or 70.

73. 73. A host cell comprising a nucleic acid molecule according to claim 69 or 70 or a vector according to claim 72.

74. 74. A method for producing a fusion protein according to any one of claims 1 to 73, wherein the fusion protein is produced starting from a nucleic acid encoding the fusion protein.

75. 75. The method of claim 74, wherein the fusion protein is produced in a bacterial or eukaryotic host organism and isolated from the host organism or a culture thereof.

76. Use of a fusion protein described in any one of claims 1 to 69, or a composition comprising such a fusion protein, to simultaneously activate downstream signaling pathways of CD137 and engage CD228-positive tumor cells.

77. A method for simultaneously activating downstream signaling pathways of CD137 and engaging CD228-positive tumor cells, comprising applying one or more fusion proteins described in any one of claims 1 to 69 or one or more compositions comprising such fusion proteins to tissue containing a tumor.

78. 70. A method of simultaneously costimulating T cells and engaging CD228-positive tumor cells, comprising applying to a tissue containing a tumor one or more fusion proteins of any one of claims 1 to 69 or one or more compositions comprising such fusion proteins.

79. 70. A method for simultaneously inducing lymphocyte activity and engaging CD228-positive tumor cells, comprising applying one or more fusion proteins of any one of claims 1 to 69 or one or more compositions comprising such fusion proteins to a tissue containing a tumor.

80. 70. A method for inducing CD137 clustering and T cell activation and directing said T cells to CD228-positive tumor cells, comprising applying to a tissue containing a tumor one or more fusion proteins of any one of claims 1 to 69 or one or more compositions comprising such fusion proteins.

81. A method for inducing a localized lymphocyte response in the vicinity of CD228-positive tumor cells, comprising applying one or more fusion proteins described in any one of claims 1 to 69 or one or more compositions comprising such fusion proteins to tissue containing a tumor.

82. 70. A method of inducing increased secretion of IL-2 and / or cytotoxic factors by T cells in the vicinity of CD228-positive tumor cells, comprising applying to a tissue containing a tumor one or more fusion proteins of any one of claims 1 to 69, or one or more compositions comprising such fusion proteins.

83. 70. A method for inducing increased secretion of cytotoxic factors by T cells in the vicinity of CD228-positive tumor cells, comprising applying to a tissue containing a tumor one or more fusion proteins of any one of claims 1 to 69 or one or more compositions comprising such fusion proteins.

84. 84. The method of claim 82 or 83, wherein the cytotoxic factor is selected from perforin, granzyme B, and granzyme A.

85. 70. A method for stimulating exhausted CD8+ T cell proliferation in the vicinity of CD228-positive tumor cells, comprising applying one or more fusion proteins of any one of claims 1 to 69 or one or more compositions comprising such fusion proteins to tissue containing a tumor.

86. 86. The method of claim 85, further comprising applying an anti-PD-1 antibody or an anti-PD-L1 antibody or one or more compositions comprising such antibodies to the tissue comprising the tumor.

87. 87. The method of claim 86, wherein the anti-PD-1 antibody or anti-PD-L1 antibody is nivolumab, pembrolizumab, cemiplimab, dostallimab, atezolizumab, avelumab, or durvalumab.

88. 70. A method for inducing an increase in mitochondrial content of CD8+ T cells and / or a decrease in mitochondrial depolarization of CD8+ T cells in the vicinity of CD228-positive tumor cells, comprising applying to a tissue comprising a tumor one or more fusion proteins of any one of claims 1 to 69 or one or more compositions comprising such fusion proteins.

89. 70. A pharmaceutical composition comprising one or more fusion proteins according to any one of claims 1 to 69.

90. 70. A method of treating cancer in a subject in need thereof, comprising administering to the subject a fusion protein of any one of claims 1 to 69 or one or more compositions comprising such a fusion protein.

91. 91. The method of claim 90, further comprising administering to the subject an anti-PD-1 antibody or an anti-PD-L1 antibody or one or more compositions comprising such antibodies.

92. 92. The method of claim 91, wherein the anti-PD-1 antibody or anti-PD-L1 antibody is nivolumab, pembrolizumab, cemiplimab, dostallimab, atezolizumab, avelumab, or durvalumab.

93. 70. A fusion protein according to any one of claims 1 to 69 for use in therapy.

94. 94. The fusion protein for use according to claim 93, wherein said use is in the treatment of cancer.

95. 70. Use of a fusion protein according to any one of claims 1 to 69 for the manufacture of a medicament.

96. 96. The use of claim 95, wherein the medicament is for the treatment of cancer.

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