Novel fusion proteins specific for CD137 and CD228

Abstract

The present disclosure provides antibodies, or antigen binding domains thereof, specific for CD228, as well as fusion proteins specific for both CD137 and CD228, which can be used to co-stimulate lymphocyte activation in a CD228 target dependent manner. Such antibodies, antigen binding domains or fusion proteins may be used in many pharmaceutical applications, for example, as anticancer agents and / or immunomodulators. The 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 disclosure further relates to nucleic acid molecules encoding such antibodies, antigen binding domains or fusion proteins. In addition, the application discloses therapeutic and / or diagnostic uses of such antibodies, antigen binding domains or fusion proteins.

Description

[0001] I. Cross-reference to Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 408,634, filed on September 21, 2022; U.S. Provisional Application No. 63 / 413,174, filed on October 4, 2022; and U.S. Provisional Application No. 63 / 496,463, filed on April 17, 2023; each of which is incorporated herein by reference in its entirety for any purpose.

[0003] II. Introduction and Overview

[0004] Cluster of differentiation 228 or CD228 (also known as melanin transferrin, MELTF, p97, and MFI2) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein that belongs to the transferrin family of iron-binding proteins and was first described as an oncofetal protein that is highly expressed on malignant melanoma cells (Rose et al., Proc Natl Acad Sci USA, 1986).

[0005] CD228 is expressed in various cancers, including melanoma, mesothelioma, thyroid cancer, lung cancer, liver cancer, pancreatic cancer, head and neck cancer, gastric cancer, colorectal cancer, urothelial carcinoma, breast cancer, and cervical cancer. Melanoma, also known as malignant melanoma, is a type of cancer that develops from melanocytes (pigment-containing cells). Melanoma is the most dangerous type of skin cancer. In 2015, 3.1 million people had active disease, and melanoma caused 59,800 deaths. Surgery can be effective for early-stage melanoma, but may not be a treatment option for disease that has metastasized to distant organs. Disseminated melanoma often spreads to lymph nodes in the area before spreading elsewhere. Attempts to improve survival by surgically removing lymph nodes are associated with numerous complications but have no overall survival benefit. Immunotherapy, chemotherapy, and radiation therapy have all been used, but are often incurable, particularly for advanced melanoma. When distant metastases are present, the cancer is generally considered incurable. The five-year survival rate for stage IV disease is 15-20%.

[0006] CD137 (also known as 4-1BB and TNFRSF9) is a co-stimulatory immune receptor and a member of the tumor necrosis factor receptor (TNFR) superfamily. It is mainly expressed on activated CD4+ and CD8+ T cells, activated B cells and natural killer (NK) cells, but can also be 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 the regulation of immune response, and is therefore the target of cancer immunotherapy. CD137 ligands (CD137L) are the only known natural ligands of CD137, which are constitutively expressed on several types of antigen presenting cells such as activated B cells, monocytes and spleen dendritic cells, and can be induced on T lymphocytes.

[0007] CD137L is a trimeric protein, which exists as a membrane-bound form and as a soluble variant. However, the ability of soluble CD137L to activate CD137 (for example, on lymphocytes expressing CD137) is limited, and requires high concentrations to induce effects (Wyzgol et al., J Immunol, 2009). The natural activation mode of CD137 is the engagement of CD137 positive cells with CD137L positive cells. Then, CD137 activation is considered to be induced by clustering of CD137L on relative cells, resulting in further downstream effects of accompanying in signal transduction (Yao et al., Nat Rev Drug Discov, 2013; Snell et al., Immunol Rev, 2011) and CD137 positive T cells via TRAF1, 2 and 3. In the case of T cells being activated by identifying their respective homologous targets, the effect triggered by the costimulation of CD137 is further enhanced activation, enhanced survival and proliferation, the generation of proinflammatory cytokines and improved killing ability.

[0008] The present disclosure provides, inter alia, novel antibodies and antigen binding domains thereof that bind CD228, as well as novel methods for simultaneously engaging CD137 and CD228 via one or more fusion proteins having the properties of binding specificity for CD137 and binding specificity for CD228.

[0009] III. Definitions

[0010] 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.

[0011] As used herein, unless otherwise indicated, "CD137" means human CD137 (huCD137). Human CD137 means the full-length protein defined by UniProt Q07011, its fragment or its variant. CD137 is also known as 4-1BB, tumor necrosis factor receptor superfamily member 9 (TNFRSF9), and is induced by lymphocyte activation (ILA). In some specific embodiments, CD137 of non-human species is used, such as cynomolgus monkey CD137 or mouse CD137.

[0012] As used herein, unless otherwise indicated, "CD228" refers to human CD228. Human CD228 refers to the full-length protein defined by UniProt P08582, its mature form, its isomer, its fragment, or its variant. Human CD228 is encoded by the MELTF gene. CD228 is also known as melanocyte transferrin, MELTF, p97, and MFI2, which terms may be used interchangeably herein. In some specific embodiments, CD228 of a non-human species is used, such as cynomolgus monkey CD228 or mouse CD228.

[0013] As used herein, "binding affinity" describes the ability of a biomolecule (e.g., polypeptide or protein) of the present disclosure (e.g., lipocalin mutein, antibody, antigen binding domain thereof, fusion protein, or any other peptide or protein) to bind to a selected target (and form a complex). Binding affinity is measured by a variety of methods known to those skilled in the art, including but not limited to fluorescence titration, enzyme-linked immunosorbent assay (ELISA)-based assays including direct and competitive ELISA, calorimetry 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 thus reported as the dissociation constant (K) measured using such methods. D ), half-maximal effect concentration (EC 50 ) or half-maximal inhibitory concentration (IC 50 ) value. Lower K D 、EC 50 or IC 50 The value reflects better (higher) binding ability (affinity). Accordingly, 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", "about the same", "substantially the same", or "substantially similar" mean that one biomolecule has a value reported as K. D 、EC 50 or IC 50The binding affinity of a molecule is the same as or similar to the binding affinity of another molecule within the experimental variability of the binding affinity measurement. Preferably, "comparable", "about the same", "substantially the same" or "substantially similar" relates to values that are within 50% deviation, more preferably within 20% deviation, and most preferably within 10% deviation of a given reference value. The experimental variability of binding affinity measurements depends on the specific method used and is known to those skilled in the art.

[0014] As used herein, the term "substantially" may also refer to the overall or near-overall extent or degree of the characteristic or property that exhibits interest. It will be understood by those of ordinary skill in the biological field that biological and chemical phenomena rarely, if ever, complete and / or proceed to completeness or achieve or avoid absolute results. The term "substantially" is therefore used in this article to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0015] As used herein, the terms "detect," "detection," "detectable," or "detecting" are understood at both a quantitative and a qualitative level, as well as combinations thereof. They therefore include quantitative, semi-quantitative, and qualitative measurements performed on the biomolecules of the present disclosure.

[0016] As used herein, "detectable affinity" generally means the affinity determined by K D 、EC 50 or IC 50 The reported binding capacity between a biomolecule and its target is at most about 10 -5 M or lower. By K D 、EC 50 or IC 50 Values reported are higher than 10 -5 The binding affinity of M is generally no longer measurable with common methods such as ELISA and SPR and is therefore of secondary importance. Thus, "detectable affinity" may refer to an affinity of about 10 -5 M or lower K D value.

[0017] As used herein, "specific for," "specific binding," "specifically bind," 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., a cellular receptor for neutrophil gelatinase-associated lipocalin). It will be understood that such specificity is not an absolute property, but a relative property, and can be determined, for example, by means of SPR, Western blot, ELISA, fluorescence-activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), immunohistochemistry (IHC), and peptide scanning.

[0018] When used in the context of a biomolecule herein (e.g., an antibody, antigen binding domain thereof, or fusion protein that binds to CD137 and / or CD228 of the present disclosure), the terms "specifically for," "specific binding," "specifically bind," 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 neither CD137 or CD228 nor a protein that is closely related to or homologous to CD137 or CD228. However, CD137 or CD228 from species other than humans, 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 the biomolecule of the present disclosure binds to another protein with a lower binding affinity than CD137 and / or CD228, i.e., shows less than 30%, preferably 20%, more preferably 10%, particularly preferably less than 9, 8, 7, 6 or 5% cross-reactivity. Whether a biomolecule specifically reacts as defined herein above can be easily tested, in particular by comparing the reaction of a biomolecule of the present disclosure with CD137 and / or CD228 and the reaction of said biomolecule with (one or more) other proteins.

[0019] As used herein, the term "lipocalin" refers to a monomeric protein weighing approximately 18-20 kDa having a cylindrical β-sheet supersecondary structural region comprising a plurality of β-strands (preferably eight β-strands designated A to H) connected in pairs by a plurality of (preferably four) loops at one end to form a ligand-binding pocket and define the entrance to the ligand-binding pocket. Preferably, the loops forming the ligand-binding pocket used in the present disclosure are loops connecting the open ends of β-strands A and B, C and D, E and F, and G and H, and are designated as loops AB, CD, EF, and GH. It is well established that the diversity of these loops in the otherwise rigid lipocalin scaffold results in a variety of different binding modes among members of the lipocalin family, each of which can accommodate 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). It is understood that the lipocalin protein family has evolved naturally to bind a wide range of ligands, sharing a rare low level of overall sequence conservation (often having less than 20% sequence identity), but still retaining a highly conserved overall folding pattern. The correspondence between positions in various lipocalins is also well known to those skilled in the art (see, for example, U.S. Patent No. 7,250,297). Proteins that fall within the definition of "lipocalin" as used herein include, but are not limited to, human lipocalins, including tear lipocalin (Tlc, Lcn1), lipocalin-2 (Lcn2) or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D (ApoD), apolipoprotein M, alpha 1-acid glycoprotein 1, alpha 1-acid glycoprotein 2, alpha 1-microglobulin, complement component 8γ, retinol binding protein (RBP), epididymal retinoic acid binding protein, immunosuppressive glycoprotein (glycodelin), odorant binding protein IIa, odorant binding protein IIb, lipocalin-15 (Lcn15), and prostaglandin D synthase.

[0020] As used herein, unless otherwise indicated, "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 that is substantially free of a signal peptide. "Mature hTlc" of the present disclosure refers to the mature form of human tear lipocalin, which does not contain a signal peptide. Mature hTlc is described by residues 19-176 of the sequence deposited in the SWISS-PROT database under accession number P31025, and its amino acid sequence is shown in SEQ ID NO: 1.

[0021] 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" means a protein that is substantially free of a signal peptide. "Mature hNGAL" of the present disclosure refers to the mature form of human neutrophil gelatinase-associated lipocalin, which does not contain a signal peptide. Mature hNGAL is described by residues 21-198 of the sequence deposited in the SWISS-PROT database under accession number P80188, and its amino acid sequence is shown in SEQ ID NO: 2.

[0022] As used herein, "native sequence" refers to a protein or polypeptide having a sequence found in nature or having a 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.

[0023] "Native sequence lipocalin" refers to a lipocalin having an amino acid sequence identical to the corresponding polypeptide derived from nature. Thus, a native sequence lipocalin may have the amino acid sequence of a naturally occurring (wild-type) lipocalin from any organism, particularly a mammal. When used in the context of a lipocalin, the term "native sequence" specifically encompasses naturally occurring truncated or secreted forms of the lipocalin, naturally occurring variant forms of the lipocalin, such as alternatively spliced forms, and naturally occurring allelic variants. The terms "native sequence lipocalin" and "wild-type lipocalin" are used interchangeably herein.

[0024] As used herein, a "mutant," a "mutant" entity (whether a protein or nucleic acid) or a "mutant" refers to an exchange, deletion or insertion of one or more amino acids or nucleotides compared to a naturally occurring (wild-type) protein or nucleic acid. The term also includes fragments of a mutant as described herein. The present disclosure specifically encompasses a lipocalin mutant (also known as a lipocalin mutant) having a cylindrical beta-sheet supersecondary structure region as described herein. The present invention relates to a lipocalin mutein (lipocalin mutein) wherein the supersecondary structure region comprises eight beta strands connected in pairs by four loops at one end to form a ligand-binding pocket and define an entrance to the ligand-binding pocket, wherein at least one amino acid located within the four loops has been mutated compared to a native sequence lipocalin. The lipocalin mutein of the present invention preferably has the function of binding to CD137 as described herein.

[0025] As used herein, the term "fragment" in conjunction with 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 is N-terminally and / or C-terminally truncated, i.e., lacks at least one N-terminal and / or C-terminal amino acid. Such fragments may include at least 10 or more, e.g., 20 or 30 or more, consecutive amino acids of the primary sequence of mature hTlc or hNGAL or the lipocalin mutein from which it is derived, and are typically detectable in immunoassays for mature hTlc or hNGAL. Such fragments may lack at most 2, at most 3, at most 4, at most 5, at most 10, at most 15, at most 20, at most 25, or at most 30 (including all numbers in between) N-terminal and / or C-terminal amino acids. As illustrative examples, such fragments may lack one, two, three or four N-terminal amino acids (His-His-Leu-Leu) and / or one or two C-terminal amino acids (Ser-Asp) of mature hTlc. It will be appreciated that the fragment is preferably a functional fragment of mature hTlc or hNGAL or a lipocalin mutant thereof, meaning that it preferably retains the binding specificity of the mature hTlc / hNGAL or lipocalin mutant thereof, preferably for CD137. As illustrative examples, such functional fragments may comprise at least the amino acids at positions 5-153, 5-150, 9-148, 12-140, 20-135 or 26-133 of the linear polypeptide sequence corresponding to mature hTlc. As another illustrative example, such functional fragments may comprise at least the amino acids at positions 13-157, 15-150, 18-141, 20-134, 25-134 or 28-134 of the linear polypeptide sequence corresponding to mature hNGAL.

[0026] With respect to the corresponding target CD137 or CD228 of the antibody of the present disclosure, its antigen binding domain or fusion protein, " fragment " refers to CD137 or CD228 or the protein domain of CD137 or CD228 that is truncated at the N-terminus and / or C-terminus. The fragment of CD137 or CD228 as described herein retains the ability of full-length CD137 or CD228 to be recognized and / or bound by the antibody of the present disclosure, its antigen binding domain or fusion protein. As an illustrative example, the fragment can be the extracellular domain of CD137. For example, such extracellular domains of CD137 can include amino acids of the extracellular subdomains of CD137, such as domain 1 (residues 24-45 of UniProt Q07011), domain 2 (residues 46-86), domain 3 (87-118) and domain 4 (residues 119-159) individually or in combination.

[0027] As used herein, the term "variant" refers to a derivative of a protein or polypeptide, including, for example, mutations by substitution, deletion, insertion, and / or chemical modification of an amino acid sequence or nucleotide sequence. In some embodiments, such mutations and / or chemical modifications do not reduce the functionality of the protein or peptide. Such substitutions may be conservative, i.e., amino acid residues are replaced by chemically similar amino acid residues. Examples of conservative substitutions are substitutions among 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 have been substituted with their respective D-stereoisomers or with amino acids other than the 20 naturally occurring amino acids (e.g., such as ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline). Such variants also include, for example, proteins or polypeptides in which one or more amino acid residues are added or deleted 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 the native sequence protein or polypeptide. The variant preferably retains the biological activity of the protein or polypeptide from which it is derived, e.g., binds to the same target.

[0028] As used herein in relation to CD137 or CD228, the term "variant" refers to CD137 or CD228, respectively, or a fragment thereof, which has one or more, e.g., 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 the native sequence of CD137 or CD228 (wild-type CD137 or CD228), e.g., CD137 as deposited by UniProt Q07011 or CD228 as deposited by UniProt P08582 as described herein. Preferably, the CD137 variant 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 variants or CD228 variants as described herein retain the ability to bind to the antibodies, antigen binding domains thereof, lipocalin muteins or fusion proteins disclosed herein that are specific for CD137 and / or CD228.

[0029] As used herein with respect to lipocalin muteins, the term "variant" relates to the lipocalin muteins of the present disclosure or fragments thereof, wherein the sequence has a mutation, including a substitution, deletion, insertion, and / or chemical modification. Variants of lipocalin muteins as described herein retain the biological activity of the lipocalin mutein from which it is derived, such as 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.

[0030] As used herein with respect to antibodies or their antigen-binding domains, the term "variant" relates to antibodies or their antigen-binding domains of the present disclosure, wherein the sequence has mutations, including substitutions, deletions, insertions, and / or chemical modifications. Variants of antibodies or their antigen-binding domains as described herein retain the biological activity of the antibody or its antigen-binding domain from which it is derived, such as binding to CD228. Generally, a variant of an antibody or its antigen-binding domain has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or 98% amino acid sequence identity with the antibody or its antigen-binding domain from which it is derived.

[0031] 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 the naturally occurring amino acid at a given position in a protein or polypeptide sequence is altered, for example, by substitution of at least one amino acid. The term "mutagenesis" also includes (additionally) modifying the length of a sequence segment by deletion or insertion of one or more amino acids. Thus, within the scope of the present disclosure, for example, the replacement of one amino acid at a selected sequence position with a stretch of three amino acids results in the addition of two amino acid residues compared to the length of the respective segment of the native protein or polypeptide amino acid sequence. Such insertions or deletions can be introduced independently of one another into any sequence segment that can be subjected to mutagenesis in the present disclosure. In an exemplary embodiment of the present disclosure, an insertion can be introduced into an amino acid sequence segment corresponding to loop AB of a native sequence lipocalin (see International Patent Publication No. WO 2005 / 019256, which is incorporated herein by reference in its entirety).

[0032] As used herein, the term "random mutagenesis" means that there is no predetermined mutation (change of amino acid) at a certain sequence position, but at least two amino acids can be incorporated at a predefined sequence position with a certain probability during the mutagenesis process.

[0033] As used herein, the term "sequence identity" or "identity" refers to a property of sequences that measures their similarity or relationship. As used in the present disclosure, the term "sequence identity" or "identity" means the percentage of pairwise identical residues relative to the number of residues in the longer of the two sequences after (homologous) alignment of a protein or polypeptide sequence of the present disclosure with a 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 result by 100.

[0034] As used herein, the term "sequence homology" or "homology" has its ordinary meaning, and homologous amino acids include identical amino acids as well as 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).

[0035] The skilled person will recognize the 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. The percentage of sequence homology or sequence identity can be determined, for example, herein 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 based on an alignment of the entire protein or polypeptide sequence including the propeptide sequence (matrix: BLOSUM 62; gap cost: 11.1; cutoff value set to 10 -3 ), preferably using the wild-type protein scaffold as the reference in the pairwise comparison. It is calculated as the percentage of the number of "positives" (homologous amino acids) indicated as results in the BLASTP program output divided by the total number of amino acids selected for alignment by the program.

[0036] Specifically, to determine whether the amino acid sequence of a lipocalin (mutant) differs from a reference (wild-type) lipocalin at a certain position in the amino acid sequence of the reference (wild-type) lipocalin, the skilled person can use means and methods well known in the art, such as manual alignment or alignment 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. Accordingly, 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 a lipocalin is the sequence of hTLc as shown in SEQ ID NO: 1 or the sequence of hNGAL as shown in SEQ ID NO: 2.

[0037] A "gap" is a gap in an alignment that is the result of an amino acid addition or deletion. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less conserved and have deletions, additions or substitutions may have a lower degree of sequence identity.

[0038] As used herein, the term "position" refers to the position of an amino acid within the amino acid sequences disclosed herein or the position of a nucleotide within the nucleic acid sequences disclosed herein. It should be understood that when the terms "correspond" or "corresponding" are used herein in the context of an amino acid sequence position of one or more lipocalin mutants, the corresponding position is not determined solely by the number of preceding nucleotides or amino acids. Accordingly, according to the present disclosure, the absolute position of a given amino acid may differ from the corresponding position due to deletions or additions of amino acids elsewhere in the (mutant or wild-type) lipocalin. Similarly, according to the present disclosure, the absolute position of a given nucleotide may differ from the corresponding position due to deletions or additions of nucleotides elsewhere in the 5'-untranslated region (UTR) of the mutant or wild-type lipocalin (including the promoter and / or any other regulatory sequences or gene regions (including exons and introns)).

[0039] A "corresponding position" according to the present disclosure may be a sequence position that is aligned with its corresponding sequence position in a pairwise or multiple sequence alignment according to the present disclosure. It is preferably understood that for a "corresponding position" according to the present disclosure, the absolute position of a nucleotide or amino acid may differ from an adjacent nucleotide or amino acid, but said adjacent nucleotides or amino acids that may have been exchanged, deleted or added may be encompassed by the same one or more "corresponding positions".

[0040] Furthermore, with respect to corresponding positions in a lipocalin mutein based on a reference sequence according to the present disclosure, it is preferably understood that the nucleotide or amino acid positions of the lipocalin mutein may structurally correspond to positions elsewhere in the reference lipocalin (wild-type lipocalin) or another lipocalin mutein, even though they may differ in absolute position numbering, as will be appreciated by the skilled person in light of the highly conserved overall folding pattern in lipocalins.

[0041] As used interchangeably herein, the terms "conjugate," "conjugation," "fuse," "fusion," or "link" refer to the joining together of two or more subunits by all forms of covalent or non-covalent bonding, including but not limited to genetic fusion, chemical conjugation, coupling via a linker or cross-linking agent, and non-covalent association.

[0042] As used herein, the term "fusion polypeptide" or "fusion protein" refers to a polypeptide or protein comprising two or more subunits. In some embodiments, the fusion protein as described herein comprises two or more subunits, at least one of which is capable of specifically binding to CD137, and a further subunit is capable of specifically binding to CD228. Within the fusion protein, these subunits may be linked by covalent or non-covalent bonding. Preferably, the fusion protein is a translational fusion between the two or more subunits. Translational fusion may be generated by genetically engineering the coding sequence of one subunit with the coding sequence of a further subunit in the reading frame. Both subunits may be interspersed with nucleotide sequences encoding a linker. However, the subunits of the fusion protein of the present disclosure may also be linked by chemical conjugation. The subunits forming the 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 C-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 can be linked in any order and may include more than one of any of the constituent subunits. If one or more subunits are part of a protein (complex) composed of more than one polypeptide chain, the term "fusion protein" may also refer to a protein comprising 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" may refer to a single polypeptide chain comprising the lipocalin mutein and the heavy or light chain of the immunoglobulin / antibody. The term "fusion protein" may also refer to the entire immunoglobulin / antibody (both light and heavy chains) as well as a lipocalin mutein fused to one or both of its heavy and / or light chains.

[0043] As used herein, the term "subunit" of the fusion proteins disclosed herein refers to a single protein or isolated polypeptide chain that can form a stable folded structure on its own and may define a unique function that provides a binding motif for a target. In some embodiments, preferred subunits of the present disclosure are lipocalin muteins. In some other embodiments, preferred subunits of the present disclosure are antibodies, such as full-length antibodies, or antigen-binding domains / fragments thereof.

[0044] The "linker" that may be included in the fusion protein of the present disclosure connects two or more subunits of the fusion protein as described herein together. The bonding can be covalent or non-covalent. Preferred covalent bonding is via a peptide bond, such as a peptide bond between amino acids. Preferred linkers are peptide linkers. Accordingly, in a preferred embodiment, the linker comprises one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. Preferred peptide linkers are described herein, including glycine-serine (GS) linkers, glycosylated GS linkers, and proline-alanine-serine polymer (PAS) linkers. In some preferred embodiments, a GS linker, such as the (G4S)3 linker as described in SEQ ID NO: 13, is used to connect the subunits of the fusion protein together. Other preferred linkers include chemical linkers.

[0045] As used herein, the term "albumin" includes all mammalian albumins, such as human serum albumin or bovine serum albumin or rat serum albumin.

[0046] As used herein, the term "organic molecule" or "small organic molecule" refers to an organic molecule comprising at least two carbon atoms, but preferably no more than 7 or 12 rotatable carbon bonds, having a molecular weight between 100 and 2,000 Daltons, preferably between 100 and 1,000 Daltons, and optionally including one or two metal atoms.

[0047] "Sample" is defined as a biological sample taken from any subject. Biological samples include, but are not limited to, blood, serum, urine, feces, semen, or tissue, including tumor tissue.

[0048] 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, domesticated animals and farm animals, as well as zoo animals, sports animals, 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.

[0049] An "effective amount" is an amount sufficient to produce beneficial or desired results. An effective amount can be administered in one or more individual administrations or dosages.

[0050] As used herein, "antibody" includes whole antibodies or any antigen-binding fragment thereof (i.e., "antigen-binding portion" or "antigen-binding domain") or single chains thereof. 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 consists of a heavy chain variable domain / region (V H or HCVR) and heavy chain constant region (C H ). The heavy chain constant region consists of three domains C H1 、C H2 and C H3 Each 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 a domain C L The VH and VL regions can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed with more conserved regions termed framework regions (FRs). H and V L It is composed of three CDRs and four FRs arranged from amino terminus to carboxyl 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 an antibody may 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.

[0051] 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 within the term "antigen-binding fragment" of an antibody include (i) fragments of V H 、V L 、C L and C H1 (ii) a F(ab')2 fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a fragment consisting of a V H 、V L 、C L and C H1 domain and C H1 and C H2 Fab' fragments consisting of the region between the domains; (iv) H and C H1Fd fragment composed of a single-arm V domain; (v) H and V L A single-chain Fv fragment consisting of a V domain, (vi) H (viii) dAb fragments comprising V domains linked in the same polypeptide chain using short linkers; ... H and V L "diabodies" (see, for example, patent documents EP404,097; WO 93 / 11161; and Holliger et al., Proc Natl Acad Sci USA, 1993); and (ix) containing only V H or V L "Domain antibody fragments", in which in some cases two or more V H The regions are covalently linked.

[0052] Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic, or homologous; or modified forms thereof (e.g., humanized, chimeric, or multispecific). Antibodies can also be fully human.

[0053] As used herein, "framework" or "FR" refers to the variable domain residues other than the hypervariable region (CDR) residues.

[0054] "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 regions of immunoglobulin heavy chains may be different, the human IgG heavy chain Fc region is generally defined as the amino acid residue at position Cys226 according to the EU index numbering of Kabat (Johnson and Wu, Nucleic Acids Res, 2000) or the segment from Pro230 to its carboxyl terminus. The C-terminal lysine in the Fc region (residue 447 according to the EU index of Kabat) may be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Accordingly, the composition of the intact antibody may include antibody populations in which all K447 residues are removed, antibody populations in which no K447 residue is removed, and antibody populations with and without a mixture of antibodies containing K447 residues. Suitable native sequence Fc regions for use in antibodies of the present disclosure include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0055] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody.

[0056] As used herein, an "isolated antibody" refers to an antibody that is substantially free of its natural environment. For example, an isolated antibody is substantially free of cellular material and other proteins from the cell or tissue from which it was derived. An "isolated antibody" further refers to an antibody that is substantially free of other antibodies having different antigenic specificities. In this context, an isolated antibody that specifically binds CD228 is substantially free of antibodies that specifically bind antigens other than CD228. However, an isolated antibody that specifically binds CD228 may have cross-reactivity with other antigens, such as CD228 molecules from other species.

[0057] As used herein, "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0058] As used herein, "humanized antibody" refers to an antibody consisting of CDRs of antibodies derived from mammals other than humans and FR regions and constant regions of human antibodies or derived from human antibodies. A humanized antibody may comprise a variable domain having a variable region amino acid sequence that, when analyzed as a whole, is closer to humans than to other species, as described by Ehrenmann et al. (2010) as described. Due to reduced antigenicity, humanized antibodies may be used as an active ingredient in therapeutic agents. As used herein, the term "therapeutic agent" or "therapeutically active agent" refers to a therapeutically useful agent. A therapeutic agent may be any agent used to prevent, improve or treat a disease, a physiological condition, a symptom, or to evaluate or diagnose the same.

[0059] As used herein, "human antibodies" include antibodies with variable regions in which both framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also 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 somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted onto human framework sequences.

[0060] 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."

[0061] As used herein, "AAFX" refers to a fusion protein comprising antibody clone X and a lipocalin mutein having the amino acid sequence of SEQ ID NO: 40 (lipocalin mutein 1). For example, "AAF30" refers to a fusion protein comprising antibody clone 30 and lipocalin mutein 1. The symbol "HC" in the name of the fusion protein indicates that the lipocalin mutein is conjugated to the antibody via the heavy chain of the antibody, and the symbol "LC" in the name of the fusion protein indicates that the lipocalin mutein is conjugated to the antibody via the light chain of the antibody. Fusion protein names containing a clone number followed by "HC" or "LC" refer to fusion proteins containing the antibody clone and lipocalin mutein 1 conjugated via the heavy or light chain of the antibody, respectively. For example, "30HC" refers to a fusion protein comprising antibody clone 30 and lipocalin mutein 1 conjugated to the antibody via the heavy chain.

[0062] An antibody or fusion protein may also be represented by its heavy and light chain sequences (e.g., a fusion protein having the sequences of SEQ ID NOs: 80 and 76), one of which, in the case of a fusion protein (e.g., SEQ ID NO: 80), contains the sequence of a lipocalin mutein. IV. Description of the Figures

[0063] Figure 1 A schematic diagram is provided showing CD137 clustering by bridging CD137-positive T cells with CD228-expressing tumor cells using the fusion proteins provided herein.

[0064] Figure 2A and 2B Shown are the CDR sequences of certain antibodies provided herein.

[0065] Figure 3 Shown are the results of an ELISA in which anti-CD228 antibodies bound to recombinant human CD228.

[0066] Figures 4A-4C Binding of anti-CD228 antibodies to cells expressing CD228 as measured by fluorescence intensity is shown. Figure 4D The EC values of each antibody for each cell line are shown. 50 value.

[0067] Figures 5A-5E Shown are the results of biolayer interferometry (BLI) assays regarding the binding kinetics and affinity of anti-CD228 antibodies to CD228.

[0068] Figures 6A-6C Shown are the results of binding assays of anti-CD228 antibodies to CD228 and certain other transferrin family members.

[0069] Figures 7A-7B and 8A-8C show the results of anti-CD228 antibody binding assays to human, murine, and cynomolgus monkey CD228.

[0070] Figure 9 Shown are the results of a cross-competition assay between various anti-CD228 antibodies.

[0071] Figure 10 Shown are the results of an antibody internalization assay using a CD228+ tumor cell line.

[0072] Figures 11A-11I An overview of the design of representative fusion proteins described in this application that are bispecific for the targets CD137 and CD228 is provided. Representative fusion proteins are prepared based on antibodies specific for CD228 and one or more lipocalin muteins specific for CD137. Figures 11A-11I As described in , one or more lipocalin muteins are genetically fused to the C-terminus and / or N-terminus of the heavy and / or light chains of a CD228-specific antibody. The resulting fusion protein can be bivalent for CD137 (e.g., Figures 11A-11D ) or tetravalent for CD137 (e.g., as depicted in Figures 11E-11H ), or may have a higher valence for CD137 (e.g., as Figure 11I (depicted in ).

[0073] Figures 12A-12D The results of ELISA experiments are shown in which the binding of representative fusion proteins to human or cynomolgus monkey CD228 ( Figures 12A-12B ) or CD137( Figures 12C-12D ). CD228 or CD137 (with C-terminal His or Fc tags) were coated onto microtiter plates and the tested agents were titrated starting at a maximum concentration of 200 nM. Bound agents under investigation were detected via anti-human IgG Fab-HRP. Data were fitted using a 4PL fit with EC as a free parameter. 50 The value and maximum signal and the slope are fixed at one. The obtained EC 50 The values are provided in Table 4.

[0074] Figure 13 Shows the results of an ELISA experiment in which, as described in Example 12, the ability of a representative fusion protein to bind both target CD228 and CD137 simultaneously was determined. Recombinant huCD228-His was coated onto microtiter plates, and then the fusion protein and a control anti-CD228 antibody were titrated starting from the highest concentration of 200 nM. Subsequently, a constant concentration of biotinylated huCD137-His was added, which was detected via ExtrAvidin-peroxidase. The data was fitted with a 4PL fit, having the EC 50 value and the maximum signal as free parameters and a slope fixed at one. The resulting EC 50 values are provided in Table 5.

[0075] Figure 14 Shows additional results of an ELISA experiment in which, as described in Example 12, the ability of a representative fusion protein to bind both target CD228 and CD137 simultaneously was determined.

[0076] Figures 15A-15B Shows the results of evaluating the target binding of the fusion protein and respective controls by flow cytometry (FACS) 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 the fluorescence intensity was used to calculate the EC 50 value using non-linear regression (shared bottom value, slope = 1). The EC 50 values are provided in Table 6.

[0077] Figures 16A-16E Shows the potential of a representative fusion protein to co-stimulate T cell activation in a CD228 target-dependent manner, as evaluated by a CD137 bioassay as described in Example 14. In the presence of various concentrations of the fusion protein or control, NFκB-luc2 / CD137 Jurkat cells were co-cultured with tumor cells expressing high (SH-4)( Figure 16A ), medium (A375)( Figure 16B ), or low (A549)( Figure 16C ) levels of CD228 and CD228-negative RPMI-7951 tumor cells ( Figure 16D ). After 4 hours, luciferase assay reagent was added and the luminescence signal was measured. Four-parameter logistic curve analysis was performed using GraphPad to calculate the EC 50 value (see the table in Figure 16E ).

[0078] Figures 17A-17E Shown are the results of an antigenic recall assay in which the fusion protein costimulated innate and adaptive immune cytokines from peripheral blood mononuclear cells (PBMCs) in response to viral peptides.

[0079] Figure 18 、 19 , 20 and 21 show the results of antigen recall assays in which the fusion proteins costimulated T cell responses and NK cell responses in response to viral peptides.

[0080] Figures 22A-22E Shown are the results of an antigenic recall assay in which the fusion protein costimulated cytotoxic effector molecules and cytokines from PBMCs in response to viral peptides.

[0081] Figures 23A-23D The ability of representative fusion proteins to costimulate T cell activation in a CD228 target-dependent manner was shown. Various tumor cell lines expressing different levels of CD228 - 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 )-inoculated into anti-human CD3 coated plates. Pan T cells and various concentrations of fusion protein or control were added and incubated for 3 days. Secreted IL-2 levels were used as readout and determined by an electrochemiluminescence-based assay as described in Example 18. Similarly, Figures 24A-24B Additional representative fusion proteins are shown for their ability to costimulate T cell activation in a CD228 target-dependent manner.

[0082] Figures 25A-25C Shown are T cell cytokine production in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines.

[0083] Figure 26 showed CD8 T cell proliferation in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines, and Figure 27 Tumor cell killing in co-cultures is shown.

[0084] Figures 28A-28D Shown is the storage stability evaluation of fusion proteins and controls after one week of incubation at 37°C in PBS, 50% human plasma (HPL) or 50% mouse plasma (MPL) as described in Example 21. The stability was evaluated with the aid of the CD137 reporter cell assay as described in Example 14.

[0085] Figures 29A-29BThe results of the pharmacokinetic analysis of the bispecific fusion protein and the parent anti-CD228 antibody in mice as described in Example 22 are provided. Male CD-1 NUDE mice (3 mice / time point) were injected intravenously with the fusion protein at a dose of 10 mg / kg. Drug levels were detected using a sandwich ELISA that detected the intact molecule via its target CD228 and biotinylated CD137. Anti-CD228 antibody plasma levels were determined using an ELISA via the target CD228 and an anti-human IgG Fc antibody. Absolute concentrations are shown in Figure 29A For C max Normalized values are shown in Figure 29B middle.

[0086] Figure 30 The pharmacokinetics of the fusion protein in cynomolgus monkeys are shown.

[0087] Figures 31A-31D , 32A-32C, and 33A-33B show the in vivo activity of the fusion proteins in a humanized xenograft model.

[0088] Figure 34A and 34B Shown is CD8 T cell proliferation in co-culture with a CD228-expressing tumor cell line engineered with an anti-CD3 scFv.

[0089] Figure 35A Shown are the CD8 T cell counts in wells to which treatment was added relative to those in wells to which no treatment was added. Figure 35B Shown by MitoSpy TM The mean fluorescence intensity (MFI) of green FM staining indicates the mitochondrial content of CD8 T cells. Figure 35C MitoSpy TM Orange CMTMros and MitoSpy TM The percentage of CD8 T cells with depolarized mitochondria as determined by green FM staining.

[0090] Figure 36A A schematic diagram of the generation of functionally exhausted cytotoxic T cells is shown. Figure 36B Shown are the percentage of dividing cells (left), IFN-γ levels (middle), and the number of viable tumor cells (right) for T cells from different passages (P0-P4) restimulated with anti-CD3 scFv-engineered CALU-1 cells.

[0091] Figure 37A Shows information about Figure 36A Figure 4. Heatmap of exhausted CD8+ T cells from pan-cancer tumor-infiltrating lymphocyte (TIL) atlas or exhausted CD8+ T cells from pan-cancer tumor-infiltrating lymphocyte (TIL) atlas. Figure 37B Shown are the expression levels of TCF7 (TCF1) (left), HAVCR2 (TIM-3) (center), and TNFRSF9 (CD137) (right) in two groups of exhausted CD8+ T cells.

[0092] Figure 38 Shown are the fold increases in carboxyfluorescein succinimidyl ester (CFSE)-low (dividing) CD8 T cell counts in treated wells relative to untreated wells. V. Specific Implementation Methods

[0093] 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 alone as antibody therapeutics, conjugated to therapeutic agents to generate antibody-drug conjugates, included as part of bispecific antibodies or multispecific antibodies, or included as part of fusion molecules. In certain embodiments, such fusion molecules include those having a binding domain that binds to a target different from 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.

[0094] As provided herein, certain fusion proteins targeting both CD137 and CD228 are designed based on the understanding of several factors by the inventors. For example, with respect to CD137, it is recognized that CD137 is expressed on multiple immune cell types and that this engagement enhances innate and adaptive immunity. It is also recognized that CD137 agonism restores the functionality of exhausted T cells, thereby re-stimulating pre-existing anti-tumor activity. In the case of CD228, the inventors recognized that it can be an excellent antigen target for tumor-targeted anti-CD137 bispecific molecules for several reasons. First, CD228 expression is highly confined to tumors, thereby minimizing the risk of toxicity. Second, the combination of two agents with different mechanisms of action has the potential to provide broader coverage and the opportunity to promote additional combination therapies.

[0095] In addition, fusion proteins targeting both CD137 and CD228 as provided herein are based on the recognition that bivalent CD137 binding agents, such as antibodies, alone may not be sufficient to cluster CD137 on T cells or NK cells and lead to effective activation, similar to the lack of activity of trivalent soluble CD137L. A second problem observed when using bivalent anti-CD137 antibodies is that active doses can in some cases induce on-target hepatotoxicity, indicating the need for a more specific tumor-targeting mode of action.

[0096] Based in part on these different insights, the present disclosure provides, inter alia, novel methods for simultaneously engaging CD137 and CD228 via one or more fusion proteins having binding specificity for CD137 and binding specificity for CD228. The binding specificity for CD228 may be provided by an antibody or antigen binding domain thereof that binds to CD228 as provided herein. The provided fusion proteins are designed to promote CD137 clustering by bridging CD137-positive T cells with CD228-expressing tumor cells located in the tumor microenvironment, as exemplified by Figure 1 As shown in . Therefore, the fusion protein is designed to allow local induction of antigen-specific T cells in the tumor microenvironment, potentially reducing peripheral toxicity. This is in contrast to, for example, urerulumab, which is a 4-1BB agonist that has activity in solid tumors but induces on-target liver toxicity at active doses.

[0097] In some aspects, the present disclosure provides antibodies or their antigen binding domains 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 preparing the CD228 binding antibodies or their antigen binding domains as well as CD137 and CD228 binding fusion proteins as described herein and compositions comprising such proteins. The CD228 binding antibodies or their antigen binding domains as well as CD137 and CD228 binding fusion proteins and compositions thereof disclosed herein may be used in methods for detecting CD137 and / or CD228 in a sample, methods for binding CD137 and / or CD228 in a subject, or methods for regulating an immune response in a subject. Such antibodies, their antigen binding domains or fusion proteins having these characteristics associated with the uses provided by the present disclosure have not been previously described.

[0098] A. Exemplary Antibodies or Antigen Binding Domains Specific for CD228

[0099] In some embodiments, an antibody or antigen-binding domain thereof that binds CD228 is provided (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof contained in any one of the fusion proteins disclosed herein), wherein the antibody or antigen-binding domain thereof comprises:

[0100] i) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 110, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 112, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 116, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 118;

[0101] 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;

[0102] 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;

[0103] 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;

[0104] 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;

[0105] 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;

[0106] 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;

[0107] 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;

[0108] 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;

[0109] 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;

[0110] 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;

[0111] 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;

[0112] 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;

[0113] 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;

[0114] 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;

[0115] 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;

[0116] 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

[0117] xviii) VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 273, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 274, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 275, and VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 279, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 280, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 281.

[0118] In some embodiments, an antibody or antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof contained in any one of the fusion proteins disclosed herein) comprises:

[0119] 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;

[0120] 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;

[0121] 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

[0122] iv) VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, and VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 259, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 260, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 261.

[0123] In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., stand-alone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any of the fusion proteins disclosed herein) comprise: 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; or 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. NO:215, and VL, which includes (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:219, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:220 and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:221.

[0124] In some embodiments, an antibody or antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof contained in any one of the fusion proteins disclosed herein) comprises:

[0125] i) 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: 122, 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: 124;

[0126] 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;

[0127] 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;

[0128] 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;

[0129] 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;

[0130] 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;

[0131] 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;

[0132] viii) 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; or

[0133] 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.

[0134] In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., stand-alone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any of the fusion proteins disclosed herein) comprise: 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 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: 225. NO:262 has an amino acid sequence that is 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, and a VL comprising an amino acid sequence that is 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.

[0135] In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., standalone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any one of the fusion proteins disclosed herein) comprise 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.

[0136] In some embodiments, antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., standalone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any of the fusion proteins disclosed herein) include:

[0137] 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;

[0138] 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;

[0139] 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;

[0140] 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;

[0141] 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;

[0142] 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;

[0143] 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;

[0144] 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

[0145] 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.

[0146] In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., stand-alone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any fusion protein disclosed herein) comprise: 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.

[0147] In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., stand-alone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any fusion protein disclosed herein) comprise: 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.

[0148] In some embodiments, an antibody or antigen-binding domain thereof that binds CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof contained in any one of the fusion proteins disclosed herein) comprises:

[0149] i) a heavy chain (HC) comprising an amino acid sequence that is 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% identical to SEQ ID NO: 126, and a light chain (LC) comprising an amino acid sequence that is 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% identical to SEQ ID NO: 128;

[0150] ii) a 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 a 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;

[0151] iii) a 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 a 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;

[0152] iv) a 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 a 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;

[0153] v) a 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 a 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;

[0154] vi) a 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 a 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;

[0155] vii) a 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 a 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;

[0156] viii) a 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 a 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

[0157] ix) a 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 a 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.

[0158] In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., standalone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any of the fusion proteins disclosed herein) comprise: a 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 a 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; or a 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: 228; NO:266 has an amino acid sequence that is 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% identical to SEQ ID NO:266, and LC comprising an amino acid sequence that is 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% identical to SEQ ID NO:268.

[0159] In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., standalone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any of the fusion proteins disclosed herein) comprise: a 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 a 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.

[0160] In some embodiments, antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., standalone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any of the fusion proteins disclosed herein) include:

[0161] 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;

[0162] ii) a HC comprising the amino acid sequence of SEQ ID NO: 146, and a LC comprising the amino acid sequence of SEQ ID NO: 148;

[0163] 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;

[0164] 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;

[0165] v) 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;

[0166] 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;

[0167] vii) a HC comprising the amino acid sequence of SEQ ID NO: 246, and a LC comprising the amino acid sequence of SEQ ID NO: 248;

[0168] 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

[0169] ix) HC comprising the amino acid sequence of SEQ ID NO: 286, and LC comprising the amino acid sequence of SEQ ID NO: 288.

[0170] In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., stand-alone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any fusion protein disclosed herein) comprise: a HC comprising the amino acid sequence of SEQ ID NO: 226, and a LC comprising the amino acid sequence of SEQ ID NO: 228; or 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.

[0171] In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., stand-alone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any fusion protein disclosed herein) comprise: 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.

[0172] In some embodiments, the antibodies provided herein that bind to CD228 (e.g., independent antibodies or antibodies contained in any one of the fusion proteins disclosed herein) are monoclonal antibodies. In some embodiments, the antibodies provided herein that bind to CD228 are humanized or chimeric antibodies. In some embodiments, the antibodies provided herein that bind to CD228 are IgG1, IgG2, IgG3, or IgG4 antibodies.

[0173] In some embodiments, the antibodies or antigen-binding domains thereof provided herein (e.g., stand-alone antibodies or antigen-binding domains thereof, or antibodies or antigen-binding domains thereof contained in any of the fusion proteins disclosed herein) that bind to CD228 have a K of 175 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 150 nM or less. DIn some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 100 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 50 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 75 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 25 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 20 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 15 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 10 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 5 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 4 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 3 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 2.5 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 2 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 1.5 nM or less. D In some embodiments, the antibodies or antigen-binding domains thereof provided herein that bind to CD228 have a K of 1 nM or less. D Values for binding to CD228.

[0174] In some embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein (e.g., an independent antibody or antigen-binding domain thereof, or an antibody or antigen-binding domain thereof contained in any one of the fusion proteins disclosed herein) bind to cynomolgus monkey CD228. In some such embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein comprise:

[0175] 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;

[0176] 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;

[0177] 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;

[0178] 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;

[0179] 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

[0180] vi) VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, and VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 259, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 260, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 261.

[0181] In some such embodiments, the antibodies or antigen binding domains thereof provided herein that bind to CD228 comprise a set of CDRs as described above and:

[0182] i) 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: 182, 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: 184;

[0183] ii) 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: 222 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: 224; or

[0184] 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.

[0185] In some such embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein comprise: 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.

[0186] In some embodiments, the antibodies or antigen binding domains thereof provided herein that bind to CD228 (e.g., independent antibodies or antigen binding domains thereof, or antibodies or antigen binding domains thereof contained in any one of the fusion proteins disclosed herein) do not bind to murine CD228, or bind to murine CD228 with an affinity that is at least 100-fold lower than that of human CD228. In some embodiments, the antibodies or antigen binding domains thereof provided herein that bind to CD228 do not bind to transferrin or lactotransferrin. In some such embodiments, the antibodies or antigen binding domains thereof provided herein that bind to CD228 comprise:

[0187] 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;

[0188] 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;

[0189] 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;

[0190] 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;

[0191] 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;

[0192] 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;

[0193] 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;

[0194] 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;

[0195] 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;

[0196] 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;

[0197] 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

[0198] xii) VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 273, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 274, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 275, and VL comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 279, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 280, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 281.

[0199] In some such embodiments, the antibodies or antigen binding domains thereof provided herein that bind to CD228 comprise a set of CDRs as described above and:

[0200] i) 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: 182, 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: 184;

[0201] 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;

[0202] 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;

[0203] 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

[0204] v) 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; or

[0205] 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.

[0206] In some such embodiments, the antibodies or antigen-binding domains thereof that bind to CD228 provided herein comprise: 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.

[0207] In some embodiments, an antibody or antigen binding domain thereof that binds to CD228 is provided (e.g., an independent antibody or antigen binding domain thereof, or an antibody or antigen binding domain thereof contained in any fusion protein disclosed herein), wherein the antibody or antigen binding domain thereof competes with any of the antibodies or antigen binding domains disclosed herein for binding to CD228. In some such embodiments, the antibody is a monoclonal antibody. In some such embodiments, the antibody is a humanized or 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 binds to CD228 with a K of 175 nM or less. DIn some embodiments, the antibody or its antigen-binding domain binds to cynomolgus monkey CD228. In some embodiments, the antibody or its antigen-binding domain does not bind to murine CD228, or binds to murine CD228 with an affinity that is at least 100-fold lower than that of human CD228. In some embodiments, the antibody or its antigen-binding domain does not bind to transferrin or lactotransferrin. In some such embodiments, the antibody, or its antigen binding domain, competes for binding to CD228 with an antibody, or its antigen binding domain, 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: 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.

[0208] 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).

[0209] As included in the fusion protein of the present invention, the antibody specifically binding to CD228 may include an Fc portion, which allows the in vivo half-life of the bispecific binding molecules of the present invention to be extended. In some embodiments, such Fc portions are preferably from human origin, more preferably the human Fc portion of an IgG1 or IgG4 antibody, even more preferably the human Fc portion of an IgG1 or IgG4 with activation or silencing effector functions. In some embodiments, silencing effector functions may be superior to activating effector functions. In some embodiments, such Fc portions are modified to silence effector functions, with mutations at positions 234 and / or 235 according to the EU index numbering of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). In some embodiments, for silencing effector functions, mutations in positions F234 and L235 of the anti-CD228 antibodies provided may be introduced. In other embodiments, for silencing effector functions, mutations in positions D265 and P329 of the anti-CD228 antibodies provided may be introduced. Numbering for both sets of potential mutations is according to the EU index of Kabat (Shields et al., J Biol Chem, 2001).In some embodiments, provided CD228 antibodies have an engineered IgG4 backbone with mutations S228P, F234A, and L235A.

[0210] Various techniques for producing antibodies and antigen-binding domains thereof 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 the blood of an animal following immunization with an antigen in a mixture with additives and adjuvants, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous cell line cultures. Examples of such techniques are described, for example, in Harlow and Lane (1999), (1988), and include those originally described by and Milstein, 1975 described hybridoma technology, trioma technology, human B cell hybridoma technology (see, for example, Li et al., Proc Natl Acad Sci USA, 2006; Kozbor and Roder, Immunol Today, 1983) and EBV hybridoma technology for producing human monoclonal antibodies (Cole et al., Cancer Res, 1984). In addition, recombinant antibodies may be obtained from monoclonal antibodies, or may be prepared from scratch using various display methods, such as phage, ribosome, mRNA or cell display. In some embodiments, a suitable system for expressing recombinant (humanized) antibodies or fragments thereof may be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, for example, U.S. Patent No. 6,080,560; Holliger and Hudson, Nat Biotechnol, 2005). Further, the technology for producing single-chain antibodies (see, in particular, U.S. Patent No. 4,946,778) can be adjusted to produce single-chain antibodies specific for the target of the present invention. Surface plasmon resonance, as employed in the BIAcore system, can be used to increase the efficiency of phage antibodies.

[0211] B. Exemplary fusion proteins of the present disclosure specific for CD137 and CD228.

[0212] 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 CD 137. In some embodiments, in the fusion proteins provided herein, the antibody or antigen-binding domain thereof is any of the antibodies or antigen-binding domains thereof disclosed herein, particularly any of the antibodies or antigen-binding domains thereof disclosed in Section IV.A above.

[0213] In some embodiments, the provided fusion proteins may also contain at least one additional subunit, such as a third subunit. For example, the fusion protein may contain a third subunit specific for CD137. In some embodiments, the third subunit may be or comprise a lipocalin mutein specific for CD137. For example, two lipocalin muteins may be fused to a first antibody subunit, one at the C-terminus and one at the N-terminus of the antibody. In some embodiments, the lipocalin mutein may be fused to a heavy chain or a light chain of an antibody.

[0214] In some embodiments, provided fusion proteins may comprise one or more additional subunits (eg, a fourth subunit, a fifth subunit, or a sixth subunit).

[0215] In some embodiments, at least one subunit may be fused to another subunit at its N-terminus and / or its C-terminus.

[0216] In some embodiments, at least one subunit can be connected to another subunit via a joint. In some further embodiments, the joint is a peptide joint, such as a glycine-serine (GS) joint, such as an unstructured GS joint or a glycosylated GS joint, or a proline-alanine-serine polymer (PAS) joint. In some embodiments, the GS joint is a (Gly4Ser)3 joint ((G4S)3) as shown in SEQ ID NO:13. Other exemplary joints are shown in SEQ ID NO:14-23. In some embodiments, the peptide joint may 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, when the first subunit comprises an antibody such as a full-length antibody, the second subunit may be connected via a peptide joint between the C-terminus of the heavy chain constant region (CH) of the second subunit and the antibody. In some further embodiments, the third subunit may be connected via a peptide linker between the N-terminus of the third subunit and the C-terminus of the light chain constant region (CL) of the antibody.

[0217] In some embodiments, a subunit may be Figures 11A-11I In some embodiments, a lipocalin mutein subunit may be fused to an antibody subunit at its N-terminus and / or its C-terminus. For example, a lipocalin mutein subunit may be fused to an antibody subunit at its N-terminus and / or its C-terminus. As a further example, a lipocalin mutein may be linked 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, preferably via a peptide linker. Figures 11A-11D ).

[0218] In some embodiments, the lipocalin mutein subunit may be fused to an antibody fragment at its N-terminus and / or its 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 an antibody, preferably via a peptide linker.

[0219] In some embodiments, when one subunit comprises an antibody, eg, 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.

[0220] 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.

[0221] In some embodiments, the fusion protein comprises an antibody (e.g., a full-length antibody) specific for CD228 and two lipocalin muteins specific for CD137, wherein each lipocalin mutein is fused at its N-terminus to the C-terminus of the antibody heavy chain via a peptide linker.

[0222] In some embodiments, the fusion protein comprises an antibody (e.g., a full-length antibody) specific for CD228 and two lipocalin muteins specific for CD137, wherein each lipocalin mutein is fused at its N-terminus to the C-terminus of the antibody light chain via a peptide linker.

[0223] In some embodiments, with respect to the fusion proteins of the present disclosure in which at least one subunit may be or comprise an antibody, such as a full-length antibody, when the fusion protein simultaneously binds to CD137 and CD228, the Fc function of the Fc region of the antibody against Fc receptor-positive cells may be simultaneously preserved.

[0224] In some embodiments, wherein at least one subunit of the fusion protein provided may be or comprise an antibody, such as a full-length antibody, the Fc function of the antibody's Fc region on Fc receptor-positive cells may be reduced or completely inhibited by protein engineering when 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, as IgG4 is known to exhibit reduced Fc-γ receptor interactions compared to IgG1. In some embodiments, to further reduce residual binding to Fc-γ receptors, mutations such as F234A and L235A may be introduced into the IgG4 backbone. In some embodiments, the S228P mutation may also 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 the native glycosylation motif.

[0225] In some embodiments, the Fc portion of an antibody included in a fusion protein of the present disclosure may 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 may become internalized and recirculate back into the bloodstream, enhancing its half-life in vivo.

[0226] In one aspect, the fusion proteins of the present disclosure bind to CD137 with high affinity. In another aspect, the fusion proteins provided bind to CD228 with high affinity. In some preferred embodiments, the fusion proteins provided bind to both CD137 and CD228 simultaneously. In some embodiments, simultaneous binding to CD137 and CD228 allows the fusion proteins provided to exhibit a sustained anti-tumor or anti-infective response.

[0227] In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD228 with a K of at most about 200 nM, at most about 180 nM, at most about 150 nM, at most about 120 nM, at most about 100 nM, at most about 70 nM, or at most about 35 nM, or even lower, such as about 30 nM or lower, about 25 nM or lower, or about 20 nM or lower. DIn some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD228 with a K value comparable to that of an antibody or variant of said antibody specific for CD228 as included in such fusion proteins. D Comparable or lower K values D The K value of the provided fusion protein is, for example, an antibody having a heavy chain and a light chain provided by SEQ ID NOs: 74 and 76 or SEQ ID NOs: 77 and 79. D The value may for example be measured in a surface plasmon resonance (SPR) assay, for example as essentially described in Example 10.

[0228] In some embodiments, the fusion proteins of the present disclosure cross-react with cynomolgus monkey CD228. In some embodiments, the fusion proteins of the present disclosure may be capable of binding to cynomolgus monkey CD228 with a K of at most about 200 nM, at most about 180 nM, at most about 150 nM, at most about 120 nM, at most about 100 nM, at most about 70 nM, or at most about 35 nM, or even lower, such as about 30 nM or lower, about 25 nM or lower, or about 20 nM or lower. D The K value of the fusion protein provided D The value may for example be measured in a surface plasmon resonance (SPR) assay, for example as essentially described in Example 10.

[0229] In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD137 with a K of at most about 10 nM or even lower, such as about 7 nM or lower, about 6 nM or lower, about 5 nM or lower, or about 4 nM or lower. D In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD137 with a K value comparable to that of a lipocalin mutein specific for CD137 included in a particular fusion protein (e.g., SEQ ID NO: 40), or a lipocalin mutein fused to the Fc region of an antibody (e.g., SEQ ID NO: 84). D Comparable or lower K values D The K value of the fusion protein provided D The value may for example be measured in an SPR assay, for example as essentially described in Example 10.

[0230] In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD228 with an EC of at most about 2 nM, at most about 1.5, or at most about 1.2 nM, or even lower, such as about 1 nM or lower, about 0.9 nM or lower, about 0.8 nM or lower, about 0.7 nM or lower, about 0.6 nM or lower, or about 0.5 nM or lower. 50In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD228 with a K value comparable to that of an antibody or variant of said antibody specific for CD228 as included in such fusion proteins. D EC values comparable or lower 50 The antibody is, for example, an antibody having heavy and light chains provided by SEQ ID NOs: 74 and 76 or SEQ ID NOs: 77 and 79. The EC of the provided fusion protein is 50 The value may be measured, for example, in an enzyme-linked immunosorbent assay (ELISA) assay, for example an ELISA assay as essentially described in Example 11.

[0231] In some embodiments, the fusion proteins of the present disclosure cross-react with cynomolgus monkey CD228. In some embodiments, provided fusion proteins may be capable of binding to cynomolgus monkey CD228 with an EC of at most about 2 nM, at most about 1.5, or at most about 1.2 nM or even lower, such as about 1 nM or lower, about 0.9 nM or lower, about 0.8 nM or lower, about 0.7 nM or lower, about 0.6 nM or lower, or about 0.5 nM or lower. 50 The EC of the provided fusion protein 50 The value can be measured, for example, in an ELISA assay, for example, as essentially described in Example 11.

[0232] In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD137 with an EC of at most about 6 nM or even lower, such as about 5 nM or lower, about 4 nM or lower, about 3 nM or lower, about 2.5 nM or lower, about 2 nM or lower, about 1.5 nM or lower, about 1 nM or lower, or about 0.8 nM or lower. 50 In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD137 and have an EC value similar to that of a lipocalin mutein specific for CD137 included in a particular fusion protein (e.g., SEQ ID NO: 40), or a lipocalin mutein fused to the Fc region of an antibody (e.g., SEQ ID NO: 84). 50 EC values comparable or lower 50 The EC of the provided fusion protein 50 The value can be measured, for example, in an ELISA assay, for example, as essentially described in Example 11.

[0233] In some embodiments, the fusion proteins of the present disclosure cross-react with cynomolgus monkey CD137. In some embodiments, provided fusion proteins may be capable of binding to cynomolgus monkey CD137 with an EC of at most about 150 nM or even lower, such as about 120 nM or lower, about 100 nM or lower, about 90 nM or lower, about 80 nM or lower, about 70 nM or lower, about 60 nM or lower, about 50 nM or lower, about 40 nM or lower, or about 30 nM or lower. 50 The EC of the provided fusion protein 50 The value can be measured, for example, in an ELISA assay, for example, as essentially described in Example 11.

[0234] In some embodiments, the fusion proteins of the present disclosure may be capable of simultaneously binding to CD137 and CD228. In some embodiments, provided fusion proteins may be capable of simultaneously binding to CD137 and CD228 with an EC of at most about 20 nM, at most about 15 nM, or at most about 10 nM or even lower, such as about 7 nM or lower, about 5 nM or lower, about 3 nM or lower, about 2.5 nM or lower, about 2 nM or lower, or about 1.5 nM or lower. 50 Simultaneous binding may for example be determined in an ELISA assay, for example an ELISA assay as essentially described in Example 12.

[0235] In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD137 expressed on cells with an EC of at most about 30 nM or even lower, such as about 20 nM or lower, about 15 nM or lower, about 10 nM or lower, about 9 nM or lower, about 7 nM or lower, about 5 nM or lower, about 3 nM or lower, about 2.5 nM or lower, or about 2 nM or lower. 50 The EC of the provided fusion protein 50 The values may be measured, for example, in a flow cytometric analysis as essentially described in Example 13. The cells expressing CD137 may be, for example, CHO cells transfected with human CD137 or cynomolgus CD137.

[0236] In some embodiments, the fusion proteins of the present disclosure may be capable of binding to CD228 expressed on cells with an EC of at most about 20 nM or even lower, such as about 10 nM or lower, about 8 nM or lower, about 6 nM or lower, about 5 nM or lower, about 4 nM or lower, about 3 nM or lower, about 2.5 nM, about 2 nM or lower, or about 1.5 nM or lower. 50 The EC of the provided fusion protein 50The value may for example be measured in a flow cytometric analysis as essentially described in Example 13. The cells expressing CD228 may for example be tumor cells expressing CD228, such as SH-4 cells.

[0237] In some embodiments, the fusion proteins of the present disclosure may be able to costimulate T cell proliferation and / or T cell responses. In some embodiments, the fusion proteins provided result in comparable or greater T cell activation than T cell activation initiated by anti-CD137 antibodies (e.g., reference antibodies of SEQ ID NOs: 26 and 27). Stimulated T cell responses or T cell activation may be measured, for example, in a CD137 bioassay as essentially described in Example 14, or in a functional T cell activation assay as essentially described in Example 18.

[0238] In some embodiments, fusion protein of the present disclosure may be able to costimulate T cell response in a CD228-dependent manner. In some embodiments, the fusion protein provided may result in the production of IL-2 by T cell local induction near CD228 positive cells (such as cells transfected by CD228 or CD228 positive tumor cells). When used in this article, "near CD228 positive cells" refers to that T cells and CD228 positive cells are close to each other by the fusion protein provided, and the fusion protein is combined with CD137 and CD228 simultaneously. T cells may be activated by the CD228-dependent fusion protein provided, for example, in the CD137 bioassays substantially described in Example 14, or in the functional T cell activation assays substantially described in Example 18, determined.

[0239] In some preferred embodiments, the fusion proteins provided may be able to costimulate T cell responses in the presence of CD228-expressing tumor cells and / or in a (CD228-positive) tumor microenvironment. In some embodiments, the fusion proteins provided may be able to costimulate T cell responses in the presence of CD228-positive tumor cells with 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. 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 as essentially described in Example 14.

[0240] In some embodiments, the fusion proteins provided are unable to costimulate T cell responses when CD228 is not present. In some embodiments, the fusion proteins provided are unable to costimulate T cell responses when cells expressing CD228 are not present. In some embodiments, the fusion proteins provided may be able to distinguish the presence of CD228 and result in better corresponding T cell activation than the CD137 antibodies shown in SEQ ID NOs: 26 and 27. The CD228-dependent effects of the fusion proteins may be determined, for example, in the CD137 bioassay essentially described in Example 14, or in the functional T cell activation assay essentially described in Example 18.

[0241] In some embodiments, for example, in the presence of CD228 positive (tumor) cells, the fusion proteins of the present disclosure may be able to induce increased IL-2 secretion. In some preferred embodiments, the fusion proteins provided may be able to induce concentration-dependent IL-2 secretion and / or confirm a trend of inducing enhanced IL-2 secretion at higher concentrations (preferably at the coating concentration). In some embodiments, the fusion proteins provided may result in increased IL-2 secretion with better efficiency compared to anti-CD137 antibodies such as SEQ ID NOs: 26 and 27 reference antibodies. IL-2 secretion may, for example, be measured in a functional T cell activation assay as essentially described in Example 18.

[0242] In some embodiments, the fusion proteins provided are capable of stimulating CD4+ and / or CD8+ T cells and / or NK cell proliferation. In some embodiments, the fusion proteins provided are capable of inducing NF-κB signaling. In some embodiments, the fusion proteins provided are capable of inducing increased secretion of cytokines such as interferon gamma, TNF-α, IL-2, IL-5, IL-10, IL-13, and IP-10 (CXCL10). In some embodiments, the fusion proteins provided are capable of inducing increased secretion of cytotoxic factors such as perforin, granzyme B, and granzyme A.

[0243] In some embodiments, the fusion proteins provided have favorable stability and / or pharmacokinetic profiles. In some embodiments, the fusion proteins provided have antibody-like pharmacokinetics. In some embodiments, the fusion proteins provided have a terminal half-life (e.g., in vivo terminal half-life in mice) of about 200 hours or longer, about 250 hours or longer, or about 300 hours or longer. In some embodiments, the fusion proteins provided have a terminal half-life longer than that of 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 profiles of the fusion proteins provided may be analyzed as described in Example 22. In some embodiments, if c max A % above 10% after 336 hours may be considered to be achieving a favorable pharmacokinetic profile or antibody-like pharmacokinetics.

[0244] In some embodiments, the fusion proteins of the present disclosure may be able to induce increased CD8+ T cell mitochondrial content and / or decreased CD8+ T cell mitochondrial depolarization. In some embodiments, the provided fusion proteins may induce increased CD8+ T cell mitochondrial content and / or decreased CD8+ T cell mitochondrial depolarization in the vicinity of CD228-positive cells (e.g., CD228-transfected cells or CD228-positive tumor cells). Increased CD8+ T cell mitochondrial content induced by the provided fusion proteins may be achieved, for example, by using polarization-independent dyes (e.g., MitoSpy TM Green FM) as essentially described in Example 26. Induction of reduced mitochondrial depolarization in CD8+ T cells by provided fusion proteins can be achieved, for example, by using polarization-dependent dyes (e.g., MitoSpy TM Orange CMTMRos) was determined as essentially described in Example 26.

[0245] In some embodiments, the fusion protein of the present invention may be able to stimulate the proliferation of exhausted CD8+ T cells and / or synergize with anti-PD-1 or anti-PD-L1 antibodies to stimulate the proliferation of exhausted CD8+ T cells. In some embodiments, the fusion protein provided may be able to stimulate the proliferation of exhausted CD8+ T cells and / or synergize with anti-PD-1 or anti-PD-L1 antibodies to stimulate the proliferation of exhausted CD8+ T cells near CD228 positive cells (e.g., CD228 transfected cells or CD228 positive tumor cells). 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 unexhausted CD8+ T cells. In some embodiments, a separate fusion protein may be able to stimulate the proliferation of exhausted CD8+ T cells. In some embodiments, the fusion protein may be able to stimulate the proliferation of exhausted CD8+ T cells in conjunction with an anti-PD-1 or anti-PD-L1 antibody; in such embodiments, an anti-PD-1 or anti-PD-L1 antibody alone may have no or only limited ability to stimulate the proliferation of exhausted CD8+ T cells, but it may be able to enhance the ability of the fusion protein to stimulate the proliferation of exhausted CD8+ T cells. In some embodiments, the anti-PD-1 or anti-PD-L1 antibody is nivolumab, pembrolizumab, cemiplizumab, dostarlimab, atezolizumab, avelumab, or durvalumab. Cell proliferation may be determined, for example, by using flow cytometry as essentially described in Example 28, using a dye such as CFSE.

[0246] In some embodiments, a fusion protein is provided comprising the amino acid sequence set forth in any one of SEQ ID NOs: 75, 76, and 78-83.

[0247] 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 shown in any one of SEQ ID NOs:75, 76, and 78-83.

[0248] In some embodiments, the fusion proteins provided comprise an amino acid sequence that has 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 shown 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.

[0249] In some embodiments, the fusion proteins provided 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, the fusion proteins provided comprise the amino acid sequences set forth in SEQ ID NOs: 80 and 76. In one embodiment, the fusion proteins provided comprise the amino acid sequences set forth in SEQ ID NOs: 82 and 79. In one embodiment, the fusion proteins provided comprise the amino acid sequences set forth in SEQ ID NOs: 75 and 81. In one embodiment, the fusion proteins provided comprise the amino acid sequences set forth in SEQ ID NOs: 78 and 83. In one embodiment, the fusion proteins provided comprise the amino acid sequences set forth in SEQ ID NOs: 96 and 97. In one embodiment, the fusion proteins provided comprise the amino acid sequences set forth in SEQ ID NOs: 98 and 99. In one embodiment, the fusion proteins provided comprise the amino acid sequences set forth in SEQ ID NOs: 100 and 101. In one embodiment, the fusion proteins provided comprise the amino acid sequences set forth in SEQ ID NOs: 102 and 103. In one embodiment, the fusion proteins provided comprise the amino acid sequences set forth in SEQ ID NOs: 104 and 105. In one embodiment, the fusion proteins provided comprise the amino acid sequences set forth in SEQ ID NOs: 106 and 107.

[0250] C. Exemplary lipocalin muteins of the present disclosure.

[0251] Lipocalins are protein-binding molecules that have evolved naturally to bind ligands. Lipocalins are found in many organisms, including vertebrates, insects, plants, and bacteria. Members of the lipocalin family (Pervaiz and Brew, FASEBJ, 1987) are typically small secreted proteins with a single polypeptide chain. They are characterized by a range of different molecular recognition properties: their binding to various major hydrophobic small molecules (e.g., retinoids, fatty acids, cholesterol, prostaglandins, biliverdin, pheromones, tastants, and odorants), as well as their binding to specific cell surface receptors and their formation of macromolecular complexes. Although they have been primarily classified as transport proteins in the past, it is now clear that lipocalins perform a variety of physiological functions. These include roles in retinol transport, olfaction, pheromone signaling, and prostaglandin synthesis. Lipocalins have also been implicated in the regulation of immune responses and mediation of cellular homeostasis (reviewed, for example, in Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996).

[0252] Lipocalins share a generally low level of overall sequence conservation, often with less than 20% sequence identity. In stark contrast, their overall folding pattern is highly conserved. The central portion of the lipocalin structure consists of a single eight-stranded antiparallel β-sheet that closes back on itself to form a continuously hydrogen-bonded β-barrel. This β-barrel forms a central cavity. One end of the barrel is sterically blocked by an N-terminal peptide segment spanning its base and three peptide loops connecting the β-strands. The other end of the β-barrel is open to the solvent and contains a target binding site formed by four flexible peptide loops (AB, CD, EF, and GH). The diversity of loops in the otherwise rigid lipocalin scaffold generates a variety of different binding modes, each capable of accommodating targets of varying size, shape, and chemical properties (reviewed, for example, in Skerra, Biochim Biophys Acta, 2000; Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996).

[0253] Lipocalin muteins according to the present disclosure may be muteins of any lipocalin. Examples of suitable lipocalins (sometimes also referred to as "reference lipocalins," "wild-type lipocalins," "reference protein scaffolds," or simply "scaffolds") in which muteins may be used include, but are not limited to, tear lipocalins (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-associated protein (A2m), 24p3 / uterocalin (24p3), von Ebner's gland protein 1 (VEGP 1), von Ebner's gland protein 2 (VEGP2), and the major allergen Can f 1 (ALL-1). In related embodiments, the lipocalin mutein is derived from the following lipocalins: human tear lipocalin (hTlc), human neutrophil gelatinase-associated lipocalin (hNGAL), human apolipoprotein D (hAPOD), and bile pigment binding protein of Pieris brassicae.

[0254] When compared to the sequence identity of another lipocalin, the amino acid sequence of the lipocalin mutein according to the present disclosure may have a high sequence identity compared to the reference (or wild-type) lipocalin from which it is derived (e.g., hTlc or hNGAL) (see also above). In this general context, the amino acid sequence of the lipocalin mutein according to the present disclosure is at least substantially similar to the amino acid sequence of the corresponding reference (wild-type) lipocalin, with the proviso that there may be gaps in the alignment that are the result of amino acid additions or deletions (as defined herein). In some embodiments, the sequence of the lipocalin mutein of the present disclosure that is substantially similar to the sequence of the corresponding reference (wild-type) lipocalin 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, to the sequence of the corresponding lipocalin, respectively. In this regard, the lipocalin muteins of the present disclosure may of course contain substitutions as described herein, which render the lipocalin mutein capable of binding to CD137.

[0255] Typically, the lipocalin muteins contain one or more mutated amino acid residues in the four loops at the open end—relative to the amino acid sequence of wild-type or reference lipocalin proteins, such as hTlc and hNGAL—that contain the ligand binding pocket and define the entrance to the ligand binding pocket (see above). As explained above, these regions are essential in determining the binding specificity of the lipocalin mutein for the desired target. In some embodiments, the lipocalin muteins of the present disclosure may also contain regions of mutated amino acid residues outside of the four loops. In some embodiments, the 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, the mutant protein derived from tear lipocalin NGAL or a homolog thereof may have 1, 2, 3, 4 or more mutated amino acid residues at any sequence position in the N-terminal region and / or in the three peptide loops BC, DE and FG arranged at the ends of the β-barrel structure, which is located opposite the native lipocalin binding pocket. In some embodiments, the mutant protein derived from tear lipocalin, NGAL or a homolog thereof may not have a mutated amino acid residue in the peptide loop DE arranged at the ends of the β-barrel structure compared to the wild-type sequence of tear lipocalin, NGAL or a homolog thereof.

[0256] In some embodiments, the lipocalin muteins according to the present disclosure may include 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 mutated amino acid residues compared to the amino acid sequence of the corresponding reference (wild-type) lipocalin, provided that such lipocalin muteins are capable of binding to CD 137. In some embodiments, the lipocalin muteins of the present disclosure include at least two, including 2, 3, 4, 5 or even more mutated amino acid residues, wherein the native amino acid residue of the corresponding reference (wild-type) lipocalin is substituted with an arginine residue.

[0257] Any type and number of mutations, including substitutions, deletions, and insertions, are contemplated, so long as the provided lipocalin mutein retains its ability to bind CD137 and / or it has at least 60% sequence identity, 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).

[0258] 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 exemplary substitutions below.

[0259] In particular, to determine whether the amino acid sequence of a lipocalin (mutant) differs from that of a reference (wild-type) lipocalin at a certain position in the amino acid sequence of the reference (wild-type) lipocalin, the skilled person can use means and methods well known in the art, for example, manually or by alignment using a computer program such as BLAST 2.0 or Clustal W, which stands for Basic Local Alignment Search Tool, or any other suitable program suitable for generating sequence alignments. Accordingly, 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 mutant serves as the "query sequence" (see also above).

[0260] Conservative substitutions are generally the following substitutions listed according to the amino acid to be mutated, each of which is followed by one or more substitutions that can be 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, Val, Met, Ala, or Pro He, Pro, Tyr or Trp; 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 also permitted and can be determined empirically or according to other known conservative substitutions or non-conservative substitutions. As a further direction, the following groups each contain amino acids that can generally be used to define conservative substitutions with respect to each other:

[0261] i) Alanine (Ala), serine (Ser), threonine (Thr), valine (Val);

[0262] ii) Aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), asparagine (Asn), histidine (His);

[0263] iii) arginine (Arg), lysine (Lys), glutamine (Gln), asparagine (Asn), and histidine (His);

[0264] iv) Isoleucine (Ile), Leu, Leucine (Leu), Methionine (Met), Valine (Val), Alanine (Ala), Phenylalanine (Phe), Threonine (Thr), Proline (Pro);

[0265] v) Isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp).

[0266] If such conservative substitutions result in a change in biological activity, more basic changes, such as the following, or as further described below with reference to amino acid classes, may be introduced and the products screened for the desired properties. Examples of such more basic 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.

[0267] In some embodiments, substantial modifications in the physical and biological properties of the lipocalin (mutein) are accomplished by selecting substitutions that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0268] 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 affecting 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.

[0269] Any cysteine residues not involved in maintaining the correct conformation of the respective lipocalin may also be substituted (generally with serine) to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bonds may be added to the lipocalin to improve its stability.

[0270] D. Exemplary CD137-specific lipocalin muteins of the present disclosure.

[0271] As noted above, lipocalins are polypeptides defined by their supersecondary structure, namely a cylindrical beta-pleated sheet supersecondary structure region comprising eight beta strands connected in pairs by four loops at one end, 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 lipocalin muteins having a cylindrical beta-pleated sheet supersecondary structure region comprising eight beta strands connected in pairs by four loops at one end, 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 wherein the lipocalin effectively binds to CD137 with detectable affinity.

[0272] In some embodiments, the lipocalin muteins disclosed herein may be or comprise a mutein of mature human tear lipocalin (hTlc). A mutein of mature hTlc may be designated herein as an "hTlc mutein." In some other embodiments, the lipocalin muteins disclosed herein are muteins of mature human neutrophil gelatinase-associated lipocalin (hNGAL). A mutein of mature hNGAL may be designated herein as an "hNGAL mutein."

[0273] In one aspect, the present disclosure includes any number of lipocalin mutants derived from a reference (wild-type) lipocalin, preferably from mature hTlc or mature hNGAL, that bind to CD137 with detectable affinity. In a related aspect, the present disclosure includes various lipocalin mutants that are capable of activating the 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 inventors have demonstrated that it is possible to have high affinity and high specificity for the non-natural target CD137. In some embodiments, random mutagenesis is performed at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more nucleotide triplets encoding certain sequence positions on wild-type lipocalin, possibly through substitution at these positions by a subset of nucleotide triplets, with the goal of generating lipocalin muteins capable of binding CD137.

[0274] In some embodiments, the lipocalin muteins of the present disclosure may have mutations, including substitutions, deletions, and insertions of amino acid residues, at one or more sequence positions corresponding to sequence positions in 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 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, such as 25, 30, 35, 40, 45, or 50, of which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 are preferred, and 9, 10, or 11 are even more preferred. However, it is preferred that the lipocalin muteins of the present disclosure are still able to bind to CD137.

[0275] In some embodiments, the lipocalin muteins of the present disclosure may lack 1, 2, 3, 4 or more amino acids at their N-terminal end and / or 1, 2 or more amino acids at their C-terminal end 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, wherein the first one, two, three or four N-terminal amino acid residues (His-His-Leu-Leu; positions 1-4) of the mature hTlc sequence 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 have been deleted (e.g., SEQ ID NOs: 32-38). In some embodiments, the present disclosure encompasses hNGAL muteins as defined above, wherein the amino acid residues (Lys-Asp-Pro, positions 46-48) of the linear polypeptide sequence of mature hNGAL have been deleted (SEQ ID NO: 43). Furthermore, the 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) except for the mutated amino acid sequence positions.

[0276] In some embodiments, the one or more mutated amino acid residues incorporated into the lipocalin muteins of the present disclosure do not substantially impede or interfere with binding activity to a designated target and folding of the mutein. Such mutations, including substitutions, deletions, and insertions, can be accomplished at the DNA level using established standard methods (Sambrook and Russell, 2001, Molecular cloning: a laboratory manual). In some embodiments, one or more mutated amino acid residues are introduced at one or more sequence positions of the linear polypeptide sequence corresponding to a reference (wild-type) lipocalin (preferably hTlc or hNGAL) via random mutagenesis by replacing the nucleotide triplets encoding the corresponding sequence positions of the reference lipocalin with a subset of the nucleotide triplets.

[0277] In some embodiments, provided lipocalin muteins that bind to CD137 with detectable affinity may include at least one amino acid substitution of a native cysteine residue by another amino acid (e.g., a serine residue). In some embodiments, lipocalin muteins that bind to CD137 with detectable affinity may include one or more non-native cysteine residues that replace one or more amino acids of a reference (wild-type) lipocalin (preferably hTlc or hNGAL). In some embodiments, lipocalin muteins according to the present disclosure include at least two amino acid substitutions of a native amino acid by a cysteine residue, thereby forming one or more cysteine bridges. In some embodiments, the cysteine bridge 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).

[0278] Generally, the lipocalin muteins of the present disclosure may have at least about 70%, including at least about 80%, such as at least about 85% amino acid sequence identity to the amino acid sequence of mature hTlc (SEQ ID NO: 1) or mature hNGAL (SEQ ID NO: 2).

[0279] In some aspects, the present disclosure provides hTlc mutant proteins that bind CD137. In this regard, the present disclosure provides hTlc mutant proteins that can bind CD137 with a K of about 300nM, 200nM, 150nM, 100nM or less. D One or more hTlc mutant proteins that bind to CD137 with a measured affinity. In some embodiments, provided hTlc mutant proteins are capable of binding to CD137 with an EC of about 250 nM, 150 nM, 100 nM, 50 nM, 20 nM, or even lower. 50 In some other embodiments, the hTlc mutein that binds CD137 may cross-react with cynomolgus monkey CD137 (cyCD137).

[0280] In some embodiments, the hTlc muteins of the present disclosure may interfere with the binding of CD137L to CD137.

[0281] In some embodiments, provided hTlc muteins may comprise a mutated amino acid residue at one or more of 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 corresponding to the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0282] In some embodiments, provided hTlc muteins may comprise mutated amino acid residues at one or more of positions 26-34, 55-58, 60-61, 65, 104-106, and 108 corresponding to the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0283] In some embodiments, provided hTlc muteins may further comprise a mutated amino acid residue at one or more of positions 101, 111, 114, and 153 corresponding to the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0284] In some embodiments, provided hTlc muteins may comprise 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, 22, 23, 24, 25, 26 or even 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). In some preferred embodiments, provided hTlc muteins are capable of binding to CD137, particularly human CD137.

[0285] In some embodiments, provided hTlc muteins may comprise mutated amino acid residues at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even 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). In some preferred embodiments, provided hTlc muteins are capable of binding to CD137, particularly human CD137.

[0286] In some embodiments, the lipocalin muteins according to the present disclosure may include at least one amino acid substitution of a native cysteine residue by, for example, a serine residue. In some embodiments, the hTlc muteins according to the present disclosure include an amino acid substitution of a native cysteine residue at position 61 and / or 153 of the linear polypeptide sequence corresponding to mature hTlc (SEQ ID NO: 1) by another amino acid (e.g., a serine residue). In this context, it should be noted that it has been found that removal of the structural disulfide bond of wild-type hTlc formed by cysteine residues 61 and 153 (at the level of a respective natural nucleic acid library) (see Breustedt et al., J Biol Chem, 2005) may provide hTlc muteins that are not only stably folded but also capable of binding to a given non-native target with high affinity. In some embodiments, the elimination of structural disulfide bonds may provide a further advantage, namely allowing the generation or deliberate introduction of non-native disulfide bonds within the muteins of the present disclosure, thereby increasing the stability of the muteins. However, hTlc muteins that bind CD137 and have a disulfide bridge formed between Cys 61 and Cys 153 are also part of the present disclosure.

[0287] In some specific embodiments, the hTlc muteins of the present disclosure may include amino acid substitutions Cys 61→Ala, Phe, Lys, Arg, Thr, Asn, Gly, Gln, Asp, Asn, Leu, Tyr, Met, Ser, Pro or Trp and / or Cys 153→Ser or Ala at positions 61 and / or 153 of the linear polypeptide sequence corresponding to mature hTlc (SEQ ID NO: 1).

[0288] In some embodiments, two or all three cysteine codons at positions 61, 101, and 153 of the linear polypeptide sequence corresponding to mature hTlc (SEQ ID NO: 1) are replaced by codons for another amino acid. Further, in some embodiments, the hTlc mutein according to the present disclosure comprises an amino acid substitution of the native cysteine residue at position 101 of the linear polypeptide sequence corresponding to mature hTlc (SEQ ID NO: 1) by a serine residue or a histidine residue.

[0289] In some embodiments, the mutant protein according to the present disclosure comprises an amino acid substitution of a cysteine residue at a position corresponding to position 28 or 105 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1). Further, in some embodiments, the mutant protein according to the present disclosure comprises an amino acid substitution of a proline residue at a position corresponding to position 111 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1). Further, in some embodiments, the mutant protein according to the present disclosure comprises an amino acid substitution of a tryptophan residue or a glutamic acid at a position corresponding to position 114 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0290] In some embodiments, provided CD137-binding hTlc muteins may comprise one or more of the following mutated amino acid residues at one or more of 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 corresponding to mature hTlc (SEQ ID NO: 1): Ala 5→Val or Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Thr 42→Ser; Gly46→Asp; Lys 52→Glu; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys 65→Arg or Asn; Thr 71→Ala; Val 85→Asp; Lys 94→Arg or Glu; Cys 101→Ser; Glu104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys121→Glu; Ala 133→Thr; Arg 148→Ser; Ser 150→Ile; and Cys 153→Ser. In some embodiments, the hTlc muteins of the present disclosure comprise 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, mutated amino acid residues at these sequence positions of mature hTlc (SEQ ID NO: 1).

[0291] In some embodiments, 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):

[0292] (a) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg111→Pro; Lys 114→Trp; and Cys 153→Ser;

[0293] (b)Ala 5→Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; 61→Ala; Lys65→Arg; Val 85→Asp; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; 153→Ser;

[0294] (c) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys 65→Asn; Lys94→Arg; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys 121→Glu; Ala 133→Thr; and Cys153→Ser;

[0295] (d) Ala 5→Val; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys65→Arg; Lys 94→Glu; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys 121→Glu; Ala 133→Thr; and Cys 153→Ser;

[0296] (e) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Thr 42→Ser; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Cys101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ser 150→Ile; and Cys 153→Ser;

[0297] (f) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Lys 52→Glu; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Thr71→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ala 133→Thr; Arg 148→Ser; Ser 150→Ile; and Cys 153→Ser; and

[0298] (g)Ala 5→Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Gly 46→Asp; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Thr 71→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ser 150→Ile; and Cys153→Ser.

[0299] In some embodiments, the residue regions of the hTlc muteins of the present disclosure that differ from the 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 beyond the mutated amino acid sequence positions.

[0300] In some embodiments, the hTlc muteins disclosed herein have at least 70% sequence identity or at least 70% sequence homology with 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 sequence homology with the amino acid sequence of mature hTlc.

[0301] In some embodiments, the hTlc mutein of the present disclosure comprises an amino acid sequence as shown in any one of SEQ ID NOs: 32-38, or a fragment or variant thereof.

[0302] In some embodiments, the hTlc 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: 32-38.

[0303] The present disclosure also includes structural homologs of hTlc mutant proteins having an amino acid sequence selected from SEQ ID NOs: 32-38, said structural homologs having an amino acid sequence homology or sequence identity of more than about 60%, preferably more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92% and most preferably more than 95% with respect to said hTlc mutant proteins.

[0304] In some aspects, the present disclosure provides hNGAL mutant proteins that bind CD137. In this regard, the present disclosure provides hNGAL mutant proteins that can bind CD137 with a K of about 800nM, 700nM, 200nM, 140nM, 100nM or lower, preferably about 70nM, 50nM, 30nM, 10nM, 5nM, 2nM or even lower. D In some embodiments, the hNGAL mutants provided can bind to CD137 with 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 less. 50 Values for binding to CD137.

[0305] In some embodiments, the provided hNGAL mutant proteins that bind CD137 may cross-react with cynomolgus monkey CD137. In some embodiments, the provided hNGAL mutant proteins can cross-react with cynomolgus monkey CD137 at a K of about 50 nM, 20 nM, 10 nM, 5 nM, 2 nM or even lower. D In some embodiments, the hNGAL mutant proteins provided can bind to cynomolgus monkey CD137 with an EC of about 100 nM, 80 nM, 50 nM, 30 nM or even lower. 50 Values for binding to cynomolgus monkey CD137.

[0306] In some embodiments, the hNGAL muteins of the present disclosure may interfere with or compete for the binding of CD137L to CD137. In some other embodiments, the hNGAL muteins of the present disclosure may be able to bind to CD137 and / or bind to the CD137 / CD137L complex in the presence of CD137L.

[0307] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more of 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 corresponding to mature hNGAL (SEQ ID NO: 2).

[0308] In some embodiments, provided hNGAL muteins may comprise 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 even 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, provided hNGAL muteins are capable of binding to CD137, particularly human CD137.

[0309] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more of 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 corresponding to mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, provided hNGAL muteins are capable of binding to CD137, particularly human CD137.

[0310] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more of positions 36, 87, and 96 corresponding to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) and at one or more of positions 28, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 94, 100, 103, 106, 125, 127, 132, and 134 corresponding to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0311] In other embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more of 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 corresponding to mature hNGAL (SEQ ID NO: 2).

[0312] In other embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more of 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 of 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).

[0313] In some embodiments, the lipocalin muteins according to the present disclosure may comprise at least one amino acid substitution of a native cysteine residue by, for example, a serine residue. In some embodiments, the hNGAL muteins according to the present disclosure may comprise an amino acid substitution of a native cysteine residue at positions 76 and / or 175 of the linear polypeptide sequence corresponding to mature hNGAL (SEQ ID NO: 2) by another amino acid (e.g., a serine residue). In this context, it should be noted that removal of the structural disulfide bond (at the level of a respective natural nucleic acid library) formed by cysteine residues 76 and 175 of wild-type hNGAL has been found to provide hNGAL muteins that are not only stably folded but also capable of binding to a given non-native target with high affinity. In some embodiments, the elimination of structural disulfide bonds may provide the further advantage of allowing the generation or deliberate introduction of non-native disulfide bonds within the muteins of the present disclosure, thereby increasing the stability of the muteins. However, hNGAL muteins that bind CD137 and have a disulfide bridge formed between Cys76 and Cys 175 are also part of the present disclosure.

[0314] In some embodiments, provided CD137-binding hNGAL muteins may comprise one or more of the following mutated amino acid residues at one or more of 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 corresponding to mature hNGAL (SEQ ID NO: 2): Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg or Lys; Gln49→Val, Ile, His, Ser, or Asn; Tyr 52→Met; Asn 65→Asp; Ser 68→Met, Ala, or Gly; Leu70→Ala, Lys, Ser, or Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Met, Arg, Thr or Asn; Trp 79→Ala or Asp; Arg 81→Met, Trp or Ser; Phe 83→Leu; Cys 87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu and Lys 134→Tyr. In some embodiments, the hNGAL muteins of the present disclosure comprise 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).

[0315] In some embodiments, provided CD137-binding hNGAL muteins may comprise one or more of the following mutated amino acid residues at one or more of 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 corresponding to mature hNGAL (SEQ ID NO: 2): Gln 20→Arg; Asn 25→Tyr or Asp; Gln 28→His; Val33→Ile; Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Glu 44→Val or Asp; Gln 49→His; Tyr52→Ser or Gly; Lys 59→Asn; Ser 68→Asp; Leu 70→Met; Phe 71→Leu; Arg 72→Leu; Lys73→Asp; Asp 77→Gln or His; Tyr 78→His; Trp 79→Ile; Ile 80→Asn; Arg 81→Trp or Gln; Thr 82→Pro; Cys87→Ser; Phe 92→Leu or Ser; Asn 96→Phe; Lys 98→Arg; Tyr100→Asp; Pro 101→Leu; Leu 103→His or Pro; Phe 122→Tyr; Lys 125→Ser; Ser 127→Ile; Tyr132→Trp; and Lys 134→Gly.

[0316] In some embodiments, provided CD137-binding hNGAL muteins may comprise one or more of the following mutated amino acid residues at one or more of 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 corresponding to mature hNGAL (SEQ ID NO: 2): Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Ser or Gly; Ser 68→Asp; Leu 70→Met; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln or His; Trp 79→Ile; Arg 81→Trp or Gln; Asn 96→Phe; Tyr 100→Asp; Leu 103→His or Pro; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly. In some embodiments, provided CD137-binding hNGAL muteins may further comprise one or more of the following mutated amino acid residues at one or more of positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 92, 98, 101, and 122 of the linear polypeptide sequence corresponding to mature hNGAL (SEQ ID NO: 2): Gln 20→Arg; Asn 25→Tyr or Asp; Val 33→Ile; Glu 44→Val or Asp; Lys 59→Asn; Phe71→Leu; Tyr 78→His; Ile 80→Asn; Thr 82→Pro; Phe 92→Leu or Ser; Lys 98→Arg; Pro 101→Leu; and Phe 122→Tyr.

[0317] In some embodiments, 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):

[0318] (a)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln49→Asn;Tyr 52→Met;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Ser;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0319] (b)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln 49→Ile;Tyr 52→Met;Asn 65→Asp;Ser 68→Met;Leu 70→Lys;Arg 72→Asp;Lys 73→Asp;Asp 77→Met;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0320] (c)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Ala;Leu 70→Ala;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0321] (d)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Ala;Leu 70→Ala;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0322] (e)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln49→Ser;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Ser;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Met;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0323] (f)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln49→Val;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Arg;Trp 79→Asp;Arg 81→Ser;Cys 87→Ser;Leu 94→Phe;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0324] (g)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln 49→His;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Ser;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0325] (h)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Ser;Phe 83→Leu;Cys 87→Ser;Leu 94→Phe;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;or

[0326] (i) Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln49→Ser;Tyr 52→Met;Asn 65→Asp;Ser 68→Ala;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Asn;Trp 79→Ala;Arg 81→Ser;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr.

[0327] In some further embodiments, the regions of residues of the hNGAL muteins of the present disclosure that differ 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) may include the wild-type (native) amino acid sequence of mature hNGAL outside of the mutated amino acid sequence positions.

[0328] 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):

[0329] (a) Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Ser; Ser 68→Asp; Leu 70→Met; Arg 72→Leu; Lys 73→Asp; 96→Phe; Tyr 100→Asp; Leu 103→His; Lys 125→Ser; Ser 127→Ile; Tyr132→Trp; and Lys 134→Gly;

[0330] (b) Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Ser; Ser 68→Asp; Leu 70→Met; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln; Trp 79→Ile; Arg 81→Trp; Phe 92→Leu; Asn 96→Phe; Lys 98→Arg; Tyr 100→Asp; Pro 101→Leu; Leu103→His; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly;

[0331] (c) Asn 25 → Tyr; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Gln; Phe 92 → Ser; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly;

[0332] (d) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Tyr 78 → His; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly;

[0333] (e) Asn 25 → Asp; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly;

[0334] (f)Val 33→Ile;Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Gly;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Phe 92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Lys125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0335] (g)Gln 20→Arg;Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Glu 44→Val;Gln 49→His;Tyr 52→Gly;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Phe 92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Phe 122→Tyr;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0336] (h)Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Ser;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Ile 80→Asn;Arg 81→Trp;Thr 82→Pro;Asn 96→Phe;Tyr 100→Asp;Pro 101→Leu;Leu103→Pro;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0337] (i) Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Gly; Lys 59→Asn; Ser 68→Asp; Leu 70→Met; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln; Trp 79→Ile; Arg 81→Trp; Phe 92→Leu; Asn 96→Phe; Tyr 100→Asp; Leu 103→His; Lys125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly; and

[0338] (j) Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Glu 44→Asp; Gln 49→His; Tyr 52→Ser; Ser 68→Asp; Leu 70→Met; Phe 71→Leu; Arg 72→Leu; Lys 73→Asp; Asp 77→His; Trp 79→Ile; Arg81→Trp; Phe 92→Leu; Asn 96→Phe; Tyr 100→Asp; Leu 103→His; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly.

[0339] In some embodiments, the regions of residues of the hNGAL muteins of the present disclosure that differ 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 include the wild-type (native) amino acid sequence of mature hNGAL outside of the mutated amino acid sequence positions.

[0340] In some embodiments, the hNGAL mutants disclosed herein have at least 70% sequence identity or at least 70% sequence homology with the sequence of mature hNGAL (SEQ ID NO: 2). As an illustrative example, the mutant of SEQ ID NO: 40 has approximately 87% amino acid sequence identity or sequence homology with the amino acid sequence of mature hNGAL.

[0341] In some embodiments, the hNGAL muteins of the present disclosure comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 39-57, or a fragment or variant thereof.

[0342] 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.

[0343] The present disclosure also includes structural homologs of hNGAL muteins having an amino acid sequence selected from SEQ ID NOs: 39-57, said structural homologs having greater than about 60%, preferably greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 92%, and most preferably greater than 95% amino acid sequence homology or sequence identity with respect to said hNGAL muteins.

[0344] In some embodiments, the present disclosure provides for the production of a molecule with a K of about 5 nM or less. D A lipocalin mutein that binds to CD137 with a measured affinity, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0345] Suitable lipocalin muteins specific for CD137 are also described in WO 2016 / 177762, which is incorporated herein by reference in its entirety.

[0346] In some embodiments, the lipocalin muteins of the present disclosure may comprise a heterologous amino acid sequence at its N-terminus or C-terminus, preferably at the C-terminus, such as a StrepII tag (SEQ ID NO: 12) or a cleavage site sequence for certain restriction enzymes, without affecting the biological activity of the lipocalin mutein (binding to its target, e.g., CD137).

[0347] In some embodiments, further modifications of the lipocalin mutein may be introduced to modulate certain properties of the mutein, such as improving folding stability, serum stability, protein resistance or water solubility or reducing aggregation tendency, or to introduce new properties to the mutein. In some embodiments, the modifications may result in two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) properties of the provided mutein being modulated.

[0348] For example, it is possible to mutate one or more amino acid sequence positions of a lipocalin mutein to introduce new reactive groups, for example, for conjugation with other compounds such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins, or for forming non-naturally occurring disulfide bonds. Conjugated compounds such as PEG and HES can in some cases increase the serum half-life of the corresponding lipocalin mutein.

[0349] In some embodiments, the reactive group of the lipocalin mutein may be naturally present in its amino acid sequence, such as a naturally occurring cysteine residue in the amino acid sequence. In some other embodiments, such reactive groups may be introduced via mutagenesis. In the case of introducing the reactive group via mutagenesis, one possibility is to mutate the amino acid at the appropriate position to a cysteine residue. Exemplary possibilities for introducing such mutations into the amino acid sequence of the hTlc mutein include the substitutions Thr 40→Cys, Glu 73→Cys, Arg 90→Cys, Asp 95→Cys, and Glu 131→Cys of the wild-type sequence of hTlc (SEQ ID NO: 1). Exemplary possibilities for introducing such mutations into the amino acid sequence of the hNGAL mutein include introducing a cysteine residue 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 moiety may be used to PEGylate or HESylate the mutein, for example, in order to increase the serum half-life of the respective lipocalin mutein.

[0350] In some embodiments, artificial amino acids may be introduced into the amino acid sequence of the lipocalin mutein in order to provide suitable amino acid side chains as new reactive groups for conjugating one of the above compounds to the lipocalin mutein. Generally, such artificial amino acids are designed to be more reactive and thus facilitate conjugation to the desired compound. Such artificial amino acids may be introduced by mutagenesis, for example using artificial tRNA, such as p-acetylphenylalanine.

[0351] In some embodiments, the lipocalin muteins of the present disclosure are fused at their N-terminus or their 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-terminus or C-terminus to a partner, which is a protein, protein domain or peptide; such as an antibody, a signal sequence and / or an affinity tag.

[0352] 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, which allow easy detection and / or purification of recombinant proteins, 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. In general, it is possible to label the lipocalin muteins of the present disclosure with any suitable chemical substance or enzyme that directly or indirectly generates a detectable compound or signal in a chemical, physical, optical or enzymatic reaction. For example, a fluorescent or radioactive label can be conjugated to the lipocalin mutein to generate fluorescence or X-rays as a detectable signal. Alkaline phosphatase, horseradish peroxidase and β-galactosidase are examples of enzyme labels that catalyze the formation of a chromogenic reaction product (which is also an optical label). In general, all labels commonly used for antibodies (except those labels specifically used for carbohydrate moieties in the Fc portion of immunoglobulins) can also be used for conjugation to the lipocalin muteins of the present disclosure.

[0353] 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 also International Patent Publication No. WO 2006 / 056464 in this regard, 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). The moiety that extends the serum half-life may be a PEG molecule, a HES molecule, a fatty acid molecule such as palmitic acid (Vajo and Duckworth, Pharmacol Rev, 2000), the Fc portion of an immunoglobulin, the C portion of an immunoglobulin, or a PEGylated lipocalin. H 3 domains, immunoglobulin C H 4 domains, albumin binding peptides, albumin binding proteins or transferrin, to name a few.

[0354] In some embodiments, if PEG is used as a conjugated partner, the PEG molecule can be substituted, unsubstituted, linear or branched. It can also be an activated polyethylene derivative. The example of a suitable compound is a PEG molecule, such as described in International Patent Publication No. WO 1999 / 64016, U.S. Patent No. 6,177,074 or U.S. Patent No. 6,403,564 about interferon, or such as described about other proteins such as asparaginase, PEG-adenosine deaminase (PEG-ADA) or PEG-superoxide dismutase (Fuertges and Abuchowski, Journal of Controlled Release, 1990). The molecular weight range of such polymers such as polyethylene glycol may be about 300 to about 70,000 daltons, including polyethylene glycol having a molecular weight of about 10,000, about 20,000, about 30,000 or about 40,000 daltons. In addition, carbohydrate oligomers and polymers such as HES can be conjugated to the muteins of the present disclosure for the purpose of serum half-life extension as described, for example, in US Pat. Nos. 6,500,930 or 6,620,413.

[0355] In some embodiments, if the Fc portion of an immunoglobulin is used for the purpose of extending the serum half-life of the lipocalin muteins of the present disclosure, SynFusion ® commercially available from Syntonix Pharmaceuticals, Inc. (MA, USA) may be used. TM The use of this Fc fusion technology allows for the production of biopharmaceuticals with longer duration of action and may, for example, consist of two copies of a mutein linked to the Fc region of an antibody to improve pharmacokinetics, solubility, and manufacturing efficiency.

[0356] Examples of albumin binding peptides that can be used to extend the serum half-life of lipocalin muteins are, for example, those having the consensus sequence Cys-Xaa1-Xaa2-Xaa3-Xaa4-Cys, wherein 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 Publication No. 2003 / 0069395 or in Dennis et al. (2002). The albumin binding protein to be fused or conjugated to the lipocalin mutein to extend serum half-life may be a bacterial albumin binding protein, an antibody, an antibody fragment including a domain antibody (e.g., see U.S. Patent No. 6,696,245), or a lipocalin mutein having binding activity for albumin. Examples of bacterial albumin binding proteins include Streptococcal protein G (Konig and Skerra, J. Immunol Methods, 1998).

[0357] In some embodiments, if the albumin binding protein is an antibody fragment, it may be a domain antibody. Domain antibodies (dAbs) are engineered to allow precise control of biophysical properties and in vivo half-life to produce an optimal safety and efficacy product profile. Domain antibodies are commercially available, for example, from Domantis Ltd. (Cambridge, UK and MA, USA).

[0358] 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 or bovine serum albumin or rat albumin. Albumin or its fragments can be recombinantly produced, as described in U.S. Patent No. 5,728,553 or European Patent Publication Nos. EP0330451 and EP0361991. Accordingly, recombinant human albumin (e.g., from Novozymes Delta Ltd., Nottingham, UK) can be used as a partner for the lipocalin muteins of the present disclosure to extend serum half-life. ) can be conjugated or fused to the lipocalin muteins of the present disclosure.

[0359] In some embodiments, if transferrin is used as a partner to extend the serum half-life of the lipocalin muteins of the present disclosure, the muteins can be genetically fused to the N-terminus or C-terminus or both of non-glycosylated transferrin. Non-glycosylated transferrin has a half-life of 14-17 days, and transferrin fusion proteins similarly have an extended half-life. Transferrin carriers also provide high bioavailability, biodistribution, and circulatory 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 TM ) is also commercially available from Novozymes Delta Ltd. (Nottingham, UK).

[0360] Another alternative approach to extend the half-life of the lipocalin muteins of the present disclosure is to fuse long, unstructured, flexible glycine-rich sequences (e.g., polyglycine having about 20 to 80 consecutive glycine residues) to the N-terminus or C-terminus of the mutein. For example, this approach disclosed in International Patent Publication No. WO 2007 / 038619 has also been referred to as "rPEG" (recombinant PEG).

[0361] E. Exemplary uses and applications of antibodies or antigen-binding domains thereof specific for CD228 and fusion proteins specific for CD137 and CD228.

[0362] In some embodiments, the fusion proteins of the present disclosure may produce a synergistic effect by dual targeting of CD137 and CD228. In some embodiments, the fusion proteins of the present disclosure may produce a local anti-tumor effect by dual targeting of CD137 and CD228. Therefore, there are many possible applications for the fusion proteins of the present disclosure in medicine.

[0363] 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, for simultaneously binding CD137 and CD228.

[0364] The present disclosure also relates to the use of one or more fusion proteins as described for complex formation with CD137 and / or CD228.

[0365] Therefore, in one aspect of the present disclosure, the fusion protein provided may be used for the detection of CD137 and / or CD228. Such uses may include the following steps: under suitable conditions, one or more of the fusion protein is contacted with a sample suspected of containing CD137 and / or CD228, thereby allowing the complex between the fusion protein and CD137 and / or CD228 to form, and detecting the complex by a suitable signal. The detectable signal can be caused by a label as described above, or by a change in the physical properties of the binding (i.e., complex formation) itself. An example is surface plasmon resonance, whose value changes during the binding of a binding partner, one of which is fixed to a surface such as gold foil.

[0366] The fusion proteins of the present disclosure may also be used to isolate CD137 and / or CD228. Such use may include the following steps: contacting one or more of the fusion proteins with a sample presumed to contain CD137 and / or CD228 under suitable conditions, thereby allowing a complex between the fusion protein and CD137 and / or CD228 to form, and separating the complex from the sample.

[0367] 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.

[0368] In addition to their use in diagnostics, in another aspect, the present disclosure also contemplates pharmaceutical compositions comprising one or more antibodies, antigen binding domains or fusion proteins thereof of the present disclosure and a pharmaceutically acceptable excipient.

[0369] In addition, in some embodiments, the antibodies, antigen binding domains thereof, or fusion proteins provided may be used in therapy, for example, as anti-tumor agents and / or anti-infective agents and / or immunomodulators. In some embodiments, the antibodies, antigen binding domains thereof, or fusion proteins provided may be used in the manufacture of drugs, for example, drugs for treating cancers including CD228-positive cancers. In some embodiments, the antibodies, antigen binding domains thereof, or fusion proteins disclosed herein may be used in methods for preventing, improving, or treating human diseases, such as cancers, including methods for treating CD228-positive cancers. In some embodiments, the fusion proteins disclosed herein may be used together with anti-PD-1 or anti-PD-L1 antibodies to prevent, improve, or treat cancers, including methods for treating CD228-positive cancers. In some embodiments, the anti-PD-1 or anti-PD-L1 antibodies are nivolumab, pembrolizumab, cemiplizumab, dotalizumab, atezolizumab, avelumab, or durvalumab. Accordingly, a method for preventing, improving or treating a human disease (e.g., cancer, including CD228-positive cancer) in a subject in need thereof is also provided, comprising administering to the subject a therapeutically effective amount of one or more antibodies, antigen-binding domains thereof, or fusion proteins thereof, or one or more compositions comprising such antibodies, antigen-binding domains thereof, or fusion proteins. Accordingly, a method for preventing, improving or treating a cancer in a subject in need thereof, including a method for CD228-positive cancer, is also provided, 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 or anti-PD-L1 antibody, or one or more compositions comprising such antibodies. In some embodiments, the anti-PD-1 or anti-PD-L1 antibody is nivolumab, pembrolizumab, cemiplizumab, dotalizumab, atezolizumab, avelumab, or durvalumab. In some embodiments, the cancer is a CD228-positive cancer.

[0370] Examples of cancers that may be treated using the antibodies, antigen-binding domains thereof, or fusion proteins of the present disclosure include lung cancer, such as non-small cell lung cancer (NSCLC), melanoma, such as cutaneous melanoma or intraocular malignant melanoma, pancreatic neoplasms or pancreatic cancer, mesothelioma, colorectal neoplasms or colorectal cancer (CRC), thyroid cancer, breast cancer, bile duct cancer, esophageal cancer, and head and neck cancer. In some embodiments, the cancer comprises a metastatic cancer.

[0371] In some embodiments, the fusion proteins of the present disclosure may simultaneously target tumor cells expressing CD228 and activate lymphocytes of the host immune system adjacent to such tumor cells. In some embodiments, the fusion proteins of the present disclosure may increase the activity of targeted anti-tumor T cells, 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 may activate the immune response in the tumor microenvironment. In some embodiments, the fusion proteins of the present disclosure may, for example, reduce the side effects of effector lymphocytes against healthy cells, i.e., off-target toxicity, by locally inhibiting oncogene activity and / or inducing lymphocyte activation.

[0372] In some embodiments, the present disclosure encompasses the use of a fusion protein of the present disclosure or a composition comprising the fusion protein provided for inducing a local lymphocyte response near CD228-positive tumor cells. Accordingly, in some embodiments, the present disclosure provides a method for inducing a local lymphocyte response near CD228-positive tumor cells, comprising applying one or more fusion proteins of the present disclosure or one or more compositions comprising such fusion proteins. "Local" means that after simultaneously binding to T cells and engaging CD228-positive tumor cells via CD137, T cells produce cytokines, particularly IL-2 and / or IFN-γ, near CD228-positive cells. Such cytokines reflect the activation of T cells, which may then 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 fusion protein provided may also be used to increase the secretion of cytotoxic factors, such as perforin, granzyme B, and granzyme A, by T cells near CD228-positive tumor cells.

[0373] In some embodiments, the present disclosure encompasses the use of fusion proteins of the present disclosure or compositions comprising such fusion proteins for costimulating T cells and / or activating downstream signaling pathways of CD137. Preferably, the fusion proteins provided costimulate T cells and / or activate downstream signaling pathways of CD137 when engaging CD228-positive tumor cells. Accordingly, the present disclosure provides methods for inducing T lymphocyte proliferation / activity and / or activating downstream signaling pathways of CD137, preferably when engaging tumor cells expressing CD228, comprising applying one or more fusion proteins of the present disclosure and / or one or more compositions comprising such fusion proteins.

[0374] In some embodiments, the present disclosure encompasses the use of the fusion proteins of the present disclosure, or compositions comprising such fusion proteins, to induce CD137 clustering and activation on T cells and direct such T cells to tumor cells in which CD228 is expressed.

[0375] In some embodiments, the present disclosure encompasses the use of fusion proteins of the present disclosure or compositions comprising such fusion proteins for inducing an increase in CD8+ T cell mitochondrial content and / or reducing 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 containing a tumor. In some embodiments, the present disclosure encompasses the use of fusion proteins of the present disclosure or compositions comprising such fusion proteins for inducing 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 containing a tumor. In some embodiments, the present disclosure encompasses the use of fusion proteins of the present disclosure or compositions comprising such fusion proteins for inducing 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 containing a tumor.

[0376] In some embodiments, the present disclosure encompasses the use of fusion proteins of the present disclosure or compositions comprising such fusion proteins for stimulating the proliferation of exhausted CD8+ T cells 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 to tissue containing a tumor. In some embodiments, the present disclosure encompasses the use of fusion proteins of the present disclosure or compositions comprising such fusion proteins for stimulating the proliferation of exhausted CD8+ T cells 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 to, and applying an anti-PD-1 or anti-PD-L1 antibody or one or more compositions comprising such antibodies to tissue containing a tumor. In some embodiments, the anti-PD-1 or anti-PD-L1 antibody is nivolumab, pembrolizumab, cemiplizumab, dotalizumab, atezolizumab, avelumab or durvalumab.

[0377] F. Generation of exemplary provided antibodies or antigen-binding domains thereof specific for CD228 and fusion proteins specific for CD137 and CD228.

[0378] In some embodiments, the present disclosure provides nucleic acid molecules (for example, DNA or RNA), which include the nucleotide sequence of the antibody, its antigen-binding domains or fusion protein provided by coding. In some embodiments, the present disclosure encompasses the vector containing the nucleic acid molecules provided. In some embodiments, the present disclosure encompasses the host cell containing the nucleic acid molecules or vector provided. Due to the degeneracy of the genetic code, some codons are allowed to be replaced by other codons specifying the same amino acids, so the present disclosure is not limited to the specific nucleic acid molecules encoding antibodies as described herein, their antigen-binding domains or fusion protein, but encompasses all nucleic acid molecules comprising the nucleotide sequence of encoding functional antibodies, their antigen-binding domains or fusion protein. In this respect, the present disclosure also relates to the nucleotide sequence of the antibody, its antigen-binding domains or fusion protein provided by coding.

[0379] If a nucleic acid molecule, such as DNA, includes sequence elements that contain information about transcriptional and / or translational regulation, and such sequences are "operably linked" to a nucleotide sequence encoding a protein, it is said to be "capable of expressing a nucleic acid molecule" or "capable of allowing expression of a nucleotide sequence". An operable linkage is one in which the regulatory sequence elements and the sequence to be expressed are linked in a manner that allows gene expression. The exact nature of the regulatory regions required for gene expression may vary among species, but in general, these regions include a promoter, which in prokaryotes contains the promoter itself (i.e., a DNA element that directs the initiation of transcription) and a DNA element that signals the initiation of translation when transcribed into RNA. Such promoter regions typically include 5' non-coding sequences involved in the initiation of transcription and translation, such as the -35 / -10 box and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequence, and 5'-capping element in eukaryotes. These regions may also include enhancer or repressor elements as well as translation signals and leader sequences for targeting the native protein to a specific compartment of the host cell.

[0380] Additionally, 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 replaced by signals that are functional in that cell.

[0381] Thus, the nucleic acid molecules of the present disclosure may be "operably linked" to one or more regulatory sequences, such as a promoter sequence, to allow expression of the nucleic acid molecule. In some embodiments, the nucleic acid molecules of the present disclosure include a promoter sequence and a transcription termination sequence. Suitable prokaryotic promoters are, for example, the tet promoter, the lacUV5 promoter, or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.

[0382] In some embodiments, a nucleic acid molecule encoding a lipocalin mutein disclosed herein may be "operably linked" to another nucleic acid molecule encoding an antibody disclosed herein to allow for expression of the fusion protein disclosed herein.

[0383] In some embodiments, the provided methods may include subjecting at least one nucleic acid molecule encoding mature hTlc to mutagenesis at the nucleotide triplets encoding one or more of 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 corresponding to hTlc (SEQ ID NO: 1) to obtain a lipocalin mutein as included in the provided fusion protein. In some embodiments, the provided methods can include subjecting at least one nucleic acid molecule encoding mature hNGAL to mutagenesis at the nucleotide triplets encoding one or more of 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 corresponding to hNGAL (SEQ ID NO: 2) to obtain a lipocalin mutein as included in the provided fusion proteins. In some embodiments, the provided methods can include subjecting at least one nucleic acid molecule encoding mature hNGAL to mutagenesis at the nucleotide triplets encoding one or more of 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 corresponding to hNGAL (SEQ ID NO: 2) to obtain a lipocalin mutein as included in the provided fusion proteins.

[0384] In addition, with respect to the hTlc muteins or hNGAL muteins of the present disclosure as included in fusion proteins, in some embodiments, the naturally occurring disulfide bonds between Cys 61 and Cys 153 or Cys 76 and Cys 175, respectively, can be removed. Accordingly, such muteins can be produced in cellular compartments with a reducing redox environment, such as in the cytoplasm of Gram-negative bacteria.

[0385] Further with respect to the hTlc muteins or hNGAL muteins provided herein as included in fusion proteins, the present disclosure also encompasses nucleic acid molecules encoding such muteins, which, in some embodiments, may include one or more additional mutations beyond the indicated sequence positions for experimental mutagenesis. For example, such mutations are often tolerated or may even prove advantageous if they contribute to improved folding efficiency, serum stability, thermal stability, or ligand binding affinity of the lipocalin mutein and / or fusion protein.

[0386] In some embodiments, the provided nucleic acid molecules may also be part of a vector or any other type of cloning vehicle, such as a plasmid, phagemid, phage, baculovirus, cosmid, or artificial chromosome.

[0387] In some embodiments, the provided nucleic acid molecules may be included in a phagemid. As used in this context, a phagemid vector refers to a vector encoding an intergenic region of a temperate phage, such as M13 or f1, or a functional portion thereof fused to a cDNA of interest. For example, in some embodiments, after repeated infection of a bacterial host cell with such a provided phagemid vector and an appropriate helper phage (e.g., M13K07, VCS-M13, or R408), a complete phage particle is produced, thereby enabling the encoded heterologous cDNA to be physically coupled to its corresponding polypeptide displayed on the phage surface (Lowman, Annu Rev Biophys Biomol Struct, 1997, Rodi and Makowski, Curr Opin Biotechnol, 1999).

[0388] According to various embodiments, in addition to the above-mentioned regulatory sequences and nucleic acid sequences encoding antibodies, antigen-binding domains thereof, or fusion proteins as described herein, the cloning vehicle may also include replication and control sequences derived from species compatible with the host cell used for expression and a selectable marker that confers a selectable phenotype on the transformed or transfected cells. A large number of suitable cloning vectors are known in the art and commercially available.

[0389] In some embodiments, the disclosure further relates to methods for producing antibodies, antigen-binding domains, or fusion proteins of the present disclosure from nucleic acids encoding antibodies, antigen-binding domains, or fusion proteins or any subunit thereof using genetic engineering methods. In some embodiments, the methods provided can be performed in vivo, wherein the antibodies, antigen-binding domains, or fusion proteins provided can be produced, for example, in bacteria or eukaryotic host organisms and then isolated from the host organisms or their cultures. It is also possible, for example, to produce antibodies, antigen-binding domains, or fusion proteins of the present disclosure in vitro using an in vitro translation system.

[0390] When producing antibody, its antigen-binding domain or fusion protein in vivo, the nucleic acid encoding such antibody, its antigen-binding domain or fusion protein may be introduced into suitable bacteria or eukaryotic host organisms using recombinant DNA techniques well known in the art. In some embodiments, the DNA molecules encoding antibodies as described herein, its antigen-binding domain or fusion protein (for example, SEQ ID NO:91-94 and SEQ ID NO:91 and 88, 93 and 90, 87 and 92 or 89 and 94 sequence pairs), and particularly cloning vectors containing the coding sequence of such antibodies, its antigen-binding domain or fusion protein, can be transformed into a host cell capable of expressing the gene. Conversion can be performed using standard techniques. Therefore, the present disclosure also relates to host cells containing nucleic acid molecules as disclosed herein.

[0391] In some embodiments, the transformed host cell may be cultured under conditions suitable for expressing the nucleotide sequence encoding the antibody, antigen binding domain thereof, or fusion protein of the present disclosure. In some embodiments, the host cell can be a prokaryotic cell, such as Escherichia coli (E. coli) or Bacillus subtilis, or a eukaryotic cell, 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.

[0392] In some embodiments, when the lipocalin muteins of the present disclosure (including lipocalin proteins contained in fusion proteins as disclosed herein) include intramolecular disulfide bonds, it may be preferred to use an appropriate signal sequence to direct the nascent protein to a cellular compartment with an oxidative redox environment. Such an oxidative environment may be provided by the periplasm of Gram-negative bacteria such as Escherichia coli, the extracellular environment of Gram-positive bacteria, or the endoplasmic reticulum lumen of eukaryotic cells, and generally favors the formation of structural disulfide bonds.

[0393] In some embodiments, it is also possible to produce the antibodies, antigen-binding domains thereof, or fusion proteins of the present disclosure in the cytosol of host cells (preferably E. coli). In this case, the provided antibodies, antigen-binding domains thereof, or fusion proteins can be obtained directly in a soluble and folded state, or recovered in the form of inclusion bodies, followed by in vitro renaturation. A further option is to use specific host strains with an oxidative intracellular environment, which may therefore allow disulfide bond formation in the cytosol (Venturi et al., J Mol Biol, 2002).

[0394] In some embodiments, the antibodies, antigen-binding domains thereof, or fusion proteins of the present disclosure as described herein may not necessarily be generated or produced, in whole or in part, using genetic engineering. Rather, such proteins may be obtained by any of a number of conventional and well-known techniques, such as common organic synthesis strategies, solid-phase assisted synthesis techniques, commercially available automated synthesizers, or by in vitro transcription and translation. For example, promising antibodies, antigen-binding domains thereof, or fusion proteins, or lipocalin muteins included in such fusion proteins, may be identified using molecular modeling, synthesized in vitro, and investigated for binding activity to a target 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).

[0395] In some embodiments, the antibodies, antigen-binding domains thereof, or fusion proteins of the present disclosure may be produced by in vitro transcription / translation using well-established methods known to those skilled in the art.

[0396] In some further embodiments, the antibodies, antigen binding domains thereof, or fusion proteins as described herein may also be prepared by conventional recombinant techniques, alone or in combination with conventional synthetic techniques.

[0397] In addition, in some embodiments, fusion proteins according to the present disclosure may be obtained by conjugating the individual subunits (e.g., antibodies and mutant proteins included in the fusion protein). For example, such conjugation can be achieved using conventional methods through all forms of covalent or non-covalent bonding.

[0398] Technicians will understand that methods that can be used to prepare antibodies, antigen-binding domains thereof or fusion proteins contemplated by the present disclosure, but whose protein or nucleic acid sequences are not clearly disclosed herein. As an overview, such modifications of amino acid sequences include, for example, site-directed mutagenesis of a single amino acid position to simplify the subcloning of protein genes or portions thereof by incorporating cleavage sites for certain restriction enzymes. Additionally, these mutations can be incorporated to further improve the affinity of the antibody, its antigen-binding domain or fusion protein for its target (e.g., CD137 and / or CD228). Furthermore, mutations can be introduced to modulate one or more properties of the protein, for example, to improve folding stability, serum stability, protein resistance or water solubility or to reduce aggregation tendency (if necessary).

[0399] Additional 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, it should be understood that although the present disclosure is specifically disclosed through exemplary embodiments and optional features, modifications and variations of the contents embodied therein disclosed herein may be employed by those skilled in the art, and such modifications and variations are considered to be within the scope of the present disclosure.

[0400] VI. Examples

[0401] Example 1: Generation of anti-CD228 antibodies

[0402] Human immunoglobulin transgenic rat strain ( OMT, Inc.) for the development of hybridoma cells expressing monoclonal antibodies. Contains a chimeric human / rat IgH locus (comprising 22 human VH, all human D, and JH segments linked in a natural configuration to a rat CH locus) along with a fully human IgL locus (12 Vκ linked to Jκ-CK and 16 Vλ linked to Jλ-Cλ). See Osborn, et al. (2013) J Immunol 190(4):1481-1490); WO 2014 / 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.

[0403] 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.

[0404] Using RPMI-7951 cells engineered to stably express human CD228, the presence of antibodies against human CD228 in the serum of transgenic rats was monitored by flow cytometry at days 33, 81, and 127. Transgenic rats with detectable immune responses were boosted five and seven days before spleen and lymph node harvest. The booster immunization before organ harvest consisted of 90 μg intravascular injection and 18 μg intraperitoneal injection of recombinant human CD228 protein suspended in phosphate-buffered saline (PBS).

[0405] The specific splenocytes and lymphocytes showing B cell lineage and for fluorescently labeled recombinant human CD228 are sorted into cell lysis buffer as individual cells by flow activated cell sorting (FACS). RNA from individual B cells is reverse transcribed into cDNA, and is amplified to known human variable regions from primer sets. Amplicon experience DNA purification is used for Sanger sequencing using appropriate sequencing primers, which is then identified and annotated by IgBLAST heavy chain and light chain sequences.

[0406] The productive heavy and light chain sequences of the human variable regions identified by single B cell cloning and Sanger sequencing were selected for gene synthesis and cloned into expression vectors using conventional cloning techniques. The expression vectors encode human IgG1, human kappa or human lambda constant regions, which are located within the framework and downstream of the human variable regions. Sanger sequencing verified the antibody expression constructs.

[0407] 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). Cell culture supernatants were collected by centrifugation 9 days after transfection. The filtered culture supernatant was spiked with 10% Triton-X (final concentration 0.1%) and placed on a rocking platform at 4°C overnight. The supernatant was loaded onto a 5 mL HiTrap MabSelectSuRe column using an Avant25 chromatography system at 2.5 mL / min. The column was then washed with 5 column volumes (CV) of internal wash buffer (1X PBS, 0.1% Triton-X), 5 CV of high salt buffer (1X PBS, 0.5 M NaCl), and 7.5 CV of standard 1X PBS (pH 7.4). The protein was eluted with 1 CV of 20 mM citrate (pH 3) and immediately buffer-exchanged into 1X PBS (pH 7.4) using a HiPrep Sephadex G-25 Desalting column. Fractionation was monitored via A280 UV to determine optimal collection of the purified protein.

[0408] Figure 2A and 2B The CDR sequences of the antibodies are shown as described by Kabat ( Figure 2A ) and IMGT( Figure 2B ) defined.

[0409] Example 2: Binding of anti-CD228 antibodies to recombinant human CD228

[0410] The binding of humanized antibodies specific for human melanin transferrin (CD228) to recombinant human CD228 (R&D Systems) was assessed by ELISA. The antibodies were titrated on a plate coated with 1 μg / ml recombinant human CD228, detected with HRP-labeled goat anti-human IgG secondary reagent (Thermo), and developed with TMB substrate (Thermo). OD450 values were read on a plate reader using SoftMaxPro software. The OD450 values for each experiment were transferred to GraphPad Prism8 for plotting and analysis. The results are shown in Figure 3 middle.

[0411] Example 3: Binding of anti-CD228 antibodies to cells

[0412] The humanized antibodies specific for human CD228 were evaluated for relative binding to cancer cell lines expressing CD228. Cells were incubated with titrations of Alexa-647 labeled humanized antibody clones, washed, and fluorescence intensity was assessed by flow cytometry on an AttuneNXT flow cytometer. MFI values were transferred to GraphPadPrism 8 for plotting and analysis. EC50 values were determined by nonlinear regression. The numbers next to the cell line names reflect the surface CD228 copy number estimated using the QIFIKIT quantitative analysis kit (Agilent).

[0413] The results are Figures 4A-4C The raw mean fluorescence intensity (MFI) values are shown in the figure. Figure 4D The EC values of each antibody for each cell line are shown. 50 value.

[0414] Example 4: Binding profile of anti-CD228 antibodies

[0415] use The RED384 system (Sartorius) was used to determine the binding kinetics and affinity of human CD228 to a C-terminal polyhistidine tag (R&D Systems) by biolayer interferometry (BLI). Anti-human antibody capture AHC (GE Healthcare) biosensors were used for analysis. Subsequently, anti-CD228 antibodies OMT8, OMT24, OMT30, OMT35, and OMT36 (IgG) were captured for 180 seconds by anti-human IgG-Fc antibodies at the chip surface in HBS-EP+ buffer at 0.5 μg / mL. After each capture step, the biosensor was washed in HBS-EB+ blank buffer. For affinity determination, dilutions of recombinant huCD228 (100 nM, 40 nM, 16 nM, 6.4 nM, 2.6 nM, and 1.0 nM) or blanks were prepared in HBS-EP+ buffer and applied to the biosensor. The binding assay was 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 Analysis Software (v12.0) evaluated the data, and the results were displayed in Figures 5A-5E A single reference was used, and a 1:1 binding model was used to fit the raw data.

[0416] Example 5: Lack of cross-reactivity of anti-CD228 antibodies with transferrin and lactotransferrin

[0417] The humanized antibody specific for human melanin transferrin (CD228) was evaluated by ELISA for binding to related transferrin family members lactotransferrin and transferrin. Antibodies were titrated on plates coated with 1 μg / mL of recombinant human melanin transferrin (CD228), lactotransferrin and transferrin, and detected with HRP-labeled goat anti-human IgG secondary reagent (Thermo) developed with TMB substrate (Thermo), and OD450 values were read on a plate reader using SoftMax Pro software. OD450 values for each experiment were transferred to GraphPad Prism 8 for plotting and analysis. Figures 6A-6C As shown in , these results indicate that the anti-CD228 antibody does not cross-react with other transferrin family members.

[0418] Example 6: Cross-reactivity of anti-CD228 antibodies with non-human CD228

[0419] The CD228-negative human melanoma cell line RPMI-7951 was engineered to express cynomolgus macaque CD228 to test the binding of anti-CD228 antibody clones. 50,000 RPMI-7951 cells were incubated with titrations of humanized antibody clones, washed with staining buffer to remove excess antibody, and incubated with 250 ng / mL fluorescent dye-labeled monoclonal antibody against human IgG1 (Thermo) to detect bound antibody. Figure 7A Shown in FIG is mean fluorescence intensity (MFI), as determined by measurement on an Attune NXT flow cytometer (Thermo).

[0420] The reactivity of CD228 antibody clones against recombinant cynomolgus monkey CD228 with a his tag was tested by ELISA. Plates were coated with 1 μg / ml of recombinant his-cynomolgus monkey CD228 in phosphate buffered saline, blocked with 250 μL of Superblock Blocking Buffer (Thermo), the antibody titrated across the plate, bound antibody was detected with HRP-labeled goat anti-human IgG (Sigma), developed with TMB substrate (Thermo), and OD450 values were read on a plate reader using SoftmaxPro software. The results are shown in Figure 7B middle.

[0421] 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 and detected with HRP-labeled goat anti-human IgG secondary reagent (Thermo) developed with TMB substrate (Thermo), and OD450 values were read on a plate reader using SoftMaxPro software. OD450 values for each experiment are shown in Figures 8A-8C middle.

[0422] These results indicate that a subset of antibody clones (OMT30, OMT35, OMT24, OMT8, OMT36, and L235) cross-react with cynomolgus monkey CD228, with minimal cross-reactivity with murine CD228.

[0423] Example 7: Anti-CD228 Antibody Cross-Competition Assay

[0424] Fluorescent dye-labeled humanized monoclonal antibodies specific for human CD228 were evaluated for competition with unlabeled humanized monoclonal antibodies for binding to HT-1080 and SK-MEL-5 tumor cell lines 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 in staining buffer, and incubated with 200 ng / mL of A647-labeled form. Bound fluorescent dye-labeled antibody was determined by flow cytometry on an Attune NXT flow cytometer. Results are presented in Figure 9 The percentage of mean fluorescence intensity compared to a control non-binding human IgG1 (Sigma) pre-incubation is shown. Clones with direct epitope or steric competition showed low percent staining values. These results indicate that antibody clones 28, 32, and 35 may share closely related epitopes, while clones 8, 11, 24, 30, and 36 likely bind to unique CD228 epitopes.

[0425] Example 8: Internalization of Antibody Clones on CD228+ Tumor Cell Lines

[0426] Humanized antibodies specific for human CD228 were evaluated for relative internalization on CD228-expressing melanoma and lung cancer cell lines SK-MEL-5 and Calu-1, respectively. Cells were incubated with 2 μg / ml of 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-labeled anti-human IgG1 antibody (Invitrogen). At the end of the time course, cells were evaluated by FACS on an Attune NXT flow cytometer for the mean fluorescence intensity of surface-bound antibody. Internalization results are shown in Table 1. Figure 10 Shown in are 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.

[0427] Example 9: Expression and analysis of representative fusion proteins

[0428] Representative antibody-lipocalin mutein fusion proteins are generated by fusing a CD228-specific antibody and a CD137-specific lipocalin mutein, such as the lipocalin mutein of SEQ ID NO: 40, via a linker, such as the unstructured (G4S)3 linker of SEQ ID NO: 13, to simultaneously bind CD228 and CD137. Two different exemplary CD228-specific antibodies are used. The first CD228-specific antibody has a heavy chain provided by SEQ ID NO: 75 (or comprises a heavy chain variable domain of SEQ ID NO: 70, or comprises heavy chain CDRs (HCDR1, HCDR2, HCDR3) of SEQ ID NOs: 58-60) and a light chain provided by SEQ ID NO: 76 (or comprises a light chain variable domain of SEQ ID NO: 71, or comprises light chain CDRs (LCDR1, LCDR2, LCDR3) of SEQ ID NOs: 61-63). The second CD228-specific antibody has a heavy chain provided by SEQ ID NO: 78 (or a heavy chain variable domain comprising SEQ ID NO: 72, or a heavy chain CDR (HCDR1, HCDR2, HCDR3) comprising SEQ ID NOs: 64-66) and a light chain provided by SEQ ID NO: 79 (or a light chain variable domain comprising SEQ ID NO: 73, or a light chain CDR (LCDR1, LCDR2, LCDR3) comprising SEQ ID NOs: 67-69). Different forms of the generated fusion proteins are described in Figures 11A-11I are depicted in, including bivalent for CD137 (e.g., Figures 11A-11D ) or tetravalent for CD137 (e.g., as Figures 11E-11H ), or with even higher valence for CD137 (e.g., as 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) are bivalent for CD137, wherein a CD137-specific lipocalin mutein is fused to each heavy chain ( Figure 11A ) or each light chain ( Figure 11B ) at the C-terminus.

[0429] The CD228-specific antibodies and all antibody-lipocalin mutein fusion proteins described in this example have 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 may also be present in all antibodies and fusion proteins described herein, including any one or more of the mutations F234A, L235A, M428L, N434S, M252Y, S254T, and T256E. The F234A and L235A mutations may be introduced to reduce ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010). 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). All antibodies were expressed without a carboxy-terminal lysine to avoid heterogeneity.

[0430] The present disclosure also embodies asymmetric antibody-lipocalin mutein fusion formats, wherein, for example, one light chain of an antibody may be fused to a lipocalin mutein while the other is not.

[0431] The constructs of exemplary fusion proteins were generated by gene synthesis and cloned into mammalian expression vectors. They were then transiently expressed in suspension-adapted CHO-K1 cells. The fusion protein concentration in the cell culture medium was measured by BLItz (ForteBio) using a protein A biosensor for kinetic assays. The titers obtained are summarized in Table 1.

[0432] Table 1: Transient expression titers

[0433]

[0434] For each construct, one liter of the above 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., the monomer content, was determined by HPLC with an Agilent AdvanceBio SEC column ( The determination was performed by analytical size exclusion chromatography using a 400 nm sieve (2.7 mm, 7.8 x 300 mm) and DPBS as running buffer at 0.8 ml / min. The yields and monomer contents after the two-step purification process are summarized in Table 2.

[0435] Table 2: Recovery rate and monomer content of transient expression

[0436]

[0437] Example 10: Binding of fusion proteins to huCD228, cyCD228 or huCD137 determined by surface plasmon resonance (SPR)

[0438] The binding kinetics and affinity of the exemplary fusion proteins for 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).

[0439] According to the manufacturer's instructions, anti-human IgG Fc antibody (GE Healthcare) is fixed to a CM5 sensor chip. Subsequently, in HBS-EP+ buffer, the fusion proteins to be tested (SEQ ID NO:80 and 76, SEQ ID NO:82 and 79, SEQ ID NO:75 and 81 and SEQ ID NO:78 and 83) are captured at the chip surface for 180 seconds at a flow rate of 10 μL / min by anti-human IgG-Fc antibody. After each capture step, the needle is washed. The hIgG1 form of two anti-CD228 antibodies included in the fusion protein (SEQ ID NO:74 / 76 and 77 / 79, respectively) is also tested as a control.

[0440] For affinity determination, dilutions or blanks of recombinant huCD228-His, cyCD228-His or huCD137-His (500nM, 125nM, 31.25nM and 7.8nM) were prepared in HBS-EP+ buffer and applied to the prepared chip surface. Binding assays were performed with a contact time of 180 seconds, a dissociation time of 1200 seconds and a flow rate of 30 μL / min. All measurements were performed at 25°C. The chip surface was regenerated by injecting 3M MgCl2 for 120 seconds. Prior to protein measurement, three start-up cycles were performed for state conditioning purposes. Data were evaluated using Biacore 8K Evaluation software (V1.1.1). Dual references were used and a 1:1 binding model was used to fit the raw data.

[0441] Table 3 summarizes the k values for representative fusion proteins and control antibodies. on 、k off and the resulting equilibrium dissociation constant (K D) values determined. 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 huCD228 and cyCD228 with nanomolar affinities similar to those of the respective antibodies. All bispecific fusion proteins bound CD137 with affinities in the single-digit nanomolar range.

[0442] Table 3: Kinetic constants and affinities of fusion proteins determined by SPR

[0443]

[0444]

[0445] Example 11: Binding of fusion proteins to CD228 or CD137 in enzyme-linked immunosorbent assay (ELISA)

[0446] Enzyme-linked immunosorbent assay (ELISA) was used to determine the binding potency of the exemplary fusion proteins to human CD228 or CD137 and cynomolgus monkey CD228 or CD137.

[0447] Recombinant huCD228-His or cyCD228-His (human or cynomolgus CD228 with a C-terminal polyhistidine tag, R&D Systems or Sino Biologics) was coated on 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 PBS-0.05% T, exemplary 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) and two anti-CD228 antibodies included in the fusion proteins (SEQ ID NOs: 74 / 76 and 77 / 79, respectively) were added to the wells at varying concentrations (maximum concentration: 200 nM) and incubated at room temperature for 1 hour, followed by another washing step. The binding molecules under investigation were detected by incubation with anti-human IgG Fab-HRP (Jackson Laboratory) diluted 1:5000 in PBS-0.1% T-2% BSA. After additional washing steps, fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and fluorescence intensity was measured using a fluorescence microplate reader.

[0448] The same ELISA setup was also used to determine the binding potency of the fusion proteins for CD137, with huCD137-His (human CD137 with a C-terminal polyhistidine tag, R&D Systems) or cyCD137-Fc (cynomolgus monkey CD137 fused to the Fc C-terminus) coated instead on the microtiter plates. The test reagents were titrated similarly, and bound reagents were detected via anti-human IgG Fab-HRP (Jackson Laboratory).

[0449] The results of the exemplary experiments are given in Figures 12A-12D is depicted, together with the fitted curve from the 4PL fit, where EC 50 The value and maximum signal are free parameters, and the slope is fixed to unity. The resulting EC 50 The values are provided in Table 4.

[0450] EC observed for human CD228 and cynomolgus monkey CD228 using 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). 50 EC values of the respective antibodies 50 The curves for the antibodies showed a plateau approximately 2-fold higher, potentially due to the accessibility of the detection antibody, as no such difference was observed when anti-human IgG Fc-HRP antibody was used for detection (data not shown). All fusion proteins showed EC values in the low single-digit nanomolar range for binding to human CD137. 50 The fusion proteins also showed cross-reactivity with cynomolgus monkey CD137. Overall, the EC values of the HC fusion proteins (SEQ ID NOs: 80 and 76, SEQ ID NOs: 82 and 79) were 50 The values were slightly better than those of the corresponding LC fusion proteins (SEQ ID NO: 75 and 81, SEQ ID NO: 78 and 83). 50 value.

[0451] Table 4: ELISA data on CD228 or CD137 binding

[0452]

[0453] Example 12: Simultaneous binding of fusion protein to CD228 and CD137 in ELISA

[0454] To demonstrate simultaneous binding of exemplary fusion proteins to CD228 and CD137, a dual binding ELISA format was used.

[0455] Recombinant huCD228-His (1 μg / mL) in PBS was coated overnight on microtiter plates at 4°C. After each incubation step, the plates were washed five times with 100 μL PBS-0.05% T. The plates were blocked with 2% BSA (w / v) in PBS-0.1% T for 1 hour at room temperature and then washed again. Test fusion proteins and control anti-CD228 antibodies at different concentrations (maximum concentration: 200 nM) were added to the wells and incubated for 1 hour at room temperature, followed by a wash step. Subsequently, biotinylated huCD137-His (huCD137-His-Bio, Sino Biological) was added to PBS-0.1% T-2% BSA at a constant concentration of 1 μg / mL 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 hour. After an additional washing step, fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well, and the fluorescence intensity was detected using a fluorescence microplate reader.

[0456] Figure 13 Dual binding data for 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) are shown in FIG, along with fitting curves from 4PL fitting, where EC 50 The value and maximum signal are free parameters, and the slope is fixed to unity. 50 The values are summarized in Table 5. In contrast to the anti-CD228 antibodies, all bispecific fusion proteins showed clear binding signals, confirming that the fusion proteins were able to simultaneously engage CD228 and CD137. Figure 14 Additional dual binding data for fusion proteins (SEQ ID NOs: 76, 79, 80, 82, 96, 97, 100, 101, 102, and 103) are shown in FIG.

[0457] Table 5: ELISA data for simultaneous target binding of both CD228 and CD137

[0458]

[0459] Example 13: Flow cytometric analysis of fusion protein binding to CD228-positive cells and cells expressing human or cynomolgus monkey CD137

[0460] Target-specific binding of the fusion proteins to cells expressing human CD228 and cells expressing human and cynomolgus monkey CD137 was assessed by flow cytometry (FACS).

[0461] Human melanoma SH-4 cells (ATCC, CRL7724) were used as huCD228-positive cells. CHO cells were stably transfected with human CD137 or cynomolgus CD137 using the Flp-In system (Life technologies) according to the manufacturer's instructions.

[0462] SH-4 cells were cultured in DMEM (PAN Biotech) supplemented with 10% fetal bovine serum (Biochrom). Transfected CHO cells were cultured in Ham's F12 medium (Life technologies) supplemented with 10% fetal bovine serum (Biochrom) and 500 μg / ml hygromycin B (Roth). Cells were cultured in cell culture flasks according to the manufacturer's instructions (37° C., 5% CO atmosphere).

[0463] For flow cytometric analysis, the respective cell lines were incubated with 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 two anti-CD228 antibodies included in the fusion proteins (SEQ ID NOs: 74 / 76 and 77 / 79, respectively), IgG4 isotype controls (SEQ ID NOs: 24 and 25), and monoclonal anti-CD137 antibodies (SEQ ID NOs: 26 and 27), which were detected using fluorescently labeled anti-human IgG antibodies or anti-human NGAL antibodies in FACS analysis as described below:

[0464] Make 5×10 4 Cells / well were incubated in ice-cold PBS (PBS-FCS) containing 5% fetal calf serum for 1 hour. A dilution series of the fusion protein and a control antibody were added to the cells and incubated on ice for 1 hour. The cells were washed twice with PBS and then incubated on ice for 30 minutes with Alexa647-labeled goat anti-hIgG antibody (Life Technologies) or patented Alexa488-labeled rabbit anti-NGAL antibody. The cells were then washed and analyzed using an iQue flow cytometer (Intellicyte Screener). The average geometric fluorescence signal was plotted and fitted using Graphpad software using nonlinear regression (shared bottom, slope = 1).

[0465] The ability of the fusion protein to bind to human CD228 and human CD137 expressed on cells was shown in Figures 15A-15BBinding affinity (EC) of the 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) to cells expressing huCD228 or huCD137 50 ) were in the single-digit (low) nanomolar range, comparable to the respective control antibodies, and the tested fusion proteins fully cross-reacted with cynomolgus monkey CD137 - in contrast to the anti-CD137 antibodies of SEQ ID NOs: 26 and 27 (summarized in Table 6). None of the fusion proteins bound to mock-transfected cells (data not shown).

[0466] Table 6: Binding affinity of fusion proteins to cells expressing human CD228, human CD137 or cynomolgus monkey CD137 ("N / A": not measured; "--": not calculated)

[0467]

[0468] Example 14: CD228-dependent T cell co-stimulation assay using CD137 reporter cells

[0469] The potential of exemplary fusion proteins to induce activation of the CD137 signaling pathway in the presence of CD228 was evaluated using a commercially available dual stably transfected Jurkat cell line expressing CD137 and luc2 genes (a humanized form of firefly luciferase, wherein luc2 expression is driven by an NFκB response element). In this bioassay, CD137 engagement results in CD137 intracellular signaling, leading to NFκB-mediated luminescence.

[0470] Tumor cells expressing high (SH-4), medium (A375), or low (A549) levels of CD228, as well as CD228-negative RPMI-7951 tumor cells, were cultured under standard conditions. The day before the assay, tumor cells were plated at 1.25 x 10 4 Cells / well were plated and allowed to adhere overnight at 37°C in a humidified 5% CO2 atmosphere.

[0471] The next day, 3.75 x 10 4NF-kB-Luc2 / CD137 Jurkat reporter cells were added to each well, followed by the addition of various concentrations ranging from 0.0002 nM to 10 nM of fusion protein, reference anti-CD137 antibodies (Urelumab, SEQ ID NOs: 26 and 27), or IgG4 fusions with CD137-specific lipocalin mutant proteins (SEQ ID NO: 84) included in the fusion protein. The plate was covered with a breathable seal and incubated at 37°C in a humidified 5% CO2 atmosphere. After 4 hours, 30 μL of Bio-Glo was added to each well. TM Reagents were added and the bioluminescent signal was quantified using a luminometer (PHERAstar). A four-parameter logistic curve analysis was performed to calculate the Figure 16E EC in the table 50 Values (shared bottom, fixed slope). To confirm the CD228 dependency of CD137 engagement by the fusion protein, the same experiment was performed in parallel in the presence of CD228 negative cells (RPMI-7951) or in the absence of target cells. The assay was performed in triplicate.

[0472] The results of representative experiments are Figures 16A-16D The data demonstrate that all tested fusion proteins induced dose-dependent CD137-mediated T cell activation in the presence of CD228-expressing tumor cells. Figure 16A ) and A375 cells ( Figure 16B ), all fusion proteins resulted in maximal activation, which was higher than that of the reference anti-CD137 antibody. Overall, the fusion molecules of SEQ ID NOs: 80 and 76 (which are based on the CD228-specific antibodies of SEQ ID NOs: 75 and 76) showed better, i.e. lower, EC values than the other fusion molecules. 50 value. And Figure 16D It was shown that CD137 activation by the fusion protein was CD228-dependent, as activation of NF-κB-Luc2 / CD137 Jurkat cells was not detected in CD228-negative cells (RPMI-7951). In contrast, reference anti-CD137 mAbs (SEQ ID NOs: 26 and 27) showed CD137-mediated T cell co-stimulation regardless of CD228 expression.

[0473] Example 15: Evaluation of PBMC responses to viral peptides in the presence of tumor cells that overexpress or lack CD228 expression

[0474] An assay was performed to evaluate the ability of the fusion protein to co-stimulate 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-cultured with engineered CD228+ or wild-type (CD228-) RPMI-7951 tumor cell lines at 10:1 in RPMI with 10% FCS and provided with viral peptides from CMV, EBV, and influenza viruses (CEF peptides). The bispecific fusion protein 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. For T cell (IFN-γ and TNF-α) and myeloid cell (IL-12 and CXCL10) responses, the expression of IFN-γ and TNF-α in the cells was assessed by immunohistochemistry. Supernatants were evaluated by multiplex cytokine array. Data are available at Figures 17A-17E The fold changes of cytokines relative to untreated control wells are shown in Figure 2. Samples from triplicate test article treatments were combined prior to cytokine measurement. These results show that, compared to antibodies, fusion proteins (e.g., AAF30(HC)) costimulate various innate and adaptive immune cytokines from PBMCs in a CD228 target-dependent and dose-dependent manner in response to viral peptides.

[0475] Example 16: Evaluation of PBMC Cell Responses to Viral Peptides in the Presence of CD228+ Tumor Cells

[0476] To evaluate the activity of the bispecific fusion protein in the antigen recall assay, PBMC (Bloodworks Northwest) cryopreserved from healthy donors were thawed in preheated RPMI 10% FCS, washed, and labeled with 1.5 mL of 10 nM CFSE PBS 3% FCS at room temperature. To quench the labeling reaction, cells were washed 2x with 12 mL of RPMI 10% FCS. The cells were counted and cultured in RPMI-complete medium (10% FCS, 1X Glutamax, 1X MEM NEAA, 1X sodium pyruvate, 1X penicillin / streptomycin (Gibco TM )) and the cell line RPMI-7951 expressing CD228 CD228 (ATCC) (modified to express human CD228) were mixed 10:1 and plated in non-adherent 96-well round-bottom plates (Sbio) at 1.5 x 10 5 CEF peptide was added to a final concentration of 100 ng / mL, and fusion protein bispecific and controls were added in triplicate at equimolar titrations. The assay was allowed to incubate for 5 days at 37°C under 5% CO2. Figure 18 、 Figure 19 and Figure 20 Representative examples of CD8 T cells, NK cells, and CD8 T cell / Treg ratio calculations from antigen recall assays in co-culture with the CD228-engineered RPMI-7951 cell line are shown, respectively. Figure 21 Representative examples of NK cell and CD8 T cell / Treg ratio calculations from antigen recall assays are shown. These results show that, compared to 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-dependent and dose-dependent manner.

[0477] Example 17: Evaluation of PBMC cytokine responses to viral peptides in the presence of CD228+ tumor cells

[0478] To evaluate the activity of the bispecific fusion protein in the antigen recall assay, PBMC (Bloodworks Northwest) cryopreserved from healthy donors were thawed in preheated RPMI 10% FCS, washed, and labeled with 1.5 mL of 10 nM CFSE PBS 3% FCS at room temperature. To quench the labeling reaction, cells were washed 2x with 12 mL of RPMI 10% FCS. The cells were counted and cultured in RPMI-complete medium (10% FCS, 1X Glutamax, 1X MEM NEAA, 1X sodium pyruvate, 1X penicillin / streptomycin (Gibco TM )) and the cell line RPMI-7951 expressing CD228 CD228 (ATCC) (modified to express human CD228), CALU-1 cells (ATCC), or H3677 cells (Seagen) were mixed at a ratio of 10:1 and plated at 1.5 x 10 5 Cells / well were distributed. CEF peptide was added to a final concentration of 100 ng / mL, and fusion protein bispecific and controls were added in triplicate at equimolar titrations. The assay was allowed to incubate for 5 days at 37°C under 5% CO2. At the end of the assay, the plates were spun down and the supernatant was collected for cytokine assessment. Cytokine use MAPHuman CD8+ T Cell Magnetic Bead Panel Premixed17Plex was used for measurement and Read out on the system. Figures 22A-22E Representative fold change averages for cytokines across three CD228-expressing cell lines are shown. Data were exported and analyzed in Microsoft Excel.These data highlight consistent changes in cytotoxic effector molecules and different cytokines upon antigen recall in the presence of the bispecific fusion protein.

[0479] Example 18: Evaluation of T cell activation in the presence of tumor cells that highly express or lack CD228 expression

[0480] Further T cell assays were used to evaluate the ability of the fusion protein to costimulate T cell activation in a CD228 target-dependent manner. In the presence of tumor cell lines with different CD228 expression levels, the fusion protein was applied to anti-CD3 stimulated T cells at different concentrations. The tumor cell lines tested included SH-4 (CD228 high), Calu-1 (CD228 high), SK-MEL-24 (CD228 high) and RPMI-7951 (CD228 negative). The IL-2 secretion level in the supernatant was used as a readout.

[0481] Following the Biochrom protocol, PBMCs from healthy volunteer donors were isolated from buffy coats by centrifugation through a Ficoll density gradient (Biocoll, 1.077 g / mL, Biochrom). Following the manufacturer's instructions, T lymphocytes were further purified from PBMCs by magnetic cell sorting using a pan-T cell purification kit (Miltenyi Biotec GmbH). Purified pan-T cells were resuspended in a buffer consisting of 90% FCS and 10% DMSO, immediately frozen, and stored in liquid nitrogen until further use.

[0482] For the assay, T cells were thawed and cultured in culture medium (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1% penicillin-streptomycin (Life Technologies) at 37° C. in a humidified 5% CO 2 atmosphere for 16 hours.

[0483] The following procedure was performed in triplicate for each experimental condition: Flat-bottom tissue culture plates were pre-coated with 0.25 μg / mL anti-human CD3 antibody at 37°C for 1 hour and then washed twice with PBS. 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. Mitomycin-treated tumor cells were then washed twice with PBS and plated at 2.5 x 10 4Cells / well were plated in culture medium to allow overnight attachment at 37° C. in a humidified 5% CO 2 atmosphere. Target cells had been previously grown under standard conditions, detached using Accutase (PAA Laboratories), and resuspended in culture medium.

[0484] On the second day, after washing the plates twice with PBS, 1.25 x 10 4 T cells / well were added to the tumor cells. A dilution series of 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), reference anti-CD137 antibodies (SEQ ID NOs: 26 and 27), IgG4 fusions with CD137-specific lipocalin muteins (SEQ ID NO: 84) included in the fusion protein, or isotype controls (SEQ ID NOs: 24 and 25) ranging from 0.0002 nM to 10 nM was added to the corresponding wells. The plates were covered with a breathable seal and incubated at 37° C. in a humidified 5% CO atmosphere for 3 days.

[0485] After 3 days of co-culture, 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. Subsequently, the wells were washed 5 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), the assay supernatants and a series of concentrations of IL-2 standards diluted in culture medium were transferred to separate wells 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 protein (Mesoscale Discovery) in PBS-T containing 0.5% casein was added and incubated for 1 hour at room temperature. After washing, 25 μL of reading buffer (Mesoscale Discovery) was added to each well and the resulting electrochemiluminescence (ECL) signal was detected by a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.

[0486] Representative data are shown in Figures 23A-23DCo-culture of 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, SEQ ID NOs: 82 and 79, SEQ ID NOs: 75 and 81, or SEQ ID NOs: 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 greater than the increase induced by the reference anti-CD137 antibodies (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 (SEQ ID NO: 84) included in the fusion protein. In addition, co-culture with CD228-negative RPMI-7951 cells did not increase IL-2 secretion levels with any of the fusion proteins, but did increase IL-2 secretion levels with the reference anti-CD137 antibodies (SEQ ID NOs: 26 and 27). Figure 23D ).

[0487] Similar assays were performed using CD228+ Calu-1 cells or CD228- SK-BR-3 cells and different but overlapping sets of fusion proteins, and Figures 24A-24B IL-2 secretion in response to stimulation by the fusion protein and urerulumab is shown. These results show that, compared to antibodies, fusion proteins (e.g., AAF35(HC)) costimulate T cell activation in a CD228 target-dependent and dose-dependent manner.

[0488] The data indicate that the functional activity of the bispecific fusion protein, measured by its ability to activate T cells or increase IL-2 secretion, is CD228 dependent.

[0489] Example 19: Evaluation of T cell cytokines produced in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines

[0490] Experiments were performed to evaluate the effect of the fusion protein on the production of cytokines and soluble 4-1BB (sCD137) from T cells that received direct T cell receptor stimulation from tumor cells expressing CD228. Healthy donor PBMCs were co-cultured with CD228+CALU-1 tumor cells (10:1 in RPMI 10% FCS) that were engineered to express surface anti-CD3 scFv to elicit 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 in 5% CO2, supernatants were collected and the cells were plated using a flow cytometry system. The multiplex array measured soluble cytokines IL-2, IL-13, and sCD137. Samples from triplicate test article treatments were pooled prior to cytokine measurement. Results are shown in Figures 25A-25C , which demonstrated that the fusion protein potentiated cytokines from direct T cell-tumor cell interactions.

[0491] Example 20: Evaluation of CD8 T cell proliferation in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines

[0492] Experiments were performed to evaluate the effect of fusion protein bispecifics on cytotoxic T cells that received direct T cell receptor stimulation from tumor cells expressing CD228. Healthy donor PBMCs were CFSE labeled and co-cultured with CD228+CALU-1 tumor cells (10:1) that were engineered to express surface anti-CD3 scFv to elicit T cell receptor engagement. Titrations of bispecific fusion proteins or controls were added in triplicate to 96-well round-bottom plates. After incubation for 72 hours at 37°C in 5% CO2, the plates were washed and stained with live / dead cell viability dyes and antibodies specific for CD3, CD4, CD8, and CD228. The plates were stained with Attune TM NxT flow cytometry was used to assess the proliferation of live CD8 T cells and CD228+ tumor cells. Figure 26 The proportion of CD8 T cells with diluted CFSE relative to wells with no added treatment is shown. Figure 27 The percentage of remaining viable tumor cells relative to wells without treatment is shown, reflecting tumor cell killing. These results show that in the presence of CD228, fusion proteins (e.g., AAF30(HC)) costimulate CD8+ T cell proliferation and tumor cell killing in a dose-dependent manner compared to antibodies.

[0493] Example 21: Storage stability evaluation of fusion protein

[0494] To evaluate storage stability, exemplary 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) 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 then incubated at 37°C for one week or stored at -20°C. Subsequently, the activity of the fusion protein was determined by a CD137 reporter cell assay as described in Example 14 using SH-4 cells expressing CD228 as target cells. Fusion proteins diluted in PBS, 50% HPL, or 50% MPL shortly before the assay served as references, and fusion proteins stored at -20°C throughout served as untreated references. 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 middle.

[0495] The fusion molecules of SEQ ID NOs: 80 and 76 and SEQ ID NOs: 75 and 81 (which are all based on the CD228-specific antibodies of SEQ ID NOs: 75 and 76) showed excellent stability under all tested conditions ( Figure 28A and 28C Fusion molecules of SEQ ID NOs: 82 and 79 and SEQ ID NOs: 78 and 83 (all based on CD228-specific antibodies of SEQ ID NOs: 78 and 79) showed excellent stability in PBS and 50% human plasma, but slightly reduced stability in 50% mouse plasma, as evidenced by an approximately threefold higher EC 50 and a plateau about 30% lower (respectively Figure 28B and 28D ).

[0496] Example 22: Pharmacokinetics of fusion protein in mice

[0497] The pharmacokinetic analysis of representative fusion proteins (SEQ ID NO:80 and 76, SEQ ID NO:82 and 79, SEQ ID NO:75 and 81 or SEQ ID NO:78 and 83) is performed in mice. Male CD-1 NUDE mice (3 mice / time point at approximately 5 weeks of age; Charles River Laboratories, Research Models and Services, Germany GmbH) are injected into the tail vein with a fusion protein of 10 mg / kg dosage. The test article is administered using a volume of 5 mL / kg as a single push. Plasma samples from mice are obtained at the time points of 5 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 hours, 4 days, 7 days, 14 days, 21 days and 28 days. Enough whole blood obtained under isoflurane anesthesia is collected to obtain each animal and at least 50 μL Li-heparin plasma at a time.

[0498] Fusion protein levels were detected in a free PK assay using a sandwich ELISA that detected the complete bispecific construct via targets CD228 and CD137. To this end, huCD228-His (human CD228 with a C-terminal polyhistidine tag) was dissolved in PBS (1 μg / mL) and coated overnight on a microtiter plate at 4°C. After each incubation step, the plate was washed five times with 80 μL PBS supplemented with 0.05% (v / v) Tween 20. The plate was blocked for 1 hour at room temperature with PBS / BSA / Tween (PBS containing 2% BSA (w / v) and 0.1% (v / v) Tween 20) and subsequently washed. Plasma samples were diluted to 20% plasma concentration in PBS / BSA / Tween / standard CD-1 mouse plasma, added to the wells, and incubated for 1 hour at room temperature. This was followed by another washing step. After 1 hour incubation with a mixture of biotinylated human CD137 and streptavidin SULFO-TAG (Mesoscale Discovery) diluted in PBS containing 2% BSA (w / v) and 0.1% (v / v) Tween 20, the binding reagent under study was detected. After an additional washing step, 35 μL of reading buffer was added to each well, and the electrochemical (ECL) signal of each well was read using a Mesoscale Discovery reader. The level of parental anti-CD228 antibody was detected in a similar manner via target CD228 and anti-human IgG Fc antibody (GE Healthcare). Data were transferred to Excel for analysis and quantification. A calibration curve with standard protein dilution was prepared to reversely calculate the concentration in plasma. Non-compartmental analysis was performed with WinNonLin software.

[0499] Figures 29A-29B The plasma concentrations of 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 over time are shown (29A: average concentration; 29B: average concentration for C max Table 7 shows the terminal half-lives of the parental anti-CD228 antibodies (SEQ ID NOs: 74 and 76 and SEQ ID NOs: 77 and 79) as reference.

[0500] The data confirmed that the fusion protein had a long, antibody-like terminal half-life in mice. Since the assay used to determine the plasma concentration of the fusion protein requires retention of binding activity against both CD228 and CD137, the results also confirmed that the bispecific molecule remained intact over a 28-day period.

[0501] Table 7: Terminal half-life in mice determined using non-compartmental analysis

[0502] SEQ ID NO Terminal half-life [hours] 80 and 76 313 82 and 79 236 75 and 81 253 78 and 83 231 74 and 76 185 77 and 79 157

[0503] Example 23: Pharmacokinetics of fusion protein in cynomolgus monkeys

[0504] 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 pharmacokinetic differences in cynomolgus monkeys. Animals received a single dose of the fusion bispecific at a dose of 1 mg / kg or 6 mg / kg. Figure 30 As shown in , the bispecific fusion proteins exhibited similar pharmacokinetic profiles with slight differences after day 14, which may be due to anti-drug antibody responses.

[0505] Example 24: Evaluation of in vivo activity of fusion proteins in a humanized xenograft model

[0506] To evaluate the in vivo activity of the bispecific fusion proteins, humanized xenograft models using the CD228+ melanoma cell lines CALU-1 (ATCC) and H3677 (SGEN, in-house) were performed. Tumor cells were implanted into 25% (Corning) and monitored until tumors reached 100 mm 3The average volume of , at this time, healthy donor peripheral blood mononuclear cells (PBMC) were adoptively transferred via tail vein injection to provide a source of human T cells. The mice were then dosed with equimolar amounts of bispecific fusion protein (10 mg / kg), antibody (non-bispecific antibody, anti-PD-1 and / or anti-4-1BB) or control (8 mg / kg) at 5-day intervals, and tumor growth was monitored. The growth curve across the Calu-1 study is shown in Figure 31A The final tumor volumes at the end of the study (day 80) are shown in Figure 31B Growth curves across the H3677 study are shown in Figure 31C The final tumor volumes at the end of the study (day 22) are shown in Figure 31D The statistics presented are the results of Tukey's analysis. These results show that fusion proteins (e.g., AAF30(HC)) reduce CD228+ tumor cell growth in vivo compared to antibodies.

[0507] To assess intratumoral PD effects from bispecific fusion-treated animals in a humanized CD228+Calu-1 xenograft model, tumors were harvested and processed into single-cell suspensions for staining of tumor and immune cell populations and analysis by flow cytometry. Figure 32A Shown are the ratios of CD8+ T cells to tumor cells, as determined by the counts of CD8+ T cells divided by the counts of CD228+ non-immune cells. Figure 32B Changes in the CD8+ / CD4+ T cell ratio are shown. Figure 32C The proportion of CD8+ T cells in active degranulation at the end of the study, as determined by surface expression of CD107a, is shown. The dashed line in the figure indicates the CD8+ / CD4+ T cell ratio in a resting PBMC sample (i.e., non-tumor model) from the same donor ( Figure 32B ) or the percentage of CD8 T cells expressing CD107a ( Figure 32C These data demonstrate that the expansion and activation of cytotoxic CD8+ T cells is enhanced after treatment with the bispecific fusion protein compared to the control. The statistical data presented are the results of Tukey's analysis.

[0508] To assess intratumoral PD effects from bispecific fusion-treated animals in a separate humanized CD228+Calu-1 xenograft model, tumors were harvested and processed into single-cell suspensions for staining of tumor and immune cell populations and analysis by flow cytometry. Figure 33A The ratio of CD8+ T cells to CD4+ T cells is shown. Figure 33BShown are changes in intracellular TCF1 expression. These data confirm that the enhanced expansion of cytotoxic CD8+ T cells and their differentiation into cells with more stem cell-like properties indicate greater anti-tumor potential, as identified by the expression of the transcription factor TCF1 after treatment with a bispecific fusion protein targeting 4-1BB. The statistical data provided are the results of Tukey's analysis.

[0509] Example 25: Evaluation of CD8 T cell proliferation in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines

[0510] As in Example 20, an experiment was performed to evaluate the effect of the fusion protein bispecific on cytotoxic T cells that received direct T cell receptor stimulation from tumor cells expressing CD228. Healthy donor PBMCs were CFSE labeled and co-cultured with CD228+A2058 and SK-MEL-5 melanoma cells (10: 1), which were engineered to express surface anti-CD3 scFv to trigger T cell receptor engagement. Titrations of bispecific fusion proteins or controls were added in triplicate to 96-well round-bottom plates. After incubation for 72 hours at 37°C in 5% CO2, the plates were washed and stained with antibodies specific for CD3, CD4, and CD8. The plates were stained with Attune TM NxT flow cytometry was used to assess the proliferation of live CD8 T cells. Figure 34A and 34B The proportion of dividing CD8 T cells relative to wells with no treatment added is shown. These results show that in the presence of CD228, fusion proteins (e.g., AAF30(HC)) co-stimulate CD8+ T cell proliferation in a dose-dependent manner compared to antibodies, control fusion proteins, and Ureluzumab.

[0511] Example 26: Evaluation of CD8 T cell proliferation and mitochondrial function in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines

[0512] As in Example 20, an experiment was performed to evaluate the effect of the fusion protein bispecific on cytotoxic T cells that received direct T cell receptor stimulation from tumor cells expressing CD228. Healthy donor PBMCs were co-cultured with CD228+CALU-1 lung cancer cells (2.5:1) that were engineered to express surface anti-CD3 scFv to elicit T cell receptor engagement. Titrations of bispecific fusion proteins or controls were added in quadruplicate to 96-well round-bottom plates. After incubation for 6 days at 37°C in 5% CO2, the plates were washed and stained with antibodies specific for CD2, CD4, and CD8. The plates were stained with Attune TMNxT flow cytometry was used to assess the expansion of live CD8 T cells. Figure 35A The CD8 T cell counts in the treated wells relative to the untreated wells are shown. To measure the mitochondrial content of CD8 T cells, cells were immunostained with 75 nM MitoSpy TM Green FM staining was performed at 37°C in 5% CO2 for 20 min. Figure 35B Shown by MitoSpy TM The mean fluorescence intensity (MFI) of green FM staining indicates the mitochondrial content of CD8 T cells. Figure 35C The percentage of CD8 T cells with depolarized mitochondria is shown, as determined by staining with the polarization-dependent dye MitoSpy TM Orange CMTMros (25 nM, 20 min at 37°C in 5% CO2) and MitoSpy TM Green FM (75 nM, 20 minutes at 37 ° C in 5% CO2) staining was determined. These results show that in the presence of CD228, the fusion protein (e.g., AAF30 (HC)) co-stimulated CD8+ T cell proliferation in a dose-dependent manner compared to a non-bispecific CD228 binding antibody or usrulumab (20H4.9) and measurably improved the metabolic fitness of activated cytotoxic T cells.

[0513] Example 27: Preparation and characterization of T cell exhaustion model

[0514] To generate a model of functionally exhausted cytotoxic T cells that receive direct T cell receptor stimulation from CD228-expressing tumor cells, CALU-1 lung cancer cells were engineered to express a membrane-bound anti-CD3 scFv fragment, and healthy donor PBMCs were serially passaged (P0-P4) on a CALU-1 cell lawn to drive progressive T cell dysfunction. Figure 36A A schematic diagram of the generation of such functionally exhausted cytotoxic T cells is shown. Cells recovered and banked from serial passages were analyzed for their ability to proliferate, secrete interferon gamma (IFN-γ), and kill tumor cells, and Figure 36B Figure 2 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 co-culture with anti-CD3 scFv-engineered CALU-1 cells. Passage 4 (P4) T cells exhibited a significant reduction in their ability to divide, secrete cytokines, and kill tumor cells.

[0515] 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. The two states resemble progenitor (P4Tpex_like) and terminally exhausted (P4Texterm_like) CD8+ T cells. The average gene expression per state was scaled for each dataset and then displayed in Figure 37A In, it is a heat map of P4 cells or exhausted CD8+ T cells from the pan-cancer tumor-infiltrating lymphocyte (TIL) map, as described in Zheng et al., Pan-cancer single-cell landscape of tumor-infiltrating T cells, Science 374:6574 (2021). In addition, Figure 37B The expression levels of TCF7 (TCF1) (left), HAVCR2 (TIM-3) (center), and TNFRSF9 (CD137) (right) in Tpex-like and P4 Texterm-like cells are shown. The similarity of Tpex-like and Texterm-like cells to true TIL CD8+ T cells was evaluated by hierarchical clustering of marker genes of exhausted T cell status, and the results confirmed significant overlap in the gene signatures shared by Tpex-like and P4 Texterm-like cells and true exhausted TILs.

[0516] Example 28: Evaluation of functional repriming of exhausted CD8 T cells in co-culture with anti-CD3 scFv engineered CD228-expressing tumor cell lines

[0517] Experiments were conducted to evaluate the effect of the fusion protein bispecific on functionally exhausted cytotoxic T cells. The exhausted T cells from Example 27 were collected, CFSE labeled, and co-cultured with CD228+ anti-CD3 scFvCALU-1 tumor cells (1: 1) again. The titration of the bispecific fusion protein, the co-stimulatory antibody for CD28 (clone CD28.2, BioLegend, San Diego, CA, catalog #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 incubation for 96 hours at 37°C in 5% CO2, the plate was washed and stained with antibodies specific for CD3, CD4, and CD8. The plate was stained with Attune TM NxT flow cytometry was used to assess the proliferation of live CD8 T cells. Figure 38The fold increase in CFSE-low (divided) CD8 T cell counts in the treated wells relative to the untreated wells is shown (dashed line = 1). These results show that 4-1BB agonists, fusion proteins (e.g., AAF30 (HC)) and 20H4.9 re-stimulate functionally exhausted CD8+ T cell proliferation in a dose-dependent manner, while the anti-PD-1 antibody nivolumab and agonist antibodies against CD28 do not increase proliferation alone. In the presence of CD228-expressing CALU-1 tumor cells, AAF30 (HC) drives increased co-stimulation compared to 20H4.9, and the proliferative effects of AAF30 (HC) and 20H4.9 on exhausted cytotoxic T cells are further amplified by blocking the PD-1 / PD-L1 pathway with the anti-PD-1 antibody nivolumab, confirming the complementarity of these mechanisms of action and supporting the clinical combination of fusion protein bispecifics with agents that block the PD-1 / PD-L1 pathway (e.g., anti-PD-1 or anti-PD-L1 antibodies).

[0518] The embodiments described illustratively in this article may be appropriately practiced in the absence of any one or more elements, one or more limitations that are not specifically disclosed in this article. Therefore, for example, the terms "comprise", "include", "contain" etc. should be understood broadly without limitation. In addition, the terms and expressions adopted in this article have been used as descriptive rather than restrictive terms, and when using such terms and expressions, it is not intended to exclude any equivalents of the features shown and described or their parts, but it should be recognized that various modifications are possible within the scope of the present invention's claims. Therefore, it should be understood that although the embodiments herein have been specifically disclosed by preferred embodiments and optional features, those skilled in the art may adopt their equivalents, modifications and variations, and 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 described herein are incorporated by reference in their entirety to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference herein. In addition, when the definition or use of a term in a reference incorporated herein by reference is inconsistent or contrary to the definition of the term provided herein, the definition of the term provided herein is applicable, and the definition of the term in the reference is not applicable. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This includes the general description of the invention with any subject proviso or negative limitation removed from the generic description, whether or not the removed material is specifically recited herein. In addition, where features are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments will be apparent from the following claims.

[0519] VII. Non-patent References

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[0571] VIII. Sequence Listing

[0572] Exemplary Sequence Listing:

[0573]

[0574]

[0575]

[0576]

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[0640]

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[0646]

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[0667]

Claims

1. A fusion protein capable of binding to both CD137 and CD228, wherein the fusion protein comprises at least two subunits in any order, wherein the first subunit comprises an antibody or antigen binding domain thereof specific for CD228, and wherein the second subunit comprises a lipocalin mutein specific for CD137. 2 . The fusion protein of claim 1 , further comprising a third subunit comprising a lipocalin mutant protein specific for CD137.

3. The fusion protein according to claim 1 or 2, wherein the fusion protein can be expressed with a K of 150 nM or less. D Values for binding to CD228.

4. The fusion protein according to any one of claims 1 to 3, wherein the fusion protein can be expressed with a K of 7 nM or less. D Values for binding to CD137.

5. The fusion protein according to claim 3 or 4, wherein the K D The values were determined by surface plasmon resonance (SPR) measurement.

6. The fusion protein according to any one of claims 1 to 5, wherein the fusion protein is capable of activating an EC of 1.5 nM or less. 50 Values for binding to CD228.

7. The fusion protein according to any one of claims 1 to 6, wherein the fusion protein is capable of inhibiting the expression of β-actin with an EC of 4 nM or less. 50 Values for binding to CD137.

8. The fusion protein according to claim 6 or 7, wherein the EC 50 The values were determined by enzyme-linked immunosorbent assay (ELISA).

9. The fusion protein of any one of claims 1 to 8, wherein the fusion protein binds to cynomolgus monkey CD228.

10. The fusion protein of any one of claims 1 to 9, wherein the fusion protein binds to cynomolgus monkey CD137.

11. The fusion protein according to any one of claims 1 to 10, wherein the fusion protein is capable of activating an EC of 10 nM or less. 50 Values represent simultaneous binding to CD137 and CD228 as measured in ELISA assays.

12. The fusion protein according to any one of claims 1 to 11, wherein the fusion protein is capable of activating an EC of 30 nM or less. 50 Values are in combination with CD137 expressed on cells, as measured in flow cytometric analysis.

13. The fusion protein according to any one of claims 1 to 12, wherein the fusion protein is capable of activating an EC of 10 nM or less. 50 Values are in combination with CD228 expressed on cells, as measured in flow cytometric analysis.

14. The fusion protein according to any one of claims 1 to 13, wherein the fusion protein is capable of binding to tumor cells expressing CD228.

15. The fusion protein according to any one of claims 1 to 14, wherein the fusion protein is capable of stimulating T cell proliferation and / or T cell response.

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

17. The fusion protein according to any one of claims 1 to 16, wherein the fusion protein is capable of stimulating the proliferation of exhausted CD8+ T cells and / or synergistically stimulating the proliferation of exhausted CD8+ T cells with anti-PD-1 or anti-PD-L1 antibodies. 18 . The fusion protein according to claim 1 , wherein the fusion protein is capable of inducing an increase in the mitochondrial content of CD8+ T cells and / or reducing the mitochondrial depolarization of CD8+ T cells.

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

20. The fusion protein according to any one of claims 1 to 19, wherein the fusion protein is capable of inducing increased secretion of cytotoxic factors.

21. The fusion protein of any one of claims 1 to 20, wherein the fusion protein is capable of costimulating a T cell response in a CD228-dependent manner.

22. The fusion protein of any one of claims 1 to 21, wherein the fusion protein is capable of co-stimulating a T cell response in a tumor microenvironment.

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

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

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 fusion protein of claim 25, wherein the amino acid sequence of the lipocalin mutein comprises one or more of the following mutated amino acid residues at one or more of 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 corresponding to the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1): Ala 5→Val or Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Thr42→Ser; Gly 46→Asp; Lys 52→Glu; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys 65→Arg or Asn; Thr 71→Ala; Val 85→Asp; Lys 94→Arg or Glu; Cys101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys114→Trp; Lys 121→Glu; Ala 133→Thr; Arg 148→Ser; Ser 150→Ile; and Cys 153→Ser.

27. The fusion protein of claim 25 or 26, wherein the amino acid sequence of the lipocalin mutein comprises one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1): (a) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg111 → Pro; Lys 114 → Trp; and Cys 153 → Ser; (b) Ala 5 → Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys65 → Arg; Val 85 → Asp; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr; and Cys 153 → Ser; (c) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Asn; Lys94 → Arg; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr; and Cys 153 → Ser; (d) Ala 5→Val; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys65→Arg; Lys 94→Glu; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys 121→Glu; Ala 133→Thr; and Cys 153→Ser; (e) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Thr 42→Ser; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Cys101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ser 150→Ile; and Cys 153→Ser; (f) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Lys 52→Glu; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Thr71→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ala 133→Thr; Arg 148→Ser; Ser 150→Ile; and Cys 153→Ser; and (g)Ala 5→Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Gly 46→Asp; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Thr 71→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ser 150→Ile; and Cys 153→Ser.

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

29. The fusion protein according to 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: 32-38, or a fragment or variant thereof.

30. 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-associated lipocalin (hNGAL) (SEQ ID NO: 2).

31. The fusion protein of claim 30, wherein the amino acid sequence of the lipocalin mutein comprises one or more of the following mutated amino acid residues at 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-associated lipocalin (hNGAL) (SEQ ID NO: 2): Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg or Lys; Gln 49 → Val, Ile, His, Ser, or Asn; Tyr 52 → Met; Asn65 → Asp; Ser 68 → Met, Ala, or Gly; Leu 70→Ala, Lys, Ser or Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Met, Arg, Thr or Asn; Trp79→Ala or Asp; Arg 81→Met, Trp or Ser; Phe83→Leu; Cys 87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; and Lys134→Tyr.

32. 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-associated lipocalin (hNGAL) (SEQ ID NO: 2).

33. The fusion protein of claim 32, wherein the amino acid sequence of the lipocalin mutein comprises one or more of the following mutated amino acid residues at 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 corresponding to mature hNGAL (SEQ ID NO: 2): Gln 20→Arg; Asn 25→Tyr or Asp; Gln 28→His; Val 33→Ile; Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Glu 44→Val or Asp; Gln 49→His; Tyr52→Ser or Gly; Lys 59→Asn; Ser 68→Asp; Leu 70→Met; Phe 71→Leu; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln or His; Tyr 78→His; Trp 79→Ile; Ile 80→Asn; Arg 81→Trp or Gln; Thr 82→Pro; Cys 87→Ser; Phe 92→Leu or Ser; Asn 96→Phe; Lys 98→Arg; Tyr100→Asp; Pro 101→Leu; Leu 103→His or Pro; Phe 122→Tyr; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly.

34. The fusion protein according to any one of claims 30 to 33, wherein the amino acid sequence of the lipocalin mutein comprises one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): (a) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Lys; Gln49→Asn; Tyr 52→Met; Ser 68→Gly; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Thr; Trp 79→Ala; Arg 81→Ser; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; and Lys 134→Tyr; (b)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln 49→Ile;Tyr 52→Met;Asn 65→Asp;Ser 68→Met;Leu 70→Lys;Arg 72→Asp;Lys 73→Asp;Asp 77→Met;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr; (c)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Ala;Leu 70→Ala;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr; (d)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Ala;Leu 70→Ala;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr; (e)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln49→Ser;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Ser;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Met;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr; (f)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln49→Val;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Arg;Trp 79→Asp;Arg 81→Ser;Cys 87→Ser;Leu 94→Phe;Asn 96→Lys;Tyr 100→Phe;Leu103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr; (g)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln 49→His;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Ser;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr; (h)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Ser;Phe 83→Leu;Cys 87→Ser;Leu 94→Phe;Asn 96→Lys;Tyr100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr; (i) Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln49→Ser;Tyr 52→Met;Asn 65→Asp;Ser 68→Ala;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Asn;Trp 79→Ala;Arg 81→Ser;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr. (j)Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr52→Ser;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly; (k)Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Ser; Ser 68→Asp; Leu 70→Met; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln; Trp 79→Ile; Arg 81→Trp; Phe 92→Leu; Asn96→Phe; Lys 98→Arg; Tyr 100→Asp; Pro 101→Leu; Leu 103→His; Lys125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly; (l)Asn 25→Tyr; Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Gly; Ser 68→Asp; Leu 70→Met; Phe 71→Leu; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln; Trp 79→Ile; Arg81→Gln; Phe 92→Ser; Asn 96→Phe; Tyr 100→Asp; Leu 103→His; Lys125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly; (m)Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Gly; Ser 68→Asp; Leu 70→Met; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln; Tyr 78→His; Trp 79→Ile; Arg 81→Trp; Phe92→Leu; Asn 96→Phe; Tyr 100→Asp; Leu 103→His; Lys 125→Ser; Ser127→Ile; Tyr 132→Trp; and Lys 134→Gly; (n)Asn 25→Asp; Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Gly; Ser 68→Asp; Leu 70→Met; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln; Trp 79→Ile; Arg 81→Trp; Phe92→Leu; Asn 96→Phe; Tyr 100→Asp; Leu 103→His; Lys 125→Ser; Ser127→Ile; Tyr 132→Trp; and Lys 134→Gly; (o)Val 33→Ile;Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln49→His;Tyr 52→Gly;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Phe 92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly; (p)Gln 20→Arg;Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Glu 44→Val;Gln 49→His;Tyr 52→Gly;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg81→Trp;Phe 92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Phe122→Tyr;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly; (q)Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Ser;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Ile 80→Asn;Arg 81→Trp;Thr82→Pro;Asn 96→Phe;Tyr 100→Asp;Pro 101→Leu;Leu 103→Pro;Lys125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly; (r)Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Gly;Lys 59→Asn;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Phe92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Lys 125→Ser;Ser127→Ile;Tyr 132→Trp;and Lys 134→Gly;and (s) Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Glu 44→Asp; Gln 49→His; Tyr 52→Ser; Ser 68→Asp; Leu 70→Met; Phe 71→Leu; Arg 72→Leu; Lys 73→Asp; Asp 77→His; Trp 79→Ile; Arg81→Trp; Phe 92→Leu; Asn 96→Phe; Tyr 100→Asp; Leu 103→His; Lys125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly.

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 the group consisting of SEQ ID NOs: 39-57.

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: 39-57, or a fragment or variant thereof.

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

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

40. according to the fusion protein described in any one of claim 1 to 39, wherein the third subunit is connected 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 at the N-terminus.

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

43. The fusion protein of any one of claims 1 to 42, wherein the antibody or antigen binding domain thereof comprises: 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 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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 VH, which includes (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 273, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 274, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 275, and VL, which includes (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 279, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 280, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

281.

44. The fusion protein of claim 43, wherein the antibody or antigen binding domain thereof comprises: 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; 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; 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 VH, which includes (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, and VL, which includes (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 259, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 260, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

261.

45. The fusion protein of claim 43 or 44, wherein the antibody or antigen binding domain thereof comprises: 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; or VH, which includes (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 213, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 214, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 215, and VL, which includes (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 219, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 220, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:

221.

46. The fusion protein of claim 43, wherein the antibody or antigen binding domain thereof comprises: 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 that has at least 95% sequence identity to SEQ ID NO: 262, and a VL comprising an amino acid sequence that has 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.

47. The fusion protein of any one of claims 43, 44, and 46, wherein the antibody or antigen binding domain thereof comprises: 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.

48. The fusion protein of any one of claims 43 to 47, wherein the antibody or antigen binding domain thereof comprises: 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.

49. The fusion protein of claim 43 or 46, wherein the antibody or antigen binding domain thereof comprises: 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.

50. The fusion protein of any one of claims 43, 44, 46, 47, and 49, wherein the antibody or antigen binding domain thereof 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.

51. The fusion protein of any one of claims 43 to 50, wherein the antibody or antigen binding domain thereof 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.

52. The fusion protein of any one of claims 43, 46, and 49, wherein the antibody or antigen binding domain thereof comprises: 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; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 146, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 148; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 166, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 168; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 186, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 188; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 206, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 208; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 228; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 246, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 248; a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 266, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 268; or A HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 286, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

288.

53. The fusion protein of any one of claims 43, 44, 46, 47, 49, 50, and 52, wherein the antibody or antigen binding domain thereof comprises: a HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 228; or A HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 266, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

268.

54. The fusion protein of any one of claims 43 to 53, wherein the antibody or antigen binding domain thereof comprises: A HC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 226, and a LC comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

228.

55. The fusion protein of any one of claims 43, 46, 49, and 52, wherein the antibody or antigen binding domain thereof comprises: 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 The HC comprises the amino acid sequence of SEQ ID NO:286, and the LC comprises the amino acid sequence of SEQ ID NO:

288.

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

268.

57. The fusion protein of any one of claims 43 to 56, wherein the antibody or antigen binding domain thereof comprises: The HC comprises the amino acid sequence of SEQ ID NO:226, and the LC comprises the amino acid sequence of SEQ ID NO:

228.

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

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

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

61. according to the fusion protein described in any one of claim 1 to 60, wherein said antibody or its antigen binding domain is with 175nM or less K D Values for binding to CD228.

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

63. The fusion protein of any one of claims 1 to 62, wherein the antibody or antigen binding domain thereof does not bind to murine CD228, or binds to murine CD228 with an affinity that is at least 100-fold lower than that of human CD228.

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

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

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. A fusion protein according to any one of claims 1 to 44, 46, 47, 49, 50, 52, 53, 55, 56 and 58 to 66, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 75, 76 and 78-83, or wherein the fusion protein comprises an amino acid sequence that has 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 shown in any one of SEQ ID NOs: 75, 76 and 78-83.

68. A fusion protein according to any one of claims 1 to 44, 46, 47, 49, 50, 52, 53, 55, 56 and 58 to 67, wherein the fusion protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or more sequence identity to the amino acid sequence shown 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. A fusion protein according to any one of claims 1 to 44, 46, 47, 49, 50, 52, 53, 55, 56 and 58 to 68, wherein the fusion protein comprises the amino acid sequence shown 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. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1 to 69.

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

72. A vector comprising the nucleic acid molecule according to claim 69 or 70.

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

74. A method of 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. 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 the fusion protein according to any one of claims 1 to 69 or a composition comprising such a fusion protein for simultaneously activating the downstream signaling pathway of CD137 and engaging CD228-positive tumor cells.

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

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

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

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

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

82. 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 one or more fusion proteins according to any one of claims 1 to 69, or one or more compositions comprising such fusion proteins, to tissue comprising the tumor.

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

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

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

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

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

88. A method for inducing 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 according to any one of claims 1 to 69 or one or more compositions comprising such fusion proteins to a tissue comprising a tumor.

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

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

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

92. The method according to claim 91, wherein the anti-PD-1 or anti-PD-L1 antibody is nivolumab, pembrolizumab, cemiplizumab, dotarizumab, atezolizumab, avelumab, or durvalumab.

93. The fusion protein of any one of claims 1 to 69 for use in therapy.

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

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

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

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