I. BACKGROUND

[0001] Lymphocyte activation gene-3, or LAG-3 (also known as cluster of differentiation 223 or CD223) is a membrane protein of the immunoglobulin supergene family. LAG-3 is structurally and genetically related to cluster of differentiation 4 (CD4), with its encoding gene located on the distal part of the short arm of chromosome 12, near the CD4 gene, suggesting that the LAG-3 gene may have evolved through gene duplication (Triebel et a!., J Exp Med, 1990). LAG-3 is not expressed on resting peripheral blood Iymphocytes but is expressed on activated T cells and natural killer (NK) cells (Triebel et al.,J Exp Med, 1990), and has also been reported to be expressed on activated B cells and plasmacytoid dendritic cells.

[0002] Like CD4, LAG-3 binds to major histocompatibility complex (MHC) class II molecules, but with a higher affinity and at a different binding site than CD4 (Huard et a\.,Proc Natl Acad Sci U S A, 1997). MHC class II engagement on dendritic cells by LAG-3 leads to changes in the cytokine and chemokine profiles of dendritic cells (Buisson and Triebel, Vaccine, 2003). Further, LAG-3 has been reported to cause maturation of dendritic cells, as demonstrated by the production of interleukin 12 (IL-12) and tissue necrosis factor alpha (TNF-a) by these cells and increases in the capacity of dendritic cells to stimulate the proliferation and interferon gamma (IFN-γ) response by allogeneic T cells (Andreae et al.,J Immunol, 2002). LAG-3 signaling and MHC class II cross-linking has been reported to inhibit early events in primary activation of human cluster of differentiation 4 positive (CD4 ⁺ ) and cluster of differentiation 8 positive (CD8 ^' ) T cells (Macon-Lemaitre and Triebel , Immunology, 2005). LAG-3 negatively regulates the cellular proliferation, activation, and homeostasis of T cells.

[0003] Therefore, like cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1 ), LAG-3 is an inhibitory immune receptor. LAG-3 ^' s prominent role as a negative regulator of T cell response has been impressively demonstrated, in particular in conjunction with PD-1 in a study based on both knockout mice and target-specific antibodies (Woo et a L, Cancer Res, 2012). In that study, dual anti-LAG- 3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Further, LAG-3/PD-1 double knock-out mice showed markedly increased survival from and clearance of multiple transplantable tumors. Additional experimental support for the powerful combined role of PD-1 and LAG-3 as inhibitory immune checkpoints was provided by the fact that the double knock-out mice were highly prone to lethal autoinflammation.

[0004] Programmed cell death protein 1 , or PD-1 (also known as cluster of differentiation 279 or CD279) is a member of the cluster of differentiation 28 (CD28) gene family and is expressed on activated T, B, and myeloid lineage cells (Sharpe et al, Nat Immunol, 2007, Greenwald et a\.,Annu Rev Immunol, 2005). PD-1 interacts with two ligands, programmed cell death 1 ligand 1 (PD-L1 ) and programmed cell death 1 ligand 2 (PD-L2). Interaction of these ligands with PD-1 plays an important role in downregulating the immune system by limiting overly-active T cells locally, which in turn prevents autoimmunity and maintains peripheral tolerance during infection or inflammation in normal tissues.

[0005] PD-1 negatively modulates T cell activation, and the inhibitory function of PD-

1 on T cell activation is linked to an immunoreceptor tyrosine-based inhibitory motif (ITIM) of its cytoplasmic domain (Parry et al., Mo/ Cell Biol, 2005, Greenwald et a\.,Annu Rev Immunol, 2005). Disruption of this inhibitory function of PD-1 can lead to autoimmunity. On the other hand, sustained negative signals by PD-1 have been implicated in T cell dysfunctions in many pathologic situations, such as chronic viral infections and tumor immune evasion.

[0006] In many cancers, PD-1 is expressed by tumor-infiltrating lymphocytes (TILs), associated with host anti-tumor immunity (Galon et a\., Science, 2006). Multiple lines of evidence have indicated that TILs are subject to PD-1 inhibitory regulation and the anti-tumor immunity is modulated by PD-1 /PD-L1 signaling. First, the PD-L1 expression is confirmed in many human and mouse tumor lines and the expression can be further upregulated by IFN-y in vitro (Dong et al., Nat Med, 2002). Second, expression of PD-L1 by tumor cells has been directly associated with their resistance to lysis by anti-tumor T cells in vitro (Blank et a\., Cancer Res, 2004, Dong et al., Nat Med, 2002). Third, PD-1 knockout mice are resistant to tumor challenge (Iwai et al.,/ni Immunol, 2005) and T cells from PD-1 knockout mice are highly effective in tumor rejection when adoptively transferred to tumor-bearing mice (Blank et a\., Cancer Res, 2004). Fourth, blocking PD-1 inhibitory signals by a monoclonal antibody can potentiate host anti-tumor immunity in mice (Hirano et a\., Cancer Res, 2005, Iwai et al.,/ni Immunol, 2005). Fifth, high degrees of PD-L1 expression in tumors (detected by immunohistochemicai staining) are associated with poor prognosis for many human cancer types (Hamanishi et a\.,Proc Natl Acad Sci U S A, 2007).

[0007] There thus exists a need for new compounds that can modulate responses of

LAG-3 ^* lymphocytes, such as T cells, NK cells, B cells, and plasmacytoid dendritic cells, and at the same time, relieve such lymphocytes of PD-1 inhibitory regulation. Such combination may have important uses in the treatment or prevention of cancer, organ transplant rejection, or treatment of autoimmune or autoinf!ammatory diseases. It is desirable to have alternative fusion proteins with a PD-1 binding moiety and a LAG-3 binding moiety that are capable of binding LAG-3 and PD-1 , respectively, with high affinity, have enhanced biostability, and can be used in pharmaceutical and/or diagnostic applications. In this regard, it is an object of the present invention to provide such fusion proteins. No such fusion proteins having these features have been previously disclosed.

[0008] In addition, it has been regarded as natural that monkey metabolism is the most similar to that of humans, and, accordingly, cynomolgus monkeys have been widely used in pharmacokinetic or drug-safety studies in the development of new therapies, including new biologies. Such studies may further be necessary pre-prerequisites to regulatory approval. Thus, it is also desirable to have fusion proteins with a LAG-3 binding moiety that is cross-reactive with both human and cynomolgus LAG-3, with a comparable binding pattern, including comparable or similar binding affinity. No such fusion proteins with LAG-3 binding lipocalin muteins having these cross-reactivity features have been previously described.

[0009] The present disclosure provides a group of novel proteins binding to both

LAG-3 and PD-1 , thereby modulating the immune response.

II. DEFINITIONS

[0010] The following list defines terms, phrases, and abbreviations used throughout the instant specification. All terms listed and defined herein are intended to encompass all grammatical forms.

[0011] As used herein, unless otherwise specified, "LAG-3" means human LAG-3

(hu LAG-3) and include variants, isoforms and species homologs of human LAG-3. LAG-3 is also known as "lymphocyte-activation gene 3", "cluster of differentiation 223", or "CD223", which are used interchangeably. Human LAG-3 means a full-length protein defined by UniProt P18627 (version 5 of 7 July 2009), a fragment thereof, or a variant thereof. Human LAG-3 is encoded by the LAG3 gene.

[0012] As used herein, unless otherwise specified, "PD-1 " means human PD-1

(huPD-1 ) and includes variants, isoforms and species homologs of human PD-1. PD-1 is also known as "programmed cell death protein 1 ", "cluster of differentiation 279 ^" or "CD279 ^" , which are used interchangeably. Human PD-1 means a full-length protein defined by UniProt Q151 16, a fragment thereof, or a variant thereof. Human PD-1 is encoded by the PDCD1 gene.

[0013] As used herein, "detectable affinity" means the ability to bind to a selected target with an affinity constant, generally measured by K _d or EC ₅₀ , of at most about 10 ^"5 M or below (a lower K _d or EC ₅₀ value reflects better binding activity). Lower affinities that are no longer measurable with common methods such as ELISA (enzyme-linked immunosorbent assay) are of secondary importance.

[0014] As used herein, "binding affinity" of a protein of the disclosure (e.g., a lipocalin mutein or an antibody) or a fusion polypeptide thereof to one or more selected targets (in the present case, LAG-3 and/or PD-1 ), can be measured (and thereby K _d values of a mutein- ligand complex be determined) by a multitude of methods known to those skilled in the art. Such methods include, but are not limited to, fluorescence titration, competitive ELISA, calorimetric methods, such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). Such methods are well established in the art and examples thereof are also detailed below.

[0015] It is also noted that the complex formation between the respective binder and its ligand is influenced by many different factors such as the concentrations of the respective binding partners, the presence of competitors, pH and the ionic strength of the buffer system used, and the experimental method used for determination of the dissociation constant K _d (for example fluorescence titration, competition ELISA or surface plasmon resonance, just to name a few) or even the mathematical algorithm which is used for evaluation of the experimental data.

[0016] Therefore, it is also clear to the skilled person that the K _d values (dissociation constant of the complex formed between the respective binder and its target/ligand) may vary within a certain experimental range, depending on the method and experimental setup that is used for determining the affinity of a particular lipocalin mutein for a given ligand. This means that there may be a slight deviation in the measured K _d values or a tolerance range depending, for example, on whether the K _d value was determined by surface plasmon resonance (SPR), by competitive ELISA, by direct ELISA, or by another method.

[0017] As used herein, a "mutein," a "mutated" entity (whether protein or nucleic acid), or "mutant" refers to the exchange, deletion, or insertion of one or more nucleotides or amino acids, compared to the naturally occurring (wild-type) nucleic acid or protein "reference" scaffold. Said term also includes fragments of a mutein and variants as described herein. Lipocalin muteins of the present disclosure, fragments or variants thereof preferably have the function of binding to LAG-3 as described herein.

[0018] The term "fragment" as used herein in connection with the muteins of the disclosure relates to proteins or peptides derived from full-length mature human tear lipocalin (hTIc or hTLPC) that are N-terminally and/or C-terminally shortened, i.e., lacking at least one of the N-terminal and/or C-terminal amino acids. Such a fragment may lack up to 2, up to 3, up to 4, up to 5, up to 10, up to 15, up to 20, up to 25, or up to 30 (including all numbers in between) of the N-terminal and/or C-terminal amino acids. As an illustrative example, such a fragment may lack 4 N-terminal and 2 C-terminal amino acids. It is understood that the fragment is preferably a functional fragment of the full-length tear lipocalin (mutein), which means that it preferably comprises the binding pocket of the full-length tear lipocalin (mutein) it is derived from. As an illustrative example, such a functional fragment may comprise at least amino acids 5-156 of the linear polypeptide sequence of native mature hTIc. Such fragments may include at least 10, more such as 20 or 30 or more consecutive amino acids of the primary sequence of mature tear lipocalin and are usually detectable in an immunoassay of the mature lipocalin.

[0019] In general, the term "fragment," as used herein with respect to the corresponding protein ligand LAG-3 and/or PD-1 of a lipocalin mutein of the disclosure or of the combination according to the disclosure and/or of a fusion protein described herein, relates to N-terminally and/or C-terminally shortened protein or peptide ligands, which retain the capability of the full-length ligand to be recognized and/or bound by a mutein according to the disclosure.

[0020] The term "mutagenesis" as used herein means that the experimental conditions are chosen such that the amino acid naturally occurring at a given sequence position of the mature lipocalin can be substituted by at least one amino acid that is not present at this specific position in the respective natural polypeptide sequence. The term "mutagenesis" also includes the (additional) modification of the length of sequence segments by deletion or insertion of one or more amino acids. Thus, it is within the scope of the disclosure that, for example, one amino acid at a chosen sequence position is replaced by a stretch of three random mutations, leading to an insertion of two amino acid residues compared to the length of the respective segment of the wild-type protein. Such an insertion or deletion may be introduced independently from each other in any of the peptide segments that can be subjected to mutagenesis in the disclosure. In one exemplary embodiment of the disclosure, an insertion of several mutations may be introduced into the loop AB of the chosen lipocalin scaffold (cf. International Patent Publication No. WO 2005/019256, which is incorporated by reference its entirety herein).

[0021] The term "random mutagenesis" means that no predetermined single amino acid (mutation) is present at a certain sequence position but that at least two amino acids can be incorporated with a certain probability at a predefined sequence position during mutagenesis.

[0022] "Identity" is a property of sequences that measures their similarity or relationship. The term "sequence identity" or "identity" as used in the present disclosure means the percentage of pair-wise identical residues— following (homologous) alignment of a sequence of a polypeptide of the disclosure with a sequence in question— with respect to the number of residues in the longer of these two sequences. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.

[0023] The term "homology" is used herein in its usual meaning and includes identical amino acids as well as amino acids which are regarded to be conservative substitutions (for example, exchange of a glutamate residue by an aspartate residue) at equivalent positions in the linear amino acid sequence of a polypeptide of the disclosure (e.g., any lipocalin mutein of the disclosure).

[0024] The percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002) (cf. Altschul et al., Nucleic Acids Res, 1997). In this embodiment, the percentage of homology is based on the alignment of the entire polypeptide sequences (matrix: BLOSUM 62; gap costs: 11 .1 ; cut-off value set to 10 ^"3 ) including the propeptide sequences, preferably using the wild-type protein scaffold as reference in a pairwise comparison. It is calculated as the percentage of numbers of "positives" (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0025] Specifically, in order to determine whether an amino acid residue of the amino acid sequence of a lipocalin (mutein) is different from a wild-type lipocalin corresponding to a certain position in the amino acid sequence of a wild-type lipocalin, a skilled artisan can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as BLAST 2.0, which stands for Basic Local Alignment Search Tool, or ClustalW, or any other suitable program which is suitable to generate sequence alignments. Accordingly, a wild-type sequence of lipocalin can serve as "subject sequence" or "reference sequence," while the amino acid sequence of a lipocalin different from the wild- type lipocalin described herein serves as "query sequence." The terms "wild-type sequence" and "reference sequence" and "subject sequence" are used interchangeably herein. A preferred wild-type sequence of lipocalin is the sequence of hTIc as shown in SEQ ID NO: 1.

[0026] "Gaps" are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of sequence identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, for example BLAST , BLAST2 , and Smith-Waterman (Smith and Waterman, J Mol Biol, 1981 ).

[0027] The term "variant" as used in the present disclosure relates to derivatives of a protein or peptide that include modifications of the amino acid sequence, for example by substitution, deletion, insertion or chemical modification. Such modifications do in some embodiments not reduce the functionality of the protein or peptide. Such variants include proteins, wherein one or more amino acids have been replaced by their respective D- stereoisomers or by amino acids other than the naturally occurring 20 amino acids, such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline. However, such substitutions may also be conservative, i.e., an amino acid residue is replaced with a chemically similar amino acid residue. Examples of conservative substitutions are the replacements among the members of the following groups: 1 ) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. The term "variant," as used herein with respect to the corresponding protein target LAG-3 and/or PD-1 of a lipocalin mutein of the disclosure or of a combination and/or fusion protein according to the disclosure, relates to LAG-3 and/or PD-1 or fragment thereof, respectively, that has one or more such as 1 , 2, 3, 4, 5 ,6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 60, 70, 80 or more amino acid substitutions, deletions and/or insertions in comparison to a wild-type LAG-3 or PD-1 protein, respectively, such as a LAG-3 or PD-1 reference protein as deposited with SwissProt/UniProt as described herein. A LAG-3 or PD-1 variant, respectively, has preferably an amino acid identity of at least 50%, 60%, 70%, 80%, 85%, 90% or 95% with a wild-type human LAG-3 or PD-1 , such as a LAG-3 or PD-1 reference protein as deposited with SwissProt/UniProt as described herein.

[0028] By a "native sequence" of a lipocalin is meant that the sequence of a lipocalin that has the same amino acid sequence as the corresponding polypeptide derived from nature. Thus, a native sequence lipocalin can have the amino acid sequence of the respective naturally-occurring lipocalin from any organism, in particular a mammal. Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally-occurring truncated or secreted forms of the lipocalin, naturally-occurring variant forms such as alternatively spliced forms and naturally-occurring allelic variants of the lipocalin. A polypeptide "variant" means a biologically active polypeptide having at least about 50%, 60%, 70%, 80% or at least about 85% amino acid sequence identity with the native sequence polypeptide. Such variants include, for instance, polypeptides in which one or more amino acid residues are added or deleted at the N- or C- terminus of the polypeptide. Generally, a variant has at least about 70%, including at least about 80%, such as at least about 85% amino acid sequence identity, including at least about 90% amino acid sequence identity or at least about 95% amino acid sequence identity with the native sequence polypeptide. As an illustrative example, the first four N-terminal amino acid residues (His-His-Leu-Leu, SEQ ID NO: 50) and the last 2 C-terminal amino acid residues (Ser-Asp) can be deleted in a hTIc mutein of the disclosure without affecting the biological function of the protein, e.g., SEQ ID NOs: 7-27, 201-214, and 240-249.

[0029] The term "position" when used in accordance with the disclosure means the position of either an amino acid within an amino acid sequence depicted herein or the position of a nucleotide within a nucleic acid sequence depicted herein. To understand the term "correspond" or "corresponding" as used herein in the context of the amino acid sequence positions of one or more lipocalin muteins, a corresponding position is not only determined by the number of the preceding nucleotides/amino acids. Accordingly, the position of a given amino acid in accordance with the disclosure which may be substituted may vary due to deletion or addition of amino acids elsewhere in a (mutant or wild-type) lipocalin. Similarly, the position of a given nucleotide in accordance with the present disclosure which may be substituted may vary due to deletions or additional nucleotides elsewhere in a mutein or wild-type lipocalin 5'-untranslated region (UTR) including the promoter and/or any other regulatory sequences or gene (including exons and introns).

[0030] Thus, for a "corresponding position" in accordance with the disclosure, it is preferably to be understood that the positions of nucleotides/amino acids may differ in the indicated number than similar neighboring nucleotides/amino acids, but said neighboring nucleotides/amino acids, which may be exchanged, deleted, or added, are also comprised by the one or more "corresponding positions".

[0031] In addition, for a corresponding position in a lipocalin mutein based on a reference sequence in accordance with the disclosure, it is preferably understood that the positions of nucleotides/amino acids structurally correspond to the positions elsewhere in a (mutant or wild-type) lipocalin, even if they may differ in the indicated number, as appreciated by the skilled in light of the highly-conserved overall folding pattern among lipocalins.

[0032] The term "albumin" includes all mammal albumins such as human serum albumin or bovine serum albumin or rat serum albumin.

[0033] The term "organic molecule" or "small organic molecule" as used herein for the non-natural target denotes an organic molecule comprising at least two carbon atoms, but preferably not more than 7 or 12 rotatable carbon bonds, having a molecular weight in the range between 100 and 2,000 Dalton, preferably between 100 and 1 ,000 Dalton, and optionally including one or two metal atoms.

[0034] The word "detect", "detection", "detectable", or "detecting" as used herein is understood both on a quantitative and a qualitative level, as well as a combination thereof. It thus includes quantitative, semi-quantitative and qualitative measurements of a molecule of interest.

[0035] A "subject" is a vertebrate, preferably a mammal, more preferably a human.

The term "mammal" is used herein to refer to any animal classified as a mammal, including, without limitation, humans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as cynomolgus monkeys, to name only a few illustrative examples. Preferably, the "mammal" herein is human.

[0036] An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations.

[0037] A "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.

[0038] A "subunit" of a fusion polypeptide disclosed herein is defined as a stretch of amino acids of the polypeptide, which stretch defines a unique functional unit of said polypeptide such as provides binding motif towards a target.

[0039] A "fusion polypeptide" as used herein refers to a composite protein, i.e., a single contiguous amino acid sequence, made up of two (or more) distinct, heterologous polypeptides that are not normally fused together in a single amino acid sequence. A "fusion polypeptide" as described herein preferably comprises two or more subunits, at least one of these subunits binds to LAG-3 and a further subunit binds to PD-1 . Within the fusion polypeptide, these subunits may be linked by covalent or non-covalent linkage. Preferably, the fusion polypeptide is a translational fusion between the two or more subunits. The translational fusion may be generated by genetically engineering the coding sequence for one subunit in a reading frame with the coding sequence of a further subunit. Both subunits may be interspersed by a nucleotide sequence encoding a linker. However, the subunits of a fusion polypeptide of the present disclosure may also be linked through chemical conjugation. If one or more of the distinct polypeptides is part of a protein (complex) that consists of more than one polypeptide chain, the term "fusion polypeptide" may also refer to the polypeptide comprising the fused sequences and all other polypeptide chain(s) of the protein (complex). As an illustrative example, where a full-length immunoglobulin is fused to a lipocalin mutein via a heavy or light chain of the immunoglobulin, the term "fusion polypeptide" may refer to the single polypeptide chain comprising the lipocalin mutein and the heavy or light chain of the immunoglobulin. The term "fusion polypeptide" may also refer to the entire immunoglobulin (both light and heavy chains) and the lipocalin mutein fused to one or both of its heavy and/or light chains.

[0040] A "linker" that may be comprised by a fusion polypeptide of the present disclosure links two or more subunits of a fusion polypeptide as described herein. The linkage can be covalent or non-covalent. A preferred covalent linkage is via a peptide bond, such as a peptide bond between amino acids. A preferred linker is a peptide linker. Accordingly, in a preferred embodiment said linker comprises of one or more amino acids, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. Preferred linkers are described herein. Other preferred linkers are chemical linkers.

III. DESCRIPTIONS OF FIGURES

[0041] Figure 1 : provides an overview of the design of representative fusion polypeptides described in this application that are bispecific for the targets PD-1 and LAG-3, or monospecific for LAG-3. Representative bispecific fusion polypeptides of Figure 1A-1 F were made based on an antibody specific for PD-1 (e.g., an antibody whereby heavy chains are provided by SEQ ID NO: 57, 59, 61 , 65, or comprise the CDR sequences of GYTFTDYE (HCDR1 , SEQ ID NO: 1 16), IDPGTGGT (HCDR2, SEQ ID NO: 1 17), and TSEKFGSNYYFDY (HCDR3; SEQ ID NO: 1 18), and light chains are provided by SEQ ID NO: 58, 60, 62, 66, or comprise the CDR squences of QTIVHSDGNTY (LCDR1 , SEQ ID NO: 134), KVS (LCDR2), and FQGSHVPLT (LCDR3, SEQ ID NO: 135) and one or more lipocalin muteins specific for LAG-3 (e.g., the lipocalin mutein of SEQ ID NO: 8, 240, 14, 246, 21 , 21 1 , or 247). The lipocalin muteins were genetically fused to either the C- or the N-terminus of either the heavy chain or the light chain of a PD-1 specific antibody as depicted in Figure 1 , resulting in the fusion polypeptides, e.g., SEQ ID NOs: 74 and 66; SEQ ID NOs: 61 and 75; SEQ ID NOs: 76 and 66; SEQ ID NOs: 61 and 77; SEQ ID NOs: 78 and 62; SEQ ID NOs: 65 and 79; SEQ ID NOs: 80 and 62; SEQ ID NOs: 65 and 81 ; SEQ ID NOs: 78 and 79; SEQ ID NOs: 57 and 84; SEQ ID NOs: 85 and 66; SEQ ID NOs: 61 and 86; SEQ ID NOs: 59 and 87; SEQ ID NOs: 88 and 60; SEQ ID NOs: 57 and 181 SEQ ID NOs: 185 and 66; SEQ ID NOs: 61 and 187; SEQ ID NOs: 189 and 62; SEQ ID NOs: 65 and 191 ; SEQ ID NOs: 193 and 62; SEQ ID NOs: 65 and 194 and SEQ ID NOs: 236 and 66. A monospecific fusion polypeptide was generated by fusing the LAG-3 specific lipocalin mutein of SEQ ID NO: 8 via an unstructured (G ₄ S) ₃ linker (SEQ ID NO: 54) to the C-terminus of the Fc region of an antibody provided in SEQ ID NO: 73. The resulting construct is provided in SEQ ID NO: 83. Figure 1 G-1 I show the design of additional fusion polypeptides that can be made based on an antibody specific for PD-1 and one or more lipocalin muteins specific for LAG-3.

[0042] Figure 2: depicts the results of an enzyme-linked immunosorbent assay

(ELISA) in which the binding to human PD-1 of representative fusion polypeptides and the PD-1 antibodies of SEQ ID NOs: 65 and 62 and SEQ ID NOs: 61 and 66. Recombinant human PD-1 -His (PD-1 with a C-terminal polyhistidine tag) was coated on a microtiter plate, and the tested agents (fusion polypeptides and PD-1 antibodies) were titrated starting with the highest concentration of 200 nM and a 1 :3 dilution series. Bound samples under study were detected via an anti-human IgG Fc antibody as described in Example 2. The data was fit with a 1 : 1 binding model with EC ₅₀ value and the maximum signal as free parameters, and a slope that was fixed to one. The resulting EC ₅₀ values are provided in Table 1.

[0043] Figure 3: shows the results of an ELISA experiment in which the binding to human LAG-3 of representative fusion polypeptides with the PD-1 antibody backbones of SEQ ID NOs: 65 and 62 and SEQ ID NOs: 61 and 66 was determined. Human LAG-3-His (LAG-3 with C-terminal polyhistidine tag) was coated on a microtiter plate, and the tested agents were titrated starting with the highest concentration of 200 nM. Bound agents under study were detected via an anti-Tic antibody as described in Example 3. The data was fit with a 1 : 1 binding model with EC ₅₀ value and the maximum signal as free parameters, and a slope that was fixed to one. The resulting EC ₅₀ values are provided in Table 2.

[0044] Figure 4: depicts the results of fluorescence-activated cell sorting (FACS) studies carried out in order to assess the specific binding of fusion polypeptides to human PD-1 (Figure 4A-4D) or human LAG-3 (Figure 4E-4H), expressed on mammalian cells as described in Example 4. The negative control combination of hlgG4 (Sigma) showed no binding. The geometric means of the fluorescence intensity were normalized to maximal mean and fit with a 1 : 1 binding model. The resulting EC ₅₀ values are provided in Table 3.

[0045] Figure 5: illustrates the results of an ELISA experiment in which the ability of representative fusion polypeptides to simultaneously bind both targets, PD-1 and LAG-3, was determined. Recombinant PD-1 -His was coated on a microtiter plate, followed by a titration of the fusion polypeptides starting with the highest concentration of 200 nM. Subsequently, a constant concentration of biotinylated human LAG-3-Fc was added, which was detected via extravidin as described in Example 5.

[0046] Figure 6: shows that the fusion polypeptides compete with major histocompatibility complex (MHC) class II molecules (LAG-3's natural ligands) for binding to LAG-3, depicted in competitive FACS studies conducted as described in Example 6. A constant concentration of huLAG-3-Fc fusion (human LAG-3 extracellular domain fused to human lgG1 Fc fragment), and a dilution series of fusion polypeptides or controls, were incubated with the MHC class II positive human cell line A375. Cell-bound huLAG-3-Fc was detected using a fluorescently labelled anti-lgG Fc antibody. The dose dependent inhibition of huLAG-3-Fc binding to MHC class II molecules by LAG-3 and PD-1 bispecific antibody- lipocalin mutein fusion polypeptides or LAG-3 monospecific Fc-lipocalin mutein fusion polypeptides were observed.

Figure 7: shows the results of representative experiments in which the ability of selected fusion polypeptides to induce T cell activation was investigated. PD-1 antibodies, including the respective PD-1 antibody building blocks, as well as the LAG-3 binding lipocalin muteins as Fc fusions were also tested alone and as cocktail. In the experiment, human peripheral blood mononuclear cells (PBMCs) stimulated with 1 nM staphylococcal enterotoxin B (SEB) were incubated with the fusion polypeptides, antibodies, lipocalin mutein Fc fusions, cocktails or controls. Levels of secreted interleukin 2 (IL-2), reflective of T cell activation, were determined by an electrochemoluminescence-based assay as readout for T cell activation and normalized to the levels of corresponding lgG4 control, as described in Example 7.

[0047] Figure 8: shows the results of representative experiments in which the ability of selected fusion polypeptides to include T cell activation was investigated. The respective PD-1 antibody building block and lipocalin mutein as Fc fusion were also tested. In the experiment, melanoma A375 cells were treated with mitomycin, coated and allowed to adhere overnight. The next day, purified T cells, were incubated on the coated cells in the presence of various concentrations of the bispecific fusion polypeptides and the controls as well as in the presence of staphylococcal enterobactin B (SEB) as further described in Example 10. Levels of secreted interleukin 2 (IL-2), reflective of T cell activation, were determined by an electrochemoluminescence-based assay as readout for T cell activation and normalized to the levels of corresponding lgG4 control, as described in Example 10.

[0048] Figure 9: shows the tumor volume after treatment with a selected fusion polypeptide, a PD-1 antibody, or vehicle control (PBS) in a humanized mouse tumor model. NOG mice were engrafted s.c. with HCC827 tumors that were allowed to grow for 10 days. Mice were then randomized into treatment (or control) groups and received 5 ^χ 10 ⁶ fresh human PBMCs intravenously and intraperitoneal injections of the fusion polypeptide, PD-1 antibody or vehicle control, as applicable, in the indicated doses on day 1 , day 4, day 8, and day 12. Tumor growth was recorded every 3 to 4 days.

IV. DETAILED DESCRIPTION OF THE DISCLOSURE

[0049] The present invention envisions a fusion polypeptide that is capable of binding both PD-1 and LAG-3, wherein the fusion polypeptide comprises at least two subunits in any order, wherein the first subunit is specific for PD-1 and the second subunit is specific for LAG-3. [0050] In some embodiments, the fusion polypeptide contains at least two subunits in any order: (1 ) a first subunit that comprises a full-length immunoglobulin or an antigen- binding domain thereof specific for PD-1 , and (2) a second subunit that comprises a lipocalin mutein specific for LAG-3.

[0051] In some embodiments, the fusion polypeptide also may contain a third subunit. For instance, the polypeptide may contain a third subunit specific for LAG-3. In some embodiments, said third subunit comprises a lipocalin mutein specific for LAG-3. For example, two lipocalin muteins may be fused to an immunoglobulin subunit, one at the C- terminus and one at the N-terminus of the immunoglobulin. In some embodiments, lipocalin muteins may be fused to the heavy chain or light chain of an immunoglobulin.

[0052] In some embodiments, one subunit can be linked to another subunit as essentially described in Figure 1. For example, one lipocalin mutein can be linked, via a peptide bond, to the C-terminus of the immunoglobulin heavy chain domain (VH), the N- terminus of the VH, the C-terminus of the immunoglobulin light chain (VL), and/or the N- terminus of the VL (Figure 1 ). In some particular embodiments, a lipocalin mutein subunit can be fused at its N-terminus and/or its C-terminus to an immunoglobulin subunit. For example, the lipocalin mutein may be linked via a peptide bond at the C-terminus of a heavy chain constant region (CH) or the C-terminus of a light chain constant region (CL) of the immunoglobulin. In some further embodiments, the peptide bond may be a linker, preferably a peptide linker. In some still further embodiments, the peptide bond may be a linker, preferably an unstructured (G ₄ S) ₃ linker, for example, as shown in SEQ ID NO: 54. A linker may have from 1 to 50 amino acids, such as 1 , 2, 3, 4, 5, 10, 1 1 , 12, 13, 14, 15, 16, 17 18, 19, 20, 25, 30, 35, 40, 45 or 50 amino acids. In some further embodiments, the linker is a glycine-serine linker, preferably an unstructured (G ₄ S) ₃ linker, for example, as shown in SEQ ID NO: 54. In some still further embodiments, a linker is a polyproline linker or a proline- alanine-serine polymer, for example, as shown in SEQ ID NOs:229-235.

[0053] In this regard, one subunit may be fused at its N-terminus and/or its C- terminus to another subunit. For example, when one subunit comprises a full-length immunoglobulin, another subunit may be linked via a peptide bond between the N-terminus of the second subunit and the C-terminus of a heavy chain constant region (CH) of said immunoglobulin. In some further embodiments, a third subunit may be linked via a peptide bond between the N-terminus of the third binding domain and the C-terminus of a light chain constant region (CL) of said immunoglobulin. In some still further embodiments, the peptide bond may be a linker, preferably a (G ₄ S) ₃ linker, or it may be a polyproline linker or a proline- alanine-serine polymer for example, as shown in any one of SEQ ID NO: 54 or 229-235.

[0054] In some embodiments, with respect to a fusion polypeptide of the disclosure, one of whose subunits comprises a full-length immunoglobulin, while the polypeptide is simultaneously engaging PD-1 and LAG-3, the Fc function of the Fc region of the full-length immunoglobulin to Fc receptor-positive cell may be preserved at the same time.

[0055] In some other embodiments with respect to a fusion polypeptide of the disclosure, one of whose subunits comprises a full-length immunoglobulin, while the polypeptide is simultaneously engaging PD-1 and LAG-3, the Fc function of the Fc region of the full-length immunoglobulin to Fc receptor-positive cell may be reduced or fully suppressed by protein engineering. This may be achieved, for example, by switching from the lgG1 backbone to lgG4, as lgG4 is known to display reduced Fc-gamma receptor interactions compared to lgG1 . To further reduce the residual binding to Fc-gamma receptors, mutations may be introduced into the lgG4 backbone such as F234A and L235A. An S228P mutation may also be introduced into the lgG4 backbone to minimize the exchange of lgG4 half-antibody. In some further embodiments, any of the M428L, N434S, M252Y, S254T, and T256E mutations may be introduced into the lgG4 backbone for enhanced binding to the neonatal Fc receptor and extended serum half-life. In some embodiments, M428L and N434S mutations may be introduced for extended serum half-life. In some other embodiments, M252Y, S254T, and T256E mutations may be introduced for extended serum half-life. In some still further embodiments, an additional N297A mutation may be present in the immunoglobulin heavy chain of the fusion polypeptide in order to remove the natural glycosylation motif.

[0056] In some embodiments, resulting from the simultaneous binding to PD-1 and

LAG-3, the fusion polypeptides of the disclosure may exhibit a durable anti-tumor or anti- infection response.

[0057] In some embodiments, the Fc portion of the immunoglobulin included in a fusion polypeptide of the disclosure may contribute to maintaining the serum levels of the fusion polypeptide, critical for its stability and persistence in the body. For example, when the Fc portion binds to Fc receptors on endothelial cells and phagocytes, the fusion polypeptide may become internalized and recycled back to the bloodstream, enhancing its half-life within the body. [0058] In some embodiments, the fusion polypeptide may be able to bind LAG-3 expressed on a cell with an EC ₅₀ value of at most about 0.1 nM or even lower, such as about 0.05 nM or lower, about 0.04 nM or lower, about 0.03 nM or lower, or about 0.02 nM or lower, for example, when measured in a fluorescence activated cell sorting (FACS) assay, such as the FACS assay as essentially described in Example 4. The cell expressing LAG-3 may, for example, be a CHO cell transfected with human LAG-3.

[0059] In some embodiments, the fusion polypeptide may be able to bind PD-1 preferably expressed on a cell with an EC ₅₀ value of at most about 10 nM or even lower, such as about 5 nM, about 1 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.25 nM, or even lower. The EC ₅ o of a fusion polypeptide may be measured, for example, in a fluorescence activated cell sorting (FACS) assay, such as the FACS assay as essentially described in Example 4. The cell expressing PD-1 in such assay may, for example, be a CHO cell transfected with human PD-1 .

[0060] In some embodiments, the fusion polypeptide may be able to bind PD-1 with an EC ₅₀ value of at most about 10 nM or even lower, such as about 5 nM, about 1 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.25 nM, or even lower. The EC ₅₀ of a fusion polypeptide may be, for example, measured in an ELISA (enzyme-linked immunosorbent assay) assay essentially as described in Example 2.

[0061] In some embodiments, a fusion polypeptide of the disclosure may be able to bind PD-1 with an EC ₅₀ value comparable to the EC ₅₀ value of the immunoglobulin specific for PD-1 as included in such fusion polypeptide, such as the antibody having the heavy and light chains provided by SEQ ID NOs: 65 and 62, SEQ ID NOs: 61 and 66, SEQ ID NOs: 57 and 58, or SEQ ID NOs: 59 and 60, for example, when said immunoglobulin and the fusion polypeptide are measured in as ELISA assay essentially as described in Example 2.

[0062] In another aspect, the fusion polypeptide may be able to bind LAG-3 with an

EC ₅₀ value of at most about 10 nM or even lower, such as about 5 nM, about 1 nM about, about 0.5 nM, about 0.1 nM, about 0.09 nM, about 0.08 nM, about 0.07 nM, about 0.06 nM, about 0.05 nM, about 0.04 nM, about 0.03 nM or even lower, for example, when the fusion polypeptide is measured in an ELISA assay essentially as described in Example 3.

[0063] In some embodiments, a fusion polypeptide of the disclosure may be able to bind LAG-3 with an EC ₅₀ value at least as good as or superior to the EC ₅₀ value of the lipocalin mutein specific for LAG-3 as included in such fusion polypeptide, such as the lipocalin mutein of SEQ ID NO: 8, 240, 14, 246, 21 , 21 1 , or 247, for example, when said lipocalin mutein and the polypeptide are measured in an ELISA assay essentially as described in Example 3.

[0064] In some embodiments, the fusion polypeptides of the disclosure specific for both PD-1 and LAG-3 may be capable of simultaneously binding of PD-1 and LAG-3, for example, when said fusion polypeptide is measured in an ELISA assay essentially described in Example 5. In some embodiments, the fusion polypeptide may be capable of simultaneously binding of PD-1 and LAG-3, with an EC ₅₀ value of at most about 100 nM, at most about 50 nM, at most about 25 nM, at most about 10 nM, at most about 5 nM, at most about 2 nM, at most about 1 .5 nM, at most about 1 nM or at most about 0.5 nM, for example, when measured in an ELISA assay essentially described in Example 5.

[0065] In some embodiments, the fusion polypeptides of disclosure are capable of inhibiting the binding of LAG-3 to major histocompatibility complex (MHC) class II, such as those expressed on antigen-presenting cells (APCs) or tumor cells. The inhibitory mode of action can, for example, be determined by a FACS analysis as essentially described in Example 6.

[0066] In some embodiments, the fusion polypeptides of the disclosure may be able to induce IL-2 production, reflective of T cell activation, in a functional T cell activation assay essentially described in Example 7 and may even demonstrate a tendency towards stronger IL-2 induction at higher concentrations, preferably coating concentrations.

[0067] In some embodiments, the fusion polypeptides of the disclosure may be able to compete with PD-L1 and/or PD-L2 for binding to PD-1. In some embodiments, the fusion polypeptides of the disclosure may be able to compete with PD-L1 and/or PD-L2 for binding to PD-1 with an IC ₅₀ value of about 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8nM, 7 nM, or even lower. In some embodiments, the fusion polypeptides of the disclosure may be able to compete with PD-L1 and/or PD-L2 for binding to PD-1 with an IC ₅₀ value that is about equal to or lower than the IC ₅₀ value of the antibody comprised in the fusion polypeptide. The ability to compete with PD-L1 and/or PD- L2 may be determined by enzyme-linked immunosorbent assay (ELISA), such as an ELISA assay, wherein the well of a microtiter plate is preferably coated with 1 μg mL of human PD- L1 -Fc or human PD-L2-Fc, and/or wherein the well is incubated with 20 mM of human PD-1 - Fc and/or different concentrations of the fusion polypeptide or the antibody, for example by an assay essentially described in Example 9. [0068] In some embodiments, the fusion polypeptide of the invention comprises an amino acid sequence shown in any one of SEQ ID NOs: 74-88, 177-181 , 185-194, and 236.

[0069] In some embodiments, the fusion polypeptide of the invention comprises an amino acid sequence with at least 75% or higher, at least 80% or higher, at least 85% or higher, at least 90% or higher, at least 95% or higher, at least 98% or higher, or at least 99% or higher sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-88, 177-181 , 185-194, and 236.

[0070] In some embodiments, the fusion polypeptide of the invention comprises the amino acids shown in SEQ ID NOs: 74 and 66, or the amino acids shown in SEQ ID NOs: 61 and 75, or the amino acids shown in SEQ ID NOs: 76 and 66, or the amino acids shown in SEQ ID NOs: 61 and 77, or the amino acids shown in SEQ ID NOs: 78 and 62, or the amino acids shown in SEQ ID NOs: 65 and 79, or the amino acids shown in SEQ ID NOs: 80 and 62, or the amino acids shown in SEQ ID NOs: 65 and 81 , or the amino acids shown in SEQ ID NOs: 78 and 79, or the amino acids shown in SEQ ID NOs: 57 and 84, or the amino acids shown in SEQ ID NOs: 85 and 66, or the amino acids shown in SEQ ID NOs: 61 and 86, or the amino acids shown in SEQ ID NOs: 59 and 87, or the amino acids shown in SEQ ID NOs: 88 and 60, or the amino acids shown in SEQ ID NOs: 57 and 181 , or the amino acids shown in SEQ ID NOs: 185 and 66, or the amino acids shown in SEQ ID NOs: 61 and 187, or the amino acids shown in SEQ ID NOs: 189 and 62, or the amino acids shown in SEQ ID NOs: 65 and 191 , or the amino acids shown in SEQ ID NOs: 193 and 62, or the amino acids shown in SEQ ID NOs: 65 and 194. In some embodiments, structural homologues of said fusion polypeptides have an amino acid sequence homology or sequence identity of more than about 60%, preferably more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92%, and most preferably more than 95% in relation to said fusion polypeptide.

[0071] In some embodiments, the fusion polypeptides of the invention comprise fusion 1 through fusion 1 1. Each of fusion 1 through fusion 1 1 , when used in the present disclosure, refers to fusion polypeptides comprising humanized antibodies that have the complementarity-determining region (CDR) sequences of the 388D4 antibody, namely the heavy chain CDRs GYTFTDYE (HCDR1 , SEQ ID NO: 1 16), IDPGTGGT (HCDR2, SEQ ID NO: 1 17), and TSEKFGSNYYFDY (HCDR3; SEQ ID NO: 1 18), and the light chain CDRs QTIVHSDGNTY (LCDR1 , SEQ ID NO: 134), KVS (LCDR2), and FQGSHVPLT (LCDR3, SEQ ID NO: 135).

A. Exemplary immunoglobulins as included in the fusion polypeptides.

[0072] In some embodiments, with respect to the fusion polypeptide, the first binding domain comprises a full-length immunoglobulin or an antigen-binding domain thereof specific for PD-1 . The immunoglobulin, for example, may be lgG1 , lgG2 or lgG4. In further embodiments, the immunoglobulin is a monoclonal antibody against PD-1 .

[0073] Illustrative examples of PD-1 -binding antibodies of the disclosure may comprise an antigen-binding region which cross-blocks or binds to the same epitope as a PD-1 -binding antibody comprising the VH and VL regions of antibodies nivolumab (also known as ONO-4538, BMS-936558, or MDX1 106, marketed as Opdivo®), pembrolizumab (also referred to as lambrolizumab or MK03475, trade name Keytruda®), PDR001 , MEDI0680 (formerly AMP-514), pidilizumab (CT-01 1 ), ENUM-388D4 (including the D4-1 , D4- 2 and D4-3 variants), or ENUM-244C8 (including each of its variants as well), all known in the art. In another particular embodiment, a PD-1 -binding antibody of the disclosure may comprise an antigen-binding region, such as any one of the three heavy chain complementarity-determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) and the three light chain CDRs (LCDR1 , LCDR2 and LCDR3) from an antibody selected from the group consisting of nivolumab, pembrolizumab, PDR001 , MEDI0680, pidilizumab, ENUM-388D4, and ENUM-244C8.

[0074] In some embodiments, the antibody binding to PD-1 or the antigen-binding domain thereof has an antigen-binding region which cross-blocks or binds to any one of the sequences selected from the group consisting of SEQ ID NOs: 146-176.

[0075] In some embodiments, the antibody binding to PD-1 have the sequence of the

PD-1 antibody of SEQ ID NOs: 65 and 62, SEQ ID NOs: 61 and 66, SEQ ID NOs: 57 and 58, or SEQ ID NOs: 59 and 60.

[0076] In some embodiments, the PD-1 antibody or the antigen-binding domain thereof have a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 104, 106, and 108, and a light chain variable region (LCVR) selected from the group consisting of SEQ ID NOs: 105, 107, and 109.

[0077] In some embodiments, the PD-1 antibody or the antigen-binding domain have a heavy chain that is any one of SEQ ID NOs: 57, 59, 61 , 63, and 65, and a light chain that is any one of SEQ ID NOs: 58, 60, 62, 64, and 66.

[0078] In some embodiments, the heavy chain and light chain pair of the PD-1 antibody comprise a HCVR and LCVR, respectively, as follows: SEQ ID NOs: 104 and 109, and SEQ ID NOs: 108 and 105.

[0079] In some embodiments, the PD-1 antibody or the antigen-binding domain thereof will have a heavy chain variable region (HCVR) with at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 104, 106, and 108, and a light chain variable region (LCVR) with at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 105, 107, and 109. In some other embodiments, the PD-1 antibody or antigen- binding domain thereof will have a heavy chain with at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 59, 61 , 63, and 65, and a light chain with at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 60, 62, 64, and 66.

[0080] In some preferred embodiments, an antibody of the disclosure specifically binding to PD-1 is nivolumab, pembrolizumab, PDR001 , MEDI0680, pidilizumab, ENUM- 388D4, or ENUM-244C8 or the antigen-binding domain thereof.

[0081] Various patent applications disclose anti-PD-1 antibodies, production thereof, and/or methods of enhancing immune responses with such anti-PD-1 antibodies, including the following: U.S. Patent Application Publication Nos. US 2003/0039653, US 2004/0213795, US 2006/01 10383, US 2007/0065427, US 2007/0122378, US 2009/0217401 , US 201 1/0008369, and US2015/0203579 and PCT International Application Publication Nos. WO 2003/099196, WO 2006/121 168, WO 2007/005874, WO 2008/156712, WO 20091 14335, WO 2010/027423, W02 01 1/1 10604, WO 2012/145493, WO 2013/014668, WO 2014/194302, WO 2015/035606, and WO 2016/106159. The disclosure of each of these applications is hereby incorporated by reference in its entirety.

[0082] A PD-1 -binding antibody of the disclosure may be any one of the anti-PD-1 antibodies disclosed in above mentioned applications.

[0083] A PD-1 -binding antibody of the disclosure may comprise an antigen-binding region which cross-blocks or binds to the same epitope as a PD-1 -binding antibody comprising the VH and VL regions of any one of the anti-PD-1 antibodies disclosed in above mentioned applications. In another particular embodiment, the PD-1 -binding antibody may comprise an antigen-binding region, such as any one of the three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) and the three light chain CDRs (LCDR1 , LCDR2 and LCDR3), from any one of the anti-PD-1 antibodies disclosed in above mentioned applications.

[0084] In some embodiments, the heavy chain variable region of the PD-1 antibody or the antigen-binding domain thereof will have the three complementarity-determining regions (CDRs) having following sequences: GYTFTDYE (HCDR1 , SEQ ID NO: 1 16), IDPGTGGT (HCDR2, SEQ ID NO: 1 17), TSEKFGSNYYFDY (HCDR3; SEQ ID NO: 1 18). In some embodiments, the heavy chain variable region of the PD-1 antibody or the antigen- binding domain thereof will have the three complementarity-determining regions (CDRs) having following sequences: GYTFTSYW (HCDR1 , SEQ ID NO: 1 19), IDPSNSET (HCDR2, SEQ ID NO: 120), ARSRGNYAYEMDY (HCDR3; SEQ ID NO: 121 ). In some embodiments, the heavy chain variable region of the PD-1 antibody or the antigen-binding domain thereof will have the three complementarity-determining regions (CDRs) having following sequences: GYTFTDYW (HCDR1 , SEQ ID NO: 122), IDTSDSYT (HCDR2, SEQ ID NO: 123), ARRDYGGFGY (HCDR3; SEQ ID NO: 124). In some embodiments, the heavy chain variable region of the PD-1 antibody or the antigen-binding domain thereof will have the three complementarity-determining regions (CDRs) having following sequences: GYTFTDYN (HCDR1 , SEQ ID NO: 125), IDPNNGDT (HCDR2, SEQ ID NO: 126), ARWRSSMDY (HCDR3; SEQ ID NO: 127). In some embodiments, the heavy chain variable region of the PD-1 antibody or the antigen-binding domain thereof will have the three complementarity- determining regions (CDRs) having following sequences: GYSITSDYA (HCDR1 , SEQ ID NO: 128), ITYSGSP (HCDR2, SEQ ID NO: 129), ARGLGGHYFDY (HCDR3; SEQ ID NO: 130). In some embodiments, the heavy chain variable region of the PD-1 antibody or the antigen-binding domain thereof will have the three complementarity-determining regions (CDRs) having following sequences: GFSLTSYG (HCDR1 , SEQ ID NO: 131 ), IWRGGNT (HCDR2, SEQ ID NO: 132), AASMIGGY (HCDR3; SEQ ID NO: 133).

[0085] In some embodiments, the light chain variable region of the PD-1 antibody or the antigen-binding domain thereof will have the three complementarity-determining regions (CDRs) having following sequences: QTIVHSDGNTY (LCDR1 , SEQ ID NO: 134), KVS (LCDR2), FQGSHVPLT (LCDR3, SEQ ID NO: 135). In some embodiments, the light chain variable region of the PD-1 antibody or the antigen-binding domain thereof will have the three complementarity-determining regions (CDRs) having following sequences: SSVSSNY (LCDR1 , SEQ ID NO: 136), STS (LCDR2), HQWSSYPP (LCDR3, SEQ ID NO: 137). In some embodiments, the light chain variable region of the PD-1 antibody or the antigen-binding domain thereof will have the three complementarity-determining regions (CDRs) having following sequences: QDISSY (LCDR1 , SEQ ID NO: 138), YTS (LCDR2), QQYSELPW (LCDR3, SEQ ID NO: 139). In some embodiments, the light chain variable region of the PD-1 antibody or the antigen-binding domain thereof will have the three complementarity- determining regions (CDRs) having following sequences: QGISNY (LCDR1 , SEQ ID NO: 140), YTS (LCDR2), QQYSNLPW (LCDR3, SEQ ID NO: 141 ). In some embodiments, the light chain variable region of the PD-1 antibody or the antigen-binding domain thereof will have the three complementarity-determining regions (CDRs) having following sequences: QSISDY (LCDR1 , SEQ ID NO: 142), YAS (LCDR2), QNGRSYPY (LCDR3, SEQ ID NO: 143). In some embodiments, the light chain variable region of the PD-1 antibody or the antigen-binding domain thereof will have the three complementarity-determining regions (CDRs) having following sequences: QSIVHSNGNTY (LCDR1 , SEQ ID NO: 144), KVS (LCDR2), FQGSHVPL (LCDR3, SEQ ID NO: 145).

[0086] In some embodiments, the PD-1 antibody or the antigen-binding domain thereof comprises a heavy chain variably region that will have the three complementarity- determining regions (CDRs) having following sequences: GYTFTDYE (HCDR1 , SEQ ID NO: 1 16), IDPGTGGT (HCDR2, SEQ ID NO: 1 17), TSEKFGSNYYFDY (HCDR3; SEQ ID NO: 1 18), and a light chain variably region that will have the three complementarity-determining regions (CDRs) having following sequences: QTIVHSDGNTY (LCDR1 , SEQ ID NO: 134), KVS (LCDR2), FQGSHVPLT (LCDR3, SEQ ID NO: 135). In some embodiments, the PD-1 antibody or the antigen-binding domain thereof comprises a heavy chain variably region that will have the three complementarity-determining regions (CDRs) having following sequences: GYTFTSYW (HCDR1 , SEQ ID NO: 1 19), IDPSNSET (HCDR2, SEQ ID NO: 120), ARSRGNYAYEMDY (HCDR3; SEQ ID NO: 121 ), and a light chain variably region that will have the three complementarity-determining regions (CDRs) having following sequences: SSVSSNY (LCDR1 , SEQ ID NO: 136), STS (LCDR2), HQWSSYPP (LCDR3, SEQ ID NO: 137). In some embodiments, the PD-1 antibody or the antigen-binding domain thereof comprises a heavy chain variably region that will have the three complementarity- determining regions (CDRs) having following sequences: GYTFTDYW (HCDR1 , SEQ ID NO: 122), IDTSDSYT (HCDR2, SEQ ID NO: 123), ARRDYGGFGY (HCDR3; SEQ ID NO: 124), and a light chain variably region that will have the three complementarity-determining regions (CDRs) having following sequences: QDISSY (LCDR1 , SEQ ID NO: 138), YTS (LCDR2), QQYSELPW (LCDR3, SEQ ID NO: 139). In some embodiments, the PD-1 antibody or the antigen-binding domain thereof comprises a heavy chain variably region that will have the three-complementarity determining regions (CDRs) having following sequences: GYTFTDYN (HCDR1 , SEQ ID NO: 125), IDPNNGDT (HCDR2, SEQ ID NO: 126), ARWRSSMDY (HCDR3; SEQ ID NO: 127), and a light chain variably region that will have the three complementarity-determining regions (CDRs) having following sequences: QGISNY (LCDR1 , SEQ ID NO: 140), YTS (LCDR2), QQYSNLPW (LCDR3, SEQ ID NO: 141 ). In some embodiments, the PD-1 antibody or the antigen-binding domain thereof comprises a heavy chain variably region that will have the three complementarity-determining regions (CDRs) having following sequences: GYSITSDYA (HCDR1 , SEQ ID NO: 128), ITYSGSP (HCDR2, SEQ ID NO: 129), ARGLGGHYFDY (HCDR3; SEQ ID NO: 130), and a light chain variably region that will have the three complementarity-determining regions (CDRs) having following sequences: QSISDY (LCDR1 , SEQ ID NO: 142), YAS (LCDR2), QNGRSYPY (LCDR3, SEQ ID NO: 143). In some embodiments, the PD-1 antibody or the antigen-binding domain thereof comprises a heavy chain variably region that will have the three complementarity- determining regions (CDRs) having following sequences: GFSLTSYG (HCDR1 , SEQ ID NO: 131 ), IWRGGNT (HCDR2, SEQ ID NO: 132), AASMIGGY (HCDR3; SEQ ID NO: 133), and a light chain variably region that will have the three complementarity-determining regions (CDRs) having following sequences: QSIVHSNGNTY (LCDR1 , SEQ ID NO: 144), KVS (LCDR2), FQGSHVPL (LCDR3, SEQ ID NO: 145).

[0087] Unless otherwise indicated, all CDR sequences disclosed herein are defined according to the IMGT method as described in Lefranc, M.-P., The Immunologist, 7, 132-136 (1999). CDR1 consists of positions 27 to 38, CDR2 consists of positions 56 to 65, CDR3 for germline V-genes consists of positions 105 to 1 16, and CDR3 for rearranged V-J-genes or V- D-J-genes consists of positions 105 to 1 17 (position preceding J-PHE or J-TRP 1 18) with gaps at the top of the loop for rearranged CDR3-IMGT with less than 13 amino acids, or with additional positions 1 12.1 , 1 1 1.1 , 1 12.2, 1 1 1 .2, etc. for rearranged CDR3-IMGT with more than 13 amino acids. The positions given in this paragraph are according to the IMGT numbering described in Lefranc, M.-P., The Immunologist, 7, 132-136 (1999).

[0088] In some other embodiments, with respect to a PD-1 -binding antibody of the disclosure, it is preferred that the antibody having silenced effector functions has mutations in positions F234 and L235, or, in positions D265 and P329, numbering according to EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). [0089] The antibody specifically binding to PD-1 as included in the fusion polypeptides of the disclosure may comprise an Fc part which allows for extending the in vivo half-life of the bispecific binding molecule of the invention. Such Fc part is preferably from human origin, more preferably a human Fc part of an lgG1 or lgG4 antibody, even more preferably an engineered human Fc part of an lgG1 or lgG4 with activating or silencing effector functions, wherein silencing effector functions are preferred over activating effector functions. Most preferably, such an Fc part is engineered to silence effector functions with a mutation at positions 234 and/or 235, numbering according to EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). In some embodiments, mutations in positions F234 and L235 of the anti-PD-1 antibody may be introduced to silence effector functions. In other embodiments, mutations in positions D265 and P329 of the anti-PD-1 antibody may be introduced, to silence effector function. Numbering for both sets of these potential mutations is according to the EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000).

[0090] Various techniques for the production of antibodies and fragments thereof are well known in the art and described, e.g., in Altshuler et al. (2010). Thus, polyclonal antibodies can be obtained from the blood of an animal following immunization with an antigen in mixture with additives and adjuvants and monoclonal antibodies can be produced by any technique which provides antibodies produced by continuous cell line cultures. Examples for such techniques are described in, e.g., Harlow and Lane (1999), (1988), and include the hybridoma technique originally described by Kohler and Milstein, 1975, the trioma technique, the human B cell hybridoma technique (see e.g. Li et al.,Proc Natl Acad Sci U S

A, 2006, Kozbor and Roder, Immunol Today, 1983) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Cancer Res, 1984). Furthermore, recombinant antibodies may be obtained from monoclonal antibodies or can be prepared de novo using various display methods such as phage, ribosomal, mRNA, or cell display. A suitable system for the expression of the recombinant (humanized) antibodies or fragments thereof may be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, e.g., US Patent No. 6,080,560; Holliger and Hudson, Nat Biotechnol, 2005). Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent No. 4,946,778) can be adapted to produce single chain antibodies specific for the target of this invention. Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies.

B. Exemplary LAG-3-specific lipocalin muteins as included in the fusion polypeptides. [0091] As used herein, a "lipocalin" is defined as a monomeric protein of approximately 18-20 kDa in weight, having a cylindrical β-pleated sheet supersecondary structural region comprising a plurality of (preferably eight) β-strands connected pair-wise by a plurality of (preferably four) loops at one end to define thereby a binding pocket. It is the diversity of the loops in the otherwise rigid lipocalin scaffold that gives rise to a variety of different binding modes among the lipocalin family members, each capable of accommodating targets of different size, shape, and chemical character (reviewed, e.g. in Skerra, Biochim Biophys Acta, 2000, Flower et a\.,Biochim Biophys Acta, 2000, F\ower,Biochem J, 1996). Indeed, the lipocalin family of proteins have naturally evolved to bind a wide spectrum of ligands, sharing unusually low levels of overall sequence conservation (often with sequence identities of less than 20%) yet retaining a highly conserved overall folding pattern. The correspondence between positions in various lipocalins is well known to one of skill in the art (see, e.g., U.S. Patent No. 7,250,297).

[0092] As noted above, a lipocalin is a polypeptide defined by its supersecondary structure, namely cylindrical β-pleated sheet supersecondary structural region comprising eight β-strands connected pair-wise by four loops at one end to define thereby a binding pocket. The present disclosure is not limited to lipocalin muteins specifically disclosed herein. In this regard, the disclosure relates to lipocalin muteins having a cylindrical β-pleated sheet supersecondary structural region comprising eight β-strands connected pair-wise by four loops at one end to define thereby a binding pocket, wherein at least one amino acid of each of at least three of said four loops has been mutated as compared to the reference sequence, and wherein said lipocalin is effective to bind LAG-3 with detectable affinity.

[0093] In one particular embodiment, a lipocalin mutein disclosed herein is a mutein of human tear lipocalin (hTIc or TLPC), also termed lipocalin-1 , human tear pre-albumin or von Ebner gland protein. The term "human tear lipocalin" or "hTIc" or "lipocalin-1 " as used herein refers to the mature human tear lipocalin with the SWISS-PROT/UniProt Data Bank Accession Number P31025 (Isoform 1 ). The amino acid sequence shown in SwissProt UniProt Data Bank Accession Number P31025 may be used as a preferred "reference sequence," more preferably the amino acid sequence shown in SEQ ID NO: 1 is used herein as "reference sequence."

[0094] In some embodiments, a lipocalin mutein binding LAG-3 with detectable affinity may include at least one amino acid substitution of a native cysteine residue of the reference sequence by another amino acid, for example, a serine residue. In some other embodiments, a lipocalin mutein binding LAG-3 with detectable affinity may include one or more non-native cysteine residues substituting one or more amino acids of a wild-type lipocalin. In a further particular embodiment, a lipocalin mutein according to the disclosure includes at least two amino acid substitutions of a native amino acid by a cysteine residue, hereby to form one or more cysteine bridges. In some embodiments, said cysteine bridge may connect at least two loop regions. The definition of these regions is used herein in accordance with (Biochim Biophys Acta, 2000), Flower (1996) and Breustedt et al. (2005). In a related embodiment, the disclosure teaches one or more lipocalin muteins that are capable of activating downstream signaling pathways of LAG-3 by binding to LAG-3.

[0095] Proteins of the disclosure, which are directed against or specific for LAG-3, include any number of specific-binding protein muteins that are based on a defined protein scaffold, preferably a lipocalin scaffold. Also preferably, the number of nucleotides or amino acids, respectively, that is exchanged, deleted or inserted is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 50. However, it is preferred that protein muteins of the disclosure is still capable of binding LAG-3.

[0096] In one aspect, the present disclosure includes various lipocalin muteins that bind LAG-3 with at least detectable affinity. In this sense, LAG-3 can be regarded as a non- natural ligand of the reference wild-type lipocalins, where "non-natural ligand" refers to a compound that does not bind to wild type lipocalin under physiological conditions. By engineering wild-type lipocalins with one or more mutations at certain sequence positions, the present disclosure shows that high affinity and high specificity for the non-natural ligand, LAG-3, is possible. In some embodiments, at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or even more nucleotide triplet(s) encoding certain sequence positions on wild type lipocalins, a random mutagenesis may be carried out through substitution at these positions by a subset of nucleotide triplets.

[0097] Further, the lipocalin muteins of the disclosure may have a mutated amino acid residue at any one or more, including at least at any 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12, of the sequence positions corresponding to certain sequence positions of the linear polypeptide sequence of the reference lipocalin.

[0098] A lipocalin mutein of the disclosure may include the wild-type (natural) amino acid sequence of the "parental" protein scaffold (such as a lipocalin scaffold) outside the mutated amino acid sequence positions. In some embodiments, a lipocalin mutein according to the disclosure may also carry one or more amino acid mutations at one or more sequence position(s) as long as such a mutation does, at least essentially not hamper or not interfere with the binding activity and the folding of the mutein. Such mutations can be accomplished very easily on DNA level using established standard methods (Sambrook and Russell2001 , Molecular cloning: a laboratory manual). Illustrative examples of alterations of the amino acid sequence are insertions or deletions as well as amino acid substitutions. Such substitutions may be conservative, i.e., an amino acid residue is replaced with an amino acid residue of chemically similar properties, in particular with regard to polarity as well as size. Examples of conservative substitutions are the replacements among the members of the following groups: 1 ) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) iso-leucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. On the other hand, it is also possible to introduce non-conservative alterations in the amino acid sequence. In addition, instead of replacing single amino acid residues, it is also possible to either insert or delete one or more continuous amino acids of the primary structure of the reference lipocalin, preferably hTIc, as long as these deletions or insertion result in a stable, folded and functional mutein. In such mutein, for instance, one or more amino acid residues are added or deleted at the N- or C- terminus of the polypeptide (for example, hTIc muteins with truncated N- and C-terminus). Generally, such a mutein may have about at least 70%, including at least about 80%, such as at least about 85% amino acid sequence identity, with the amino acid sequence of hTIc (SEQ ID NO: 1 ). As an illustrative example, the present disclosure also encompasses hTIc muteins as defined above, in which the first four N-terminal amino acid residues of the sequence of mature human tear lipocalin (His-His-Leu-Leu; SEQ ID NO: 50; positions 1-4) and/or the last two C-terminal amino acid residues (Ser-Asp; positions 157-158) of the linear polypeptide sequence of the mature human tear lipocalin have been deleted (see, e.g., SEQ ID NOs: 7- 27, 201-214, and 240-249).

[0099] The amino acid sequence of a lipocalin mutein disclosed herein has a high sequence identity to the reference lipocalin, preferably hTIc, when compared to sequence identities with other lipocalins. In this general context, the amino acid sequence of a lipocalin mutein of the disclosure is at least substantially similar to the amino acid sequence of the reference lipocalin, with the proviso that possibly there are gaps (as defined below) in an alignment that are the result of additions or deletions of amino acids. A respective sequence of a lipocalin mutein of the disclosure, being substantially similar to the sequences of the reference lipocalin, has, in some embodiments, at least 70% identity or sequence homology, at least 75% identity or sequence homology, at least 80% identity or sequence homology, at least 82% identity or sequence homology, at least 85% identity or sequence homology, at least 87% identity or sequence homology, or at least 90% identity or sequence homology including at least 95% identity or sequence homology, to the sequence of the reference lipocalin, with the proviso that the altered position or sequence is retained and that one or more gaps are possible.

[00100] As used herein, a lipocalin mutein of the disclosure "specifically binds" a target (for example, LAG-3) if it is able to discriminate between that target and one or more reference targets, since binding specificity is not an absolute, but a relative property. "Specific binding" can be determined, for example, in accordance with western blots, ELISA, FACS, RIA (radioimmunoassay), ECL (electrochemiluminescence), IRMA (immunoradiometric assay), IHC (ImmunoHistoChemistry), and peptide scans.

[00101] In one aspect, the present disclosure provides LAG-3-binding hTIc muteins.

[00102] In this regard, the disclosure provides one or more hTIc muteins that are capable of binding LAG-3 with an affinity measured by an EC ₅₀ or K _d of about 300 nM or lower, about 80 nM or lower, about 60 nM or lower, about 40 nM or lower, about 20 nM or lower, about 15 nM or lower, about 10 nM or lower, about 8 nM or lower, about 6 nM or lower, about 4 nM or lower, about 2 nM or lower, about 1.5 nM or lower, about 1 nM or lower, about 0.2 nM or lower, about 0.1 nM or lower, about 0.05 nM or lower, or even lower.

[00103] In some embodiments, such hTIc mutein comprises mutated amino acid residue(s) 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, 27, 28, 29, 30 or more positions corresponding to positions 5, 7-8, 10, 14, 16, 25-34, 44, 46, 52-53, 55-56, 58, 60-61 , 63, 65-66, 69-70, 73, 79-80, 84-86, 89-90, 93, 96-98, 101 , 105-106, 108, 110-114, 121 , 124, 148-150, and 152-154 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ).

[00104] In some particular embodiments, such hTIc muteins may contain mutated amino acid residue(s) 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, 27, 28, 29, 30 or more positions corresponding to positions 5, 7-8, 10, 16, 26-34, 44, 46, 53, 56, 58, 60-61 , 63, 65, 69-70, 73, 79-80, 85, 89-90, 93, 96-98, 101 , 105- 106, 108, 11 1 , 1 14, 124, 148-150, and 152-154 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ).

[00105] In some particular embodiments, such hTIc muteins may contain mutated amino acid residue(s) at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or 22 positions corresponding to positions 14, 25-26, 28, 31-32, 52, 55, 58, 66, 79, 84, 86, 101 , 105-106, 108, 110, 112-1 14, and 121 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ).

[00106] In some particular embodiments, such hTIc muteins may include mutated amino acid residue(s) 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, or 26 positions corresponding to positions 5, 8, 26-34, 56, 58, 60-61 , 65, 69, 85, 101 , 105-106, 108, 11 1 , 1 14, and 153-154 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ).

[00107] In some particular embodiments, such hTIc muteins may include mutated amino acid residue(s) 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 positions corresponding to positions 14, 25-26, 28, 32, 52, 55, 58, 66, 79, 84, 86, 101 , 105, 106, 108, 110, 112, 114, and 121 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ).

[00108] In some particular embodiments, such hTIc muteins may include mutated amino acid residue(s) at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or 22 positions corresponding to positions 26-34, 56, 58, 60-61 , 65, 70, 101 , 105-106, 108, 111 , 114, and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ).

[00109] In some particular embodiments, such hTIc muteins may include mutated amino acid residue(s) at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 positions corresponding to positions 26-34, 56, 58, 60-61 , 63, 65, 101 , 105-106, 108, 111 , 1 14, 149, and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ).

[00110] In some particular embodiments, such hTIc muteins may include mutated amino acid residue(s) 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 27positions corresponding to positions 5, 7-8, 10, 16, 44, 46, 63, 65, 69-70, 73, 80, 84, 89-90, 93, 96-98, 113, 124, 148-150, 152, or 154 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ).

[00111] In some further embodiments, the hTIc muteins may comprise at least 1 , 2, 3, 4, 5, or 6 mutated amino acid residue(s) at one or more sequence positions corresponding to sequence positions 5, 8, 65, 69, 85, and 154 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ), and wherein said polypeptide binds LAG-3. [00112] In some further embodiments, the hTIc muteins may comprise at least 1 , 2, or 3 mutated amino acid residue(s) at one or more sequence positions corresponding to sequence positions 63, 65, and 149 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ), and wherein said polypeptide binds LAG-3.

[00113] In some further embodiments, the hTIc muteins may comprise at least 1 , 2, 3, or 4 mutated amino acid residue(s) at one or more sequence positions corresponding to sequence positions 26, 84, 106, and 1 12 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ), and wherein said polypeptide binds LAG-3.

[00114] In some still further embodiments, the disclosure relates to a polypeptide, wherein said polypeptide is a hTIc mutein, in comparison with the linear polypeptide sequence of hTIc (SEQ ID NO: 1 ), comprising at least 1 mutated amino acid residue(s) at the sequence position 84, and wherein said polypeptide binds LAG-3.

[00115] In some embodiments, a lipocalin mutein according to the disclosure may include at least one amino acid substitution of a native cysteine residue by, e.g., a serine residue. In some embodiments, a hTIc mutein according to the disclosure includes an amino acid substitution of a native cysteine residue at positions 61 and/or 153 by another amino acid such as a serine residue. In this context, it is noted that it has been found that removal of the structural disulfide bond (on the level of a respective naive nucleic acid library) of wild- type tear lipocalin that is formed by the cysteine residues 61 and 153 may provide tear lipocalin muteins that are not only stably folded but are also able to bind a given non-natural ligand with high affinity. In some particular embodiments, the Tic mutein according to the disclosure includes the amino acid substitutions Cys 61→ Ala, Phe, Lys, Arg, Thr, Asn, Gly, Gin, Asp, Asn, Leu, Tyr, Met, Ser, Pro or Trp, and/or Cys 153 → Ser or Ala. Such substitutions have proven useful to prevent the formation of the naturally occurring disulphide bridge linking Cys 61 and Cys 153, and thus to facilitate handling of the mutein. However, hTIc muteins that bind LAG-3 and that have the disulphide bridge formed between Cys 61 and Cys 153 are also part of the present disclosure.

[00116] In some embodiments, the elimination of the structural disulfide bond may provide further advantage of allowing for the (spontaneous) generation or deliberate introduction of non-natural artificial disulfide bonds into muteins of the disclosure, thereby increasing the stability of the muteins. For example, in some embodiments, either two or all three of the cysteine codons at position 61 , 101 and 153 are replaced by a codon of another amino acid. Further, in some embodiments, a hTIc mutein according to the disclosure includes an amino acid substitution of a native cysteine residue at position 101 by a serine residue or a histidine residue.

[00117] However, hTIc muteins that bind LAG-3 and that have the disulfide bridge formed between Cys 61 and Cys 153 are also part of the present disclosure. In some particular embodiments, hTIc muteins that do not include mutated amino acids at positions 61 and 153 and have the disulfide bond formed between Cys 61 and Cys 153. In some further particular embodiments, the hTIc muteins with mutated amino acid(s) at position(s) 61 and/or 153 are subjected to further mutagenesis to restore the natural disulfide bond by back mutating positions 61 and/or 153 to the native cysteine.

[00118] In some embodiments, a mutein according to the disclosure includes an amino acid substitution of a native amino acid by a cysteine residue at positions 28 or 105 with respect to the amino acid sequence of hTIc (SEQ ID NO: 1 ).

[00119] Further, in some embodiments, a mutein according to the disclosure includes an amino acid substitution of a native arginine residue at positions 1 1 1 by a proline residue with respect to the amino acid sequence of hTIc (SEQ ID NO: 1 ). Further, in some embodiments, a mutein according to the disclosure includes an amino acid substitution of a native lysine residue at positions 1 14 by a tryptophan residue or a glutamic acid with respect to the amino acid sequence of hTIc (SEQ ID NO: 1 ).

[00120] In some embodiments, a lipocalin mutein according to the disclosure may include one or more amino acid mutated to an asparagine residue to introduce one or more glycosylation sites. In some preferred embodiments, a mutein according to the disclosure includes an amino acid mutation at position 12 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ). For example, a mutein according to the disclosure may have the following mutated amino acid residue with respect to the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ): Asp 12→ Asn.

[00121] In some embodiments, a mutein according to the disclosure includes an amino acid substitution at position 5 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ). For example, a mutein according to the disclosure may have the following mutated amino acid residue with respect to the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ): Ala 5→Thr.

[00122] Further, in some embodiments, a mutein according to the disclosure may include at least one amino acid substitution of a native negatively charged residue by neutural residue, wherein the native negatively charged residue is not involved in binding to LAG-3, and wherein the substitution results in an increased isoelectric point (pi) of the mutein. In some particular embodiments, such native negatively charged residues and positions include Asp 7, Glu 9, Asp 12, Glu 45, Asp 72, Glu 73, Asp 80, and Asp 95 with respect to the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ). In some particular embodiments, such neutural amino acid residues include Asn, Arg, and Lys. In some further particular embodiments, a mutein according to the disclosure includes one or more of the following mutated amino acid residues at position 7, 9, 12, 45, 72, 73, 80, and 95 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ): Asp 7→ Asn, Arg, or Lys; Glu 9→ Gin, Arg, or Lys; Asp 12→ Asn or Arg; Glu 45→ Arg; Asp 72→ Asn, Arg, or Lys; Glu 73→ Arg; Asp 80→ Gly; and Asp 95→ Asn, Arg, or Lys.

[00123] In some embodiments, a LAG-3-binding hTIc mutein according to the disclosure includes, at one or more positions corresponding to positions 5, 7-8, 10, 14, 16, 25-34, 44, 46, 52-53, 55-56, 58, 60-61 , 63, 65-66, 69-70, 73, 79-80, 84-86, 89-90, 93, 96-98, 101 , 105-106, 108, 110-1 14, 121 , 124, 148-150, and 152-154 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ), one or more of the following mutated amino acid residues: Ala 5→ Thr; Asp 7→ Gly; Glu 8→ Gin; lie 10→ Phe; Ser 14→ Pro; Thr 16→ Met; Asp 25→ Ser; Arg 26→ Ser, Asp, Glu, Ala, or Gly; Glu 27→ Asp; Phe 28→ Cys or Asp; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie or Leu; Asn 32→ Asp, Met, or Thr; Leu 33→ Asp; Glu 34→ Val; Leu 44→ His; Gly 46→ Asp; Lys 52→ Arg; Val 53→ Ala; Met 55→ Val; Leu 56→ Asp; Ser 58→ Phe or Asp; Arg 60→ Phe; Cys 61→ Trp; Glu 63→ Asp; Lys 65→ Glu; Ala 66→ Asn; Glu 69→ Gly; Lys 70→ Arg; Glu 73→ Ala; Ala 79→ Thr or Glu; Asp 80→ Gly; His 84→ Tyr; Val 85→ Ala or Asp; Ala 86→ Asp; lie 89→ Ser or Asn; Arg 90→ Ser; Val 93→ Glu; His 96→ Asn; Tyr 97→ His; lie 98→ Val; Cys 101→ Ser or Phe; Leu 105→ Cys or Gly; His 106→ Ala, Gin, Glu, Lys, or Pro; Lys 108→ Tyr or Thr; Val 110→ Gly or Asn; Arg 111→ Pro; Gly 112→ Met, Val, or Leu; Val 113→ Ala or Leu; Lys 1 14→ Trp or Ala; Lys 121→ Thr; Leu 124→ Gin; Arg 148→ Trp; Gin 149→ Leu; Ser 150→ Gly; Thr 152→ Pro; Cys 153→ Ser; and Ser 154→ Ala. In some embodiments, a hTIc mutein according to the disclosure includes two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or even more such as 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 or even more mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1 )-

[00124] In some embodiments, a LAG-3-binding hTIc mutein according to the disclosure includes, at one or more positions corresponding to positions 14, 25-26, 28, 31- 32, 52, 55, 58, 66, 79, 84, 86, 101 , 105-106, 108, 1 10, 112-114, and 121 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ), one or more of the following mutated amino acid residues: Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Ser, Asp, Glu, Ala, or Gly; Phe 28→ Asp; Met 31→ Leu; Asn 32→ Met or Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Tyr or Leu; Ala 86→ Asp; Cys 101→ Phe; Leu 105 → Gly; His 106→ Gin, Glu, Lys, or Pro; Lys 108→ Thr; Val 110→ Gly or Asn; Gly 1 12→ Met, Val, or Leu; Val 113 → Ala or Leu; Lys 114 → Ala; Lys 121 → Thr. In some embodiments, a hTIc mutein according to the disclosure includes two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or even more such as 13, 14, 15, 16, 17, 18, 19, 20, 21 , or 22 mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1 ).

[00125] In some embodiments, a LAG-3-binding hTIc mutein according to the disclosure includes, at one or more positions corresponding to positions 5, 7-8, 10, 16, 26- 34, 44, 46, 53, 56, 58, 60-61 , 63, 65-66, 69-70, 73, 79-80, 85, 89-90, 93, 96-98, 101 , 105- 106, 108, 110-111 , 114, 121 , 124, 148-150, and 152-154 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ), one or more of the following mutated amino acid residues: Ala 5→ Thr; Asp 7→ Gly; Glu 8→ Gin; lie 10→ Phe; Thr 16→ Met; Arg 26→ Ser; Glu 27 → Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33 → Asp; Glu 34→ Val; Leu 44→ His; Gly 46→ Asp; Val 53→ Ala; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Glu 63→ Asp; Lys 65→ Glu; Glu 69→ Gly; Lys 70→ Arg; Glu 73→ Ala; Ala 79→ Thr; Asp 80→ Gly; Val 85→ Ala or Asp; lie 89→ Ser or Asn; Arg 90→ Ser; Val 93→ Glu; His 96→ Asn; Tyr 97→ His; lie 98→ Val; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111 → Pro; Lys 114→ Trp; Leu 124→ Gin; Arg 148→ Trp; Gin 149→ Leu; Ser 150→ Gly; Thr 152→ Pro; Cys 153→ Ser; and Ser 154→ Ala. In some embodiments, a hTIc mutein according to the disclosure includes two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or even more such as 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 or even more mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1 ).

[00126] some embodiments, a LAG-3-binding hTIc mutein according to the disclosure includes, at one or more positions corresponding to positions 5, 7-8, 10, 16, 26-34, 44, 46, 53, 56, 58, 60-61 , 63, 65, 69-70, 73, 79-80, 85, 89-90, 93, 96-98, 101 , 105-106, 108, 111 , 114, 124, 148-150, and 152-154 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ), one or more of the following mutated amino acid residues: Ala 5→ Thr; Asp 7→ Gly; Glu 8→ Gin; lie 10→ Phe; Thr 16→ Met; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 44 → His; Gly 46→ Asp; Val 53→ Ala; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Glu 63→ Asp; Lys 65→ Glu; Glu 69→ Gly; Lys 70→ Arg; Glu 73→ Ala; Ala 79→ Thr; Asp 80→ Gly; Val 85→ Ala or Asp; lie 89→ Ser or Asn; Arg 90→ Ser; Val 93→ Glu; His 96 → Asn; Tyr 97→ His; lie 98→ Val; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108 → Tyr; Arg 111 → Pro; Lys 114→ Trp; Leu 124→ Gin; Arg 148→ Trp; Gin 149→ Leu; Ser 150→ Gly; Thr 152→ Pro; Cys 153→ Ser; and Ser 154→ Ala. In some embodiments, a hTIc mutein according to the disclosure includes two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, or even more such as 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 or even more mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1 )-

[00127] In some embodiments, the LAG-3 binding hTIc muteins include the following amino acid mutations in comparison with the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ): Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31 → lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111→ Pro; Lys 1 14→ Trp; Cys 153→ Ser; and further one or more, including 2, 3, 4, 5, 6, or 7, or even more, of the following amino acid mutations: Ala 5→ Thr; Asp 7→ Gly; Glu 8→ Gin; lie 10→ Phe; Thr 16→ Met; Leu 44→ His; Gly 46→ Asp; Val 53→ Ala; Glu 63→ Asp; Lys 65→ Glu; Glu 69→ Gly; Lys 70→ Arg; Glu 73→ Ala; Ala 79→ Thr; Asp 80 → Gly; Val 85→ Ala or Asp; lie 89→ Ser or Asn; Arg 90→ Ser; Val 93→ Glu; His 96→ Asn; Tyr 97→ His; lie 98→ Val; Leu 124→ Gin; Arg 148→ Trp; Gin 149→ Leu; Ser 150→ Gly; Thr 152→ Pro; and Ser 154→ Ala.

[00128] In some embodiments, the LAG-3 binding hTIc muteins include the following amino acid mutations in comparison with the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ): Ser 14→ Pro; Asp 25→ Ser; Phe 28→ Asp; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; Ala 86→ Asp; Cys 101→ Phe; Leu 105→ Gly; Lys 108→ Thr; Lys 114→ Ala; Lys 121→ Thr; and one or more, including 2, 3, 4, 5, 6, 7, or even more, of the following amino acid mutations: Arg 26→ Ser, Asp, Glu, Ala, or Gly; Met 31 → Leu; Asn 32→ Thr; Leu 56→ Asp; His 84→ Tyr or Leu; His 106→ Gin, Glu, Lys, or Pro; Val 110→ Gly or Asn; Gly 112→ Met, Val or Leu; Val 113→ Ala or Leu.

[00129] In some embodiments, the LAG-3 binding hTIc muteins include the following amino acid mutations in comparison with the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ): Ser 14→ Pro; Asp 25→ Ser; Phe 28→ Asp; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; Ala 86→ Asp; Cys 101→ Phe; Leu 105→ Gly; Lys 108→ Thr; Lys 114→ Ala; Lys 121→ Thr; and one or more, including 2, 3, 4, 5, 6, 7, or even more, of the following amino acid mutations: Arg 26→ Ser, Asp, Glu, Ala, or Gly; Met

31→ Leu; Asn 32→ Thr; His 84→ Tyr or Leu; His 106→ Gin, Glu, Lys, or Pro; Val 110→ Gly or Asn; Gly 112→ Met, Val or Leu; Val 113→ Ala or Leu.

[00130] In some embodiments, the LAG-3 binding hTIc muteins include one of the following amino acid mutations in comparison with the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ): Ser 14→ Pro; Asp 25→ Ser; Phe 28→ Asp; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; Ala 86→ Asp; Cys 101→ Phe; Leu 105→ Gly; Lys 108→ Thr; Lys 114→ Ala; Lys 121→ Thr; and one or more, including 2, 3, 4, 5, 6, 7 of the following amino acid mutations: Arg 26→ Ser, Asp, Glu, or Ala; Met 31 → Leu; Asn

32→ Thr; Leu 56→ Asp; His 84→ Tyr or Leu; His 106→ Glu, Lys, or Pro; Val 110→ Asn; Gly 112→ Val or Leu; Val 113→ Ala or Leu.

[00131] In some additional embodiments, the LAG-3 binding hTIc muteins include one of the following sets of amino acid mutations in comparison with the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ):

(a) Ala 5→ Thr; Glu 8→ Gin; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31 → lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Lys 65→ Glu; Glu 69→ Gly; Val 85→ Ala; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 11 1→ Pro; Lys 114→ Trp; Cys 153→ Ser; and Ser 154→ Ala;

(b) Ala 5→ Thr; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Gly 46→ Asp; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Lys 65→ Glu; Val 85→ Ala; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111→ Pro; Lys 1 14→ Trp; Ser 150→ Gly; and Cys 153→ Ser;

(c) Asp 7→ Gly; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Val 85→ Asp; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111 → Pro; Lys 114→ Trp; Arg 148→ Trp; Thr 152→ Pro; and Cys 153→ Ser; Ala 5→ Thr; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Val 53→ Ala; Leu 56 → Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Lys 65→ Glu; Ala 79→ Thr; Tyr 97→ His; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111→ Pro; Lys 114→ Trp; and Cys 153→ Ser;

Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Glu 63→ Asp; Val 85→ Asp; Arg 90→ Ser; His 96→ Asn; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111 → Pro; Lys 1 14→ Trp; Leu 124→ Gin; and Cys 153→ Ser;

Thr 16→ Met; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 44→ His; Leu 56 → Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Lys 65→ Glu; lie 89→ Ser; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111 → Pro; Lys 1 14→ Trp; and Cys 153→ Ser;

Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Glu 63→ Asp; Lys 65→ Glu; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111→ Pro; Lys 114→ Trp; Gin 149→ Leu; and Cys 153→ Ser;

Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Lys 65→ Glu; Lys 70→ Arg; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111 → Pro; Lys 114→ Trp; and Cys 153→ Ser;

Ala 5→ Thr; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Lys 65→ Glu; Asp 80→ Gly; lie 89→ Asn; lie 98→ Val; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→Tyr; Arg 111 → Pro; Lys 114→ Trp; and Cys 153→ Ser;

lie 10→ Phe; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Lys 65→ Glu; Glu 73→ Ala; lie 89→ Asn; Val 93→ Glu; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111→ Pro; Lys 114→ Trp; and Cys 153→ Ser; Ala 5→ Thr; Glu 8→ Gin; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31 → lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Lys 65→ Glu; Glu 69→ Gly; Val 85→ Ala; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111→ Pro; Lys 114→ Trp; Cys 153→ Ser; and Ser 154→ Ala;

Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108 → Tyr; Arg 111→ Pro; Lys 114→ Trp; and Cys 153→ Ser;

Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Asp; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Tyr; Ala 86 → Asp; Cys 101 → Phe; Leu 105→ Gly; Lys 108→ Thr; Val 110→ Gly; Gly 1 12→ Met; Lys 114→ Ala; and Lys 121→ Thr;

Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Glu; Phe 28→ Asp; Met 31→ Leu; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84 → Tyr; Ala 86→ Asp; Cys 101 → Phe; Leu 105→ Gly; His 106→ Gin; Lys 108→ Thr; Val 110→ Gly; Gly 112→ Met; Lys 114→ Ala; and Lys 121→ Thr;

Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Glu; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Tyr; Ala 86 → Asp; Cys 101→ Phe; Leu 105→ Gly; His 106→ Glu; Lys 108→ Thr; Val 110→ Gly; Gly 112→ Val; Lys 114→ Ala; and Lys 121→ Thr;

Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Asp; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Tyr; Ala 86 → Asp; Cys 101→ Phe; Leu 105→ Gly; His 106→ Gin; Lys 108→ Thr; Val 110→ Gly; Gly 112→ Leu; Lys 114→ Ala; and Lys 121→ Thr;

Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Ser; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Tyr; Ala 86 → Asp; Cys 101 → Phe; Leu 105→ Gly; His 106→ Gin; Lys 108→ Thr; Val 110→ Gly; Gly 112→ Met; Lys 114→ Ala; and Lys 121→ Thr;

Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Ala; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Tyr; Ala 86 → Asp; Cys 101 → Phe; Leu 105→ Gly; His 106→ Lys; Lys 108→ Thr; Val 1 10→ Gly; Gly 112→ Met; Lys 114→ Ala; and Lys 121→ Thr;

Ser 14→ Pro; Asp 25→ Ser; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; Ala 86→ Asp; Cys 101→ Phe; Leu 105→ Gly; His 106→ Gin; Lys 108→ Thr; Val 110→ Asn; Gly 112→ Met; Val 1 13 →Ala; Lys 114→Ala; and Lys 121→Thr;

(t) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Gly; Phe 28→ Asp; Met 31→ Leu; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84 → Tyr; Ala 86→ Asp; Cys 101 → Phe; Leu 105→ Gly; His 106→ Pro; Lys 108→ Thr; Val 110→ Gly; Gly 112→ Met; Lys 114→ Ala; and Lys 121→ Thr;

(u) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Asp; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Leu; Ala 86 → Asp; Cys 101→ Phe; Leu 105→ Gly; His 106→ Gin; Lys 108→ Thr; Val 110→ Gly; Gly 112→ Met; Val 113→ Leu; Lys 114→ Ala; and Lys 121→ Thr;

(v) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Gly; Phe 28→ Asp; Asn 32→ Met; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; Ala 86→ Asp; Cys 101 → Phe; Leu 105→ Gly; His 106→ Gin; Lys 108→ Thr; Val 110→ Gly; Gly 112 → Met; Lys 114→ Ala; and Lys 121→ Thr; or

(w) Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Glu 63→ Asp; Lys 65→ Glu; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 111→ Pro; Lys 114→ Trp; and Gin 149→ Leu.

[00132] In some additional embodiments, the LAG-3 binding hTIc muteins include one of the following sets of amino acid mutations in comparison with the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1 ):

(a) Ala 5→ Thr; Glu 8→ Gin; Lys 65→ Glu; Glu 69→ Gly; Val 85→ Ala; and Ser 154 →Ala;

(b) Ala 5→ Thr; Gly 46→ Asp; Lys 65→ Glu; Val 85→ Ala; and Ser 150→ Gly;

(c) Asp 7→ Gly; Val 85→ Asp; Arg 148→ Trp; and Thr 152→ Pro;

(d) Ala 5→ Thr; Val 53→ Ala; Lys 65→ Glu; Ala 79→ Thr; and Tyr 97→ His

(e) Glu 63→Asp; Val 85→ Asp; Arg 90→ Ser; His 96→ Asn; and Leu 124→ Gin;

(f) Thr 16→ Met; Leu 44→ His; Lys 65→ Glu; and lie 89→ Ser;

(g) Glu 63→ Asp; Lys 65→ Glu; and Gin 149→ Leu;

(h) Lys 65→ Glu and Lys 70→ Arg;

(i) Ala 5→ Thr; Lys 65→ Glu; Asp 80→ Gly; lie 89→ Asn; and lie 98→ Val;

G) lie 10→ Phe; Lys 65→ Glu; Glu 73→ Ala; lie 89→ Asn; and Val 93→ Glu; (k) Arg 26→Asp; Asn 32→Thr; His 84→ Tyr; Val 110→ Gly; and Gly 112→ Met;

(I) Arg 26→ Glu; Met 31 → Leu; Asn 32→ Thr; His 84→ Tyr; His 106→ Gin; Val 110 → Gly; and Gly 112→ Met;

(m) Arg 26→ Glu; Asn 32→ Thr; His 84→ Tyr; His 106→ Glu; and Gly 112→ Val;

(n) Arg 26→ Asp; Asn 32→ Thr; His 84→ Tyr; His 106→ Gin; Val 110→ Gly; and Gly 1 12→ Leu;

(o) Arg 26→ Ser; Asn 32→ Thr; His 84→ Tyr; His 106→ Gin; Val 110→ Gly; and Gly 1 12→ Met;

(p) Arg 26→ Ala; Asn 32→ Thr; His 84→ Tyr; His 106→ Lys; Val 110→ Gly; and Gly 1 12→ Met;

(q) Asn 32→ Thr; His 106→ Gin; Val 110→ Asn; Gly 112→ Met; and Val 113→ Ala;

(r) Arg 26→ Gly; Met 31→ Leu; Asn 32→ Thr; His 84→ Tyr; His 106→ Pro; Val 110→ Gly; and Gly 112→ Met; or

(s) Arg 26→ Asp; Asn 32→ Thr; His 84→ Leu; His 106→ Gin; Val 1 10→ Gly; Gly 112 →Met; and Val 113→ Leu.

[00133] In some additional embodiments the LAG-3 binding hTIc mutein includes the following amino acid mutation in comparison with the linear polypeptide sequence of the hTIc (SEQ ID NO: 1 ): insertion of Pro between positions 156 and 157.

[00134] In the residual region, i.e., the region differing from sequence positions 5, 7-8, 10, 14, 16, 25-34, 44, 46, 52-53, 55-56, 58, 60-61 , 63, 65-66, 69-70, 73, 79-80, 84-86, 89-90, 93, 96-98, 101 , 105-106, 108, 110-114, 121 , 124, 148-150, 152-154, and 157 a hTIc mutein of the disclosure may include the wild-type (natural) amino acid sequence of mature hTIc (SEQ ID NO: 1 ) outside the mutated amino acid sequence positions or mutated amino acid residues at such positions.

[00135] Unless otherwise indicated, the position of a residue of a hTIc mutein described herein is numbered in comparison with the linear polypeptide sequence of the hTIc (SEQ ID NO: 1 ).

[00136] In still further embodiments, a hTIc mutein according to the current disclosure has at least 70% sequence identity or at least 70% sequence homology to the sequence of mature hTIc (SEQ ID NO: 1 ). As an illustrative example, the mutein of the SEQ ID NO: 8 has an amino acid sequence identity or a sequence homology of approximately 81.8% with the amino acid sequence of mature hTIc (SEQ ID NO: 1 ).

[00137] In further particular embodiments, a hTIc mutein of the disclosure comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 7-27, 201-214, and 240- 249 or a fragment or variant thereof.

[00138] In further particular embodiments, a hTIc mutein of the disclosure has at least 75%, at least 80%, at least 85% or higher, at least 90% or higher, at least 95% or higher, at least 97.5% or higher, at least 98% or higher or at least 99% or higher sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-17, 19-27, 201-214, and 240-249.

[00139] The disclosure also includes structural homologues of a hTIc mutein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-27, 201-214, and 240-249 which structural homologues have an amino acid sequence homology or sequence identity of more than about 60%, preferably more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92%, and most preferably more than 95% in relation to said hTIc mutein.

[00140] A hTIc mutein according to the present disclosure can be obtained by means of mutagenesis of a naturally occurring form of mature hTIc (SEQ ID NO: 1 ). In some embodiments of the mutagenesis, a substitution (or replacement) is a conservative substitution. Nevertheless, any substitution— including non-conservative substitution or one or more from the exemplary substitutions below— is envisaged as long as the lipocalin mutein retains its capability to bind to LAG-3, and/or it has a sequence identity to the then substituted sequence in that it is at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or higher sequence identity to the amino acid sequence of mature hTIc (SEQ ID NO: 1 ).

[00141] In some particular embodiments, the present disclosure provides a lipocalin mutein that binds human LAG-3 with an affinity measured by a K _d of about 10 nM or lower, 5 nM or lower, 4 nM or lower, 3 nM or lower, 2 nM or lower, 1 nM or lower, 0.5 nM or lower, 0.1 nM or lower or 0.05 nM or lower. In some embodiments, the lipocalin mutein has at least 90% or higher, such as 95% or higher, 97.5% or higher, 98% or higher, or 99% or higher sequence identity to the amino acid sequence of any one of SEQ ID NOs: 7 and 18.

[00142] In one embodiment, the lipocalin muteins of the disclosure are fused at its N- terminus and/or its C-terminus to a fusion partner which is a protein domain that extends the serum half-life of the mutein. In further particular embodiments, the protein domain is an Fc part of an immunoglobulin, a C _H 3 domain of an immunoglobulin, a C _H 4 domain of an immunoglobulin, an albumin binding peptide or an albumin binding protein.

[00143] In another embodiment, the lipocalin muteins of the disclosure are conjugated to a compound that extends the serum half-life of the mutein. More preferably, the muteins are conjugated to a compound selected from the group consisting of a polyalkylene glycol molecule, a polyethylene glycol molecule, a hydroxyethyl starch, an Fc part of an immunoglobulin, a C _H 3 domain of an immunoglobulin, a C _H 4 domain of an immunoglobulin, an albumin binding peptide, and an albumin binding protein.

[00144] In yet another embodiment, the current disclosure relates to a nucleic acid molecule comprising a nucleotide sequence encoding a lipocalin mutein disclosed herein. The disclosure encompasses a host cell containing said nucleic acid molecule.

C. Exemplary uses, applications and production of fusion polypeptides specific for LAG -3 and PD-1.

[00145] It has been reported that LAG-3 plays an important role in promoting regulatory T cell (Treg) activity and in negatively regulating T cell activation and proliferation (Workman and Vignali,J Immunol, 2005). Both natural and induced Treg express elevated level of LAG-3, which is required for their maximal suppressive function (Huang et al., Immunity, 2004, Camisaschi et al.,J Immunol, 2010). Furthermore, ectopic expression of LAG-3 on CD4 ⁺ effector T cells reduces their proliferative capacity and confers on their regulatory potential against third party T cells (Huang et al., Immunity, 2004). Recent studies have also shown that high LAG-3 expression on exhausted lymphocytic choriomeningitis virus (LCMV)-specific CD8 ⁺ T cells contributes to their unresponsive state and limits CD8 ⁺ T cell antitumor responses (Grosso et al.,J Clin Invest, 2007, Blackburn et a\.,Nat Immunol, 2009). In fact, LAG-3 maintained tolerance to self and tumor antigens via direct effects on CD8 ⁺ T cells in two murine models (Grosso et al., J Clin Invest, 2007).

[00146] Immune tolerance observed in the setting of tumor development and tumor recurrence, however, seems to be mediated by the co-expression of various T cell negative regulatory receptors, not solely by LAG-3. Data from chronic viral infection models (Lyford- Pike et al., Cancer Res, 2013, Grosso et al.,J Clin Invest, 2007, Blackburn et a\.,Nat Immunol, 2009), knock-out mice (Woo et al., Cancer Res, 2012, Bettini et al., J Immunol, 2011 , Okazaki et al.,J Exp Med, 201 1 ), tumor recurrence models (Goding et al.,J Immunol, 2013) and, to a more limited extent, human cancer patients (Gandhi et al., Blood, 2006, Matsuzaki et al.,Proc Natl Acad Sci U S A, 2010, Goding et al.,J Immunol, 2013) support a model wherein T cells that are continuously exposed to antigens become progressively inactivated through a process termed "exhaustion". Exhausted T cells are characterized by the expression of T cell negative regulatory receptors, predominantly PD-1 , and LAG-3, whose action is to limit the cell's ability to proliferate, produce cytokines, and kill target cells and/or to increase Treg activity. However, the timing and sequence of expression of these molecules in the development and recurrence of tumors have not been fully characterized.

[00147] PD-1 is a cell surface signaling receptor that plays a critical role in the regulation of T cell activation and tolerance (Keir et a\.,Annu Rev Immunol, 2008). It is a type I transmembrane protein and together with BTLA, CTLA-4, ICOS, and CD28, comprise the CD28 family of T cell co-stimulatory receptors. PD-1 is primarily expressed on activated T cells, B cells, and myeloid cells (Dong et al., Nat Med, 1999). It is also expressed on natural killer (NK) cells (Terme et a\., Cancer Res, 2011 ).

[00148] Binding of PD-1 by its ligands, PD-L1 and PD-L2, results in phosphorylation of the tyrosine residue in the proximal intracellular immune receptor tyrosine inhibitory domain, followed by recruitment of the phosphatase SHP-2, eventually resulting in down-regulation of T cell activation. One important role of PD-1 is to limit the activity of T cells in peripheral tissues at the time of an inflammatory response to infection, thus limiting the development of autoimmunity (Pardoll, Λ/af Rev Cancer, 2012). Evidence of this negative regulatory role comes from the finding that PD-1 -deficient mice develop lupus-like autoimmune diseases including arthritis and nephritis, along with cardiomyopathy (Nishimura et a\., Science, 2001 , Nishimura et al., Immunity, 1999). In the tumor setting, the consequence is the development of immune resistance within the tumor microenvironment. PD-1 is highly expressed on tumor-infiltrating lymphocytes, and its ligands are up-regulated on the cell surface of many different tumors (Dong et a\.,Nat Med, 2002). Multiple murine cancer models have demonstrated that binding of ligand to PD-1 results in immune evasion. In addition, blockade of this interaction results in anti-tumor activity (Hamid et a\.,N Engl J Med, 2013, Topalian et a\.,N Engl J Med, 2012).

[00149] There is a strong synergy between the PD-1 and LAG-3 inhibitory pathways intolerance to both self and tumor antigens, therefore, dual blockade of the targets represents a promising combinatorial strategy for cancer (Woo et al., Cancer Res, 2012).

[00150] By simultaneously targeting immune checkpoints PD-1 and LAG-3, the fusion polypeptide of the disclosure may generate a durable anti-tumor and/or anti-infection response, increase anti-tumor lymphocyte cell activity, and enhance anti-tumor immunity, thereby produce synergistic anti-tumor results.

[00151] Numerous possible applications for the fusion polypeptides of the disclosure, therefore, exist in medicine. In some embodiments, fusion polypeptides of the disclosure may produce synergistic effects through dual-targeting of PD-1 and LAG-3.

[00152] In one aspect, the disclosure relates to the use of the fusion polypeptides disclosed herein for detecting PD-1 and LAG-3 in a sample as well as a corresponding method of diagnosis.

[00153] In another aspect, the disclosure features the use of one or more fusion polypeptides disclosed herein or of one or more compositions comprising such polypeptides for simultaneously binding of PD-1 and LAG-3.

[00154] The present disclosure also involves the use of one or more fusion polypeptides as described for complex formation with PD-1 and LAG-3.

[00155] Therefore, in a still further aspect of the disclosure, the disclosed one or more fusion polypeptides are used for the detection of PD-1 and LAG-3. Such use may include the steps of contacting one or more said fusion polypeptides, under suitable conditions, with a sample suspected of containing PD-1 and LAG-3, thereby allowing the formation of a complex between the fusion polypeptides and PD-1 and LAG-3, and detecting the complex by a suitable signal. The detectable signal can be caused by a label, as explained above, or by a change of physical properties due to the binding, i.e., the complex formation, itself. One example is surface plasmon resonance, the value of which is changed during binding of binding partners from which one is immobilized on a surface such as a gold foil.

[00156] The fusion polypeptides disclosed herein may also be used for the separation of PD-1 and LAG-3. Such use may include the steps of contacting one or more said fusion polypeptides, under suitable conditions, with a sample supposed to contain PD-1 and LAG-3, thereby allowing the formation of a complex between the fusion polypeptides and PD-1 and LAG-3, and separating the complex from the sample.

[00157] In still another aspect, the present disclosure features a diagnostic or analytical kit comprising a fusion polypeptide according to the disclosure.

[00158] In addition to their use in diagnostics, in yet another aspect, the disclosure contemplates a pharmaceutical composition comprising a fusion polypeptide of the disclosure and a pharmaceutically acceptable excipient. [00159] Furthermore, the present disclosure provides fusion polypeptides that simultaneously bind PD-1 and LAG-3 for use as anti-infection and/or anti-cancer agents and immune modulators. The fusion polypeptides of the present disclosure are envisaged to be used in a method of treatment or prevention of human diseases, such as a variety of tumors and autoinflammation in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of one or more fusion polypeptides of the disclosure.

[00160] Examples of cancers that may be treated using the fusion polypeptides of the disclosure, include liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular 20 cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of 25 childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, hematologic malignancies 30 including, for example, multiple myeloma, B cell lymphoma, Hodgkin lymphoma/primary mediastinal B-cell lymphoma, non- Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combinations of said cancers. The present invention is also applicable to the treatment of metastatic cancers.

[00161] In one embodiment, the human patient suffers from non-small cell lung cancer (NSCLC) or virally-related cancer (e.g., a human papillomavirus (HPV)-related tumor) or gastric adenocarcinoma. In a particular embodiment, the HPV-related tumor is HPV+ head and neck cancer (HNC). In another particular embodiment, the gastric adenocarcinoma is associated with Epstein-Barr virus (EBV) infection. [00162] In another embodiment, the present disclosure also relates to nucleic acid molecules (DNA and RNA) that include nucleotide sequences encoding the fusion polypeptides disclosed herein. In yet another embodiment, the disclosure encompasses a host cell containing said nucleic acid molecule. Since the degeneracy of the genetic code permits substitutions of certain codons by other codons specifying the same amino acid, the disclosure is not limited to a specific nucleic acid molecule encoding a fusion polypeptide as described herein but encompasses all nucleic acid molecules that include nucleotide sequences encoding a functional polypeptide. In this regard, the present disclosure also relates to nucleotide sequences encoding the fusion polypeptides of the disclosure.

[00163] In some embodiments, a nucleic acid molecule encoding a lipocalin mutein disclosed in this application, such as DNA, may be "operably linked" to another nucleic acid molecule encoding an immunoglobulin of the disclosure to allow expression of a fusion polypeptide disclosed herein. In this regard, an operable linkage is a linkage in which the sequence elements of one nucleic acid molecule and the sequence elements of another nucleic acid molecule are connected in a way that enables expression of the fusion polypeptide as a single polypeptide.

[00164] The disclosure also relates to a method for the production the fusion polypeptides of the disclosure starting from the nucleic acid coding for the polypeptides or any subunits therein by means of genetic engineering methods. In some embodiments, the method can be carried out in vivo, wherein the fusion polypeptide can, for example, be produced in a bacterial or eukaryotic host organism, and then isolated from this host organism or its culture. It is also possible to produce a fusion polypeptide of the disclosure in vitro, for example, by using an in vitro translation system.

[00165] When producing the fusion polypeptide in vivo, a nucleic acid encoding such polypeptide is introduced into a suitable bacterial or eukaryotic host organism by means of recombinant DNA technology (as already outlined above). For this purpose, the host cell is first transformed with a cloning vector that includes a nucleic acid molecule encoding a fusion polypeptide as described herein using established standard methods. The host cell is then cultured under conditions, which allow expression of the heterologous DNA and thus the synthesis of the corresponding polypeptide. Subsequently, the polypeptide is recovered either from the cell or the cultivation medium.

[00166] In one embodiment of the disclosure, the method includes subjecting at least one nucleic acid molecule encoding fusion polypeptides to mutagenesis at nucleotide triplets coding for at least one, sometimes even more, of the sequence positions corresponding to the sequence positions 5, 7-8, 10, 14, 16, 25-34, 44, 46, 52-53, 55-56, 58, 60-61 , 63, 65- 66, 69-70, 73, 79-80, 84-86, 89-90, 93, 96-98, 101 , 105-106, 108, 110-114, 121 , 124, 148-150, 152-154, and 157 of the linear polypeptide sequence of hTIc (SEQ ID NO: 1 ), as included in the fusion polypeptides.

[00167] In addition, with respect to hTIc muteins of the disclosure as included in the fusion polypeptides, the naturally occurring disulfide bond between Cys 61 and Cys 153 may be removed. Accordingly, such muteins can be produced in a cell compartment having a reducing redox milieu, for example, in the cytoplasm of Gram-negative bacteria.

[00168] The disclosure also includes nucleic acid molecules encoding the lipocalin muteins of the disclosure, which include additional mutations outside the indicated sequence positions of experimental mutagenesis. Such mutations are often tolerated or can even prove to be advantageous, for example, if they contribute to an improved folding efficiency, serum stability, thermal stability or ligand binding affinity of the lipocalin muteins.

[00169] A nucleic acid molecule disclosed in this application may be "operably linked" to one or more regulatory sequence(s) to allow expression of this nucleic acid molecule.

[00170] A nucleic acid molecule, such as DNA, is referred to as "capable of expressing a nucleic acid molecule" or "able to allow expression of a nucleotide sequence" if it includes sequence elements that contain information regarding to transcriptional and/or translational regulation, and such sequences are "operably linked" to the nucleotide sequence encoding the polypeptide. An operable linkage is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression. The precise nature of the regulatory regions necessary for gene expression may vary among species, but in general these regions include a promoter, which, in prokaryotes, contains both the promoter per se, i.e., DNA elements directing the initiation of transcription, as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation. Such promoter regions normally include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35/-10 boxes and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5'-capping elements in eukaryotes. These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell. [00171] In addition, the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactory functional in a particular host cell, then they may be substituted with signals functional in that cell.

[00172] Therefore, a nucleic acid molecule of the disclosure can include a regulatory sequence, such as a promoter sequence. In some embodiments, a nucleic acid molecule of the disclosure includes a promoter sequence and a transcriptional termination sequence. Suitable prokaryotic promoters are, for example, the tet promoter, the lacUV5 promoter or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.

[00173] The nucleic acid molecules of the disclosure can also be part of a vector or any other kind of cloning vehicle, such as a plasmid, a phagemid, a phage, a baculovirus, a cosmid, or an artificial chromosome.

[00174] In one embodiment, the nucleic acid molecule is included in a phasmid. A phasmid vector denotes a vector encoding the intergenic region of a temperate phage, such as M13 or f1 , or a functional part thereof fused to the cDNA of interest. After superinfection of the bacterial host cells with such an phagemid vector and an appropriate helper phage (e.g., M13K07, VCS-M13 or R408) intact phage particles are produced, thereby enabling physical coupling of the encoded heterologous cDNA to its corresponding polypeptide displayed on the phage surface (Lowman,/\nni/ Rev Biophys Biomol Struct, 1997, Rodi and Makowski, Curr Opin Biotechnol, 1999).

[00175] Such cloning vehicles can include, aside from the regulatory sequences described above and a nucleic acid sequence encoding a fusion polypeptide as described herein, replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art, and are commercially available.

[00176] The DNA molecule encoding a fusion polypeptide as described herein, for example, SEQ ID NOs: 89-103, 183, 195-200, 237 (fusion polypeptides), SEQ ID NOs: 28- 49, 215-228, and 250-260 (LAG-3 specific lipocalin muteins that can be included in fusion polypeptides), SEQ ID NOs: 67-72 (PD-1 specific antibody light chains and heavy chains that can be included in fusion polypeptides), SEQ ID NOs: 110-115 (PD-1 specific heavy chain variable regions and light chain variable regions that can be included in fusion polypeptides), SEQ ID NO: 184 (LAG-3 specific lipocalin mutein fused to the Fc region of an antibody), and in particular a cloning vector containing the coding sequence of such a polypeptide can be transformed into a host cell capable of expressing the gene. Transformation can be performed using standard techniques. Thus, the disclosure is also directed to a host cell containing a nucleic acid molecule as disclosed herein.

[00177] The transformed host cells are cultured under conditions suitable for expression of the nucleotide sequence encoding a fusion polypeptide of the disclosure. Suitable host cells can be prokaryotic, such as Escherichia coli (£. coli) or Bacillus subtilis, or eukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) or primary mammalian cells.

[00178] In some embodiments, where a lipocalin mutein of the disclosure, including as comprised in a fusion polypeptide disclosed herein, includes intramolecular disulfide bonds, it may be preferred to direct the nascent polypeptide to a cell compartment having an oxidizing redox milieu using an appropriate signal sequence. Such an oxidizing environment may be provided by the periplasm of Gram-negative bacteria such as £. coli, in the extracellular milieu of Gram-positive bacteria or the lumen of the endoplasmic reticulum of eukaryotic cells and usually favors the formation of structural disulfide bonds.

[00179] In some embodiments, it is also possible to produce a fusion polypeptide of the disclosure in the cytosol of a host cell, preferably £ coli. In this case, the polypeptide can either be directly obtained in a soluble and folded state or recovered in the form of inclusion bodies, followed by renaturation in vitro. A further option is the use of specific host strains having an oxidizing intracellular milieu, which may thus allow the formation of disulfide bonds in the cytosol (Venturi et al.,J Mol Biol, 2002).

[00180] In some embodiments, a fusion polypeptide of the disclosure as described herein may be not necessarily generated or produced only by use of genetic engineering. Rather, such polypeptide can also be obtained by chemical synthesis such as Merrifield solid phase polypeptide synthesis or by in vitro transcription and translation. It is, for example, possible that promising fusion polypeptides and/or lipocalin muteins included in such fusion polypeptides are identified using molecular modeling, synthesized in vitro, and investigated for the binding activity for the target(s) of interest. Methods for the solid phase and/or solution phase synthesis of proteins are well known in the art . [00181] In another embodiment, a fusion polypeptide of the disclosure may be produced by in vitro transcription/translation employing well-established methods known to those skilled in the art.

[00182] The skilled worker will appreciate methods useful to prepare fusion polypeptides contemplated by the present disclosure but whose protein or nucleic acid sequences are not explicitly disclosed herein. As an overview, such modifications of the amino acid sequence include, e.g., directed mutagenesis of single amino acid positions in order to simplify sub-cloning of a polypeptide gene or its parts by incorporating cleavage sites for certain restriction enzymes. In addition, these mutations can also be incorporated to further improve the affinity of a fusion polypeptide for its targets (e.g., PD-1 and LAG-3). Furthermore, mutations can be introduced to modulate certain characteristics of the polypeptide such as to improve folding stability, serum stability, protein resistance or water solubility or to reduce aggregation tendency, if necessary. For example, naturally occurring cysteine residues may be mutated to other amino acids to prevent disulfide bridge formation.

[00183] The fusion polypeptides of the disclosure may be prepared by any of the many conventional and well-known techniques such as synthetic strategies, solid phase-assisted synthesis techniques or by commercially available automated synthesizers. On the other hand, they may also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques. A fusion polypeptide according to the present disclosure may be obtained by combining compounds as defined in chapters (A) and (B) hereinabove.

[00184] Additional objects, advantages, and features of this disclosure will become apparent to those skilled in the art upon examination of the following Examples and the attached Figures thereof, which are not intended to be limiting. Thus, it should be understood that although the present disclosure is specifically disclosed by exemplary embodiments and optional features, modification and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art and that such modifications and variations are considered to be within the scope of this disclosure.

[00186] The present invention may further be characterized by the following items:

Item 1 . A fusion polypeptide that is capable of binding both PD-1 and LAG-3, wherein the fusion polypeptide comprises at least two subunits in any order, wherein the first subunit is specific for PD-1 and the second subunit is specific for LAG-3. Item 2. The fusion polypeptide of item 1 , wherein the first subunit comprises a full-length immunoglobulin or an antigen-binding domain thereof having binding specificity for PD-1 , and wherein the second subunit comprises a lipocalin mutein having binding specificity for LAG-3.

Item 3. The fusion polypeptide of item 1 or 2, wherein the fusion polypeptide is able to bind LAG-3 expressed on a cell with an EC ₅₀ value of at most about 0.1 nM.

Item 4. The fusion polypeptide of any one of items 1 -3, wherein the fusion polypeptide is able to bind LAG-3 expressed on a cell with an EC ₅₀ value of at most about 0.05 nM.

Item 5. The fusion polypeptide of any one of items 1 -4, wherein the fusion polypeptide is able to bind LAG-3 expressed on a cell with an EC ₅₀ value of at most about 0.04 nM.

Item 6. The fusion polypeptide of any one of items 1 -5, wherein the fusion polypeptide is able to bind LAG-3 expressed on a cell with an EC ₅₀ value of at most about 0.03 nM.

Item 7. The fusion polypeptide of any one of items 3-6, wherein the EC ₅₀ value is determined by fluorescence activated cell sorting (FACS).

Item 8. The fusion polypeptide of any one of items 1-7, wherein the second subunit is a LAG-3-specific lipocalin mutein comprising at least two mutated amino acid residues at the sequence positions 5, 7-8, 10, 14, 16, 25-34, 44, 46, 52-53, 55-56, 58, 60- 61 , 63, 65-66, 69-70, 73, 79-80, 84-86, 89-90, 93, 96-98, 101 , 105-106, 108, 1 10-1 14, 121 , 124, 148-150, 152-154, and 156-157 of the linear polypeptide sequence of human tear lipocalin (SEQ ID NO: 1 ).

Item 9. The fusion polypeptide of any one of items 1-8, wherein the second subunit is a LAG-3-specific lipocalin mutein comprising at least one mutated amino acid residues at the sequence positions 14, 25-26, 28, 31-32, 52, 55, 58, 66, 79, 84, 86, 101 , 105-106, 108, 110, 112-114, and 121 of the linear polypeptide sequence of human tear lipocalin (SEQ ID NO: 1 ).

Item 10. The fusion polypeptide of any one of items 1-8, wherein the second subunit is a LAG-3-specific lipocalin mutein comprising at least one mutated amino acid residues at the sequence positions 5, 7-8, 10, 16, 26-34, 44, 46, 53, 56, 58, 60-61 , 63, 65- 66, 69-70, 73, 79-80, 85, 89-90, 93, 96-98, 101 , 105-106, 108, 110-111 , 114, 121 , 124, 148-150, 152-154, and 156-157 of the linear polypeptide sequence of human tear lipocalin (SEQ ID NO: 1 ).

Item 1 1 . The fusion polypeptide of any one of items 1-8, wherein the second subunit is a LAG-3-specific lipocalin mutein comprising at least one mutated amino acid residues at the sequence positions 5, 7-8, 10, 16, 44, 46, 63, 65, 69-70, 73, 80, 84, 89-90, 93, 96-98, 113, 124, 148-150, 152, 154, and 156-157 of the linear polypeptide sequence of hTIc (SEQ ID NO: 1 ).

Item 12. The fusion polypeptide of any one of items 1-1 1 , wherein the second subunit is a LAG-3-specific lipocalin mutein comprising at least one of the following amino acid residue mutations in comparison with the linear polypeptide sequence of the human tear lipocalin (SEQ ID NO: 1 ): Ala 5→ Thr; Asp 7→ Gly; Glu 8→ Gin; lie 10→ Phe; Ser 14→ Pro; Thr 16→ Met; Asp 25→ Ser; Arg 26→ Ser, Asp, Glu, Ala, or Gly; Glu 27→ Asp; Phe 28→ Cys or Asp; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie or Leu; Asn 32→ Asp, Met or Thr; Leu 33→ Asp; Glu 34→ Val; Leu 44→ His; Gly 46→ Asp; Lys 52→ Arg; Val 53→ Ala; Met 55→ Val; Leu 56→ Asp; Ser 58 → Phe or Asp; Arg 60→ Phe; Cys 61 → Trp; Glu 63→ Asp; Lys 65→ Glu; Ala 66 → Asn; Glu 69→ Gly; Lys 70→ Arg; Glu 73→ Ala; Ala 79→ Thr or Glu; Asp 80→ Gly; His 84→ Tyr or Leu; Val 85→ Ala or Asp; Ala 86→ Asp; lie 89→ Ser or Asn; Arg 90→ Ser; Val 93→ Glu; His 96→ Asn; Tyr 97→ His; lie 98→ Val; Cys 101 → Ser or Phe; Leu 105→ Cys or Gly; His 106→ Ala, Gin, Glu, Lys, or Pro; Lys 108 → Tyr or Thr; Val 1 10→ Gly or Asn; Arg 1 1 1 → Pro; Gly 1 12→ Met, Val, or Leu; Val 1 13→ Ala or Leu; Lys 1 14→ Trp or Ala; Lys 121→ Thr; Leu 124→ Gin; Arg 148→ Trp; Gin 149→ Leu; Ser 150→ Gly; Thr 152→ Pro; Cys 153→ Ser; Ser 154→ Ala; insertion of Pro between positions 156 and 157.

Item 13. The fusion polypeptide of any one of items 1-12, wherein the second subunit is a LAG-3-specific lipocalin mutein comprising at least one of the following mutated amino acid residues in comparison with the linear polypeptide sequence of human tear lipocalin (SEQ ID NO: 1 ): Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Ser, Asp, Glu, Ala, or Gly; Phe 28→ Asp; Met 31→ Leu; Asn 32→ Met or Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Tyr or Leu; Ala 86→ Asp; Cys 101 → Phe; Leu 105→ Gly; His 106→ Gin, Glu, Lys, or Pro; Lys 108→ Thr; Val 1 10→ Gly or Asn; Gly 1 12→ Met, Val, or Leu; Val 1 13→ Ala or Leu; Lys 1 14→ Ala; Lys 121→ Thr.

Item 14. The fusion polypeptide of any one of items 1-12, wherein the second subunit is a LAG-3-specific lipocalin mutein comprising at least one of the following mutated amino acid residues in comparison with the linear polypeptide sequence of human tear lipocalin (SEQ ID NO: 1 ): Ala 5→ Thr; Asp 7→ Gly; Glu 8→ Gin; lie 10→ Phe; Thr 16→ Met; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31 → lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 44 → His; Gly 46→ Asp; Val 53→ Ala; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Glu 63→ Asp; Lys 65→ Glu; Glu 69→ Gly; Lys 70→ Arg; Glu 73 → Ala; Ala 79→ Thr; Asp 80→ Gly; Val 85→ Ala or Asp; lie 89→ Ser or Asn; Arg 90→ Ser; Val 93→ Glu; His 96→ Asn; Tyr 97→ His; lie 98→ Val; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 1 1 1 → Pro; Lys 1 14→ Trp; Leu 124→ Gin; Arg 148→ Trp; Gin 149→ Leu; Ser 150→ Gly; Thr 152→ Pro; Cys 153→ Ser; Ser 154→ Ala; insertion of Pro between positions 156 and 157.

Item 15. The fusion polypeptide of any one of items 1-12, wherein the second subunit is a LAG-3-specific lipocalin mutein comprising the following amino acid mutations: Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31 → lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108 → Tyr; Arg 1 1 1 → Pro; Lys 1 14→ Trp; Cys 153→ Ser; and one or more of the following amino acid mutations: Ala 5→ Thr; Asp 7→ Gly; Glu 8→ Gin; lie 10→ Phe; Thr 16→ Met; Leu 44→ His; Gly 46→ Asp; Val 53→ Ala; Glu 63→ Asp; Lys 65→ Glu; Glu 69→ Gly; Lys 70→ Arg; Glu 73→ Ala; Ala 79→ Thr; Asp 80→ Gly; Val 85→ Ala or Asp; lie 89→ Ser or Asn; Arg 90→ Ser; Val 93→ Glu; His 96→ Asn; Tyr 97→ His; lie 98→ Val; Leu 124→ Gin; Arg 148→ Trp; Gin 149→ Leu; Ser 150→ Gly; Thr 152→ Pro; Ser 154→ Ala; insertion of Pro between positions 156 and 157.

Item 16. The fusion polypeptide of any one of items 1-12, wherein the second subunit is a LAG-3-specific lipocalin mutein comprising the following amino acid mutations: Ser 14→ Pro; Asp 25→ Ser; Phe 28→ Asp; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; Ala 86→ Asp; Cys 101→ Phe; Leu 105→ Gly; Lys 108→ Thr; Lys 1 14→ Ala; Lys 121 → Thr; and one or more of the following amino acid mutations: Arg 26→ Ser, Asp, Glu, or Ala; Met 31 → Leu; Asn 32→ Thr; Leu 56→ Asp; His 84→ Tyr or Leu; His 106→ Glu, Lys, or Pro; Val 1 10→ Asn; Gly 1 12→ Val or Leu; Val 1 13→ Ala or Leu. Item 17. The fusion polypeptide of any one of items 1-12, wherein the second subunit is a LAG-3-specific lipocalin mutein comprising one of the following sets of amino acid residue mutations in comparison with the linear polypeptide sequence of the human tear lipocalin (SEQ ID NO: 1 ):

(a) Ala 5→ Thr; Glu 8→ Gin; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29 → Phe; Glu 30→ Trp; Met 31 → lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Lys 65→ Glu; Glu 69→ Gly; Val 85→ Ala; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 1 1 1 → Pro; Lys 1 14→ Trp; Cys 153→ Ser; Ser 154 → Ala; insertion of Pro between positions 156 and 157;

(b) Ala 5→ Thr; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31 → lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Gly 46 → Asp; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Lys 65→ Glu; Val 85→ Ala; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 1 1 1 → Pro; Lys 1 14→ Trp; Ser 150→ Gly; Cys 153→ Ser; insertion of Pro between positions 156 and 157;

(c) Asp 7→ Gly; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31 → lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56 → Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Val 85→ Asp; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 1 1 1 → Pro; Lys 1 14→ Trp; Arg 148→ Trp; Thr 152→ Pro; Cys 153→ Ser; insertion of Pro between positions 156 and 157;

(d) Ala 5→ Thr; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Val 53→ Ala; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Lys 65→ Glu; Ala 79→ Thr; Tyr 97→ His; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 1 1 1 → Pro; Lys 1 14→ Trp; Cys 153→ Ser; insertion of Pro between positions 156 and 157;

(e) Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp;

Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Glu 63→ Asp; Val 85→ Asp; Arg 90 → Ser; His 96→ Asn; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108 → Tyr; Arg 1 1 1→ Pro; Lys 1 14→ Trp; Leu 124→ Gin; Cys 153→ Ser;

(f) Thr 16→ Met; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe;

Glu 30→ Trp; Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 44→ His; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Lys 65 → Glu; lie 89→ Ser; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108 → Tyr; Arg 1 1 1→ Pro; Lys 1 14→ Trp; Cys 153→ Ser;

(g) Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp;

Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Glu 63→ Asp; Lys 65→ Glu; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 1 1 1 → Pro; Lys 1 14 → Trp; Gin 149 → Leu; Cys 153 → Ser; insertion of Pro between positions 156 and 157;

(h) Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp;

Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Lys 65→ Glu; Lys 70→ Arg; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 1 1 1 → Pro; Lys 1 14→ Trp; Cys 153→ Ser;

(i) Ala 5→ Thr; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31 → lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56 → Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Lys 65→ Glu; Asp 80→ Gly; lie 89→ Asn; lie 98→ Val; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 1 1 1 → Pro; Lys 1 14→ Trp; Cys 153→ Ser; insertion of Pro between positions 156 and 157;

(j) lie 10→ Phe; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp; Met 31 → lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56 → Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61→ Trp; Lys 65→ Glu; Glu 73→ Ala; lie 89→ Asn; Val 93→ Glu; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 1 1 1→ Pro; Lys 1 14→ Trp; Cys 153→ Ser;

(k) Ala 5→ Thr; Glu 8→ Gin; Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29 → Phe; Glu 30→ Trp; Met 31 → lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Lys 65→ Glu; Glu 69→ Gly; Val 85→ Ala; Cys 101→ Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 1 1 1 → Pro; Lys 1 14→ Trp; Cys 153→ Ser; Ser 154 → Ala; insertion of Pro between positions 156 and 157;

(I) Arg 26→ Ser; Glu 27→ Asp; Phe 28→ Cys; Pro 29→ Phe; Glu 30→ Trp;

Met 31→ lie; Asn 32→ Asp; Leu 33→ Asp; Glu 34→ Val; Leu 56→ Asp; Ser 58→ Phe; Arg 60→ Phe; Cys 61 → Trp; Cys 101 → Ser; Leu 105→ Cys; His 106→ Ala; Lys 108→ Tyr; Arg 1 1 1→ Pro; Lys 1 14→ Trp; Cys 153→ Ser; (m) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Asp; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Tyr; Ala 86→ Asp; Cys 101 → Phe; Leu 105→ Gly; Lys 108→ Thr; Val 1 10→ Gly; Gly 1 12→ Met; Lys 1 14→ Ala; Lys 121→Thr;

(n) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Glu; Phe 28→ Asp; Met 31 → Leu;

Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Tyr; Ala 86→ Asp; Cys 101 → Phe; Leu 105→ Gly; His 106→ Gin; Lys 108→ Thr; Val 1 10→ Gly; Gly 1 12→ Met; Lys 1 14→ Ala; Lys 121→Thr;

(o) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Glu; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84 → Tyr; Ala 86→ Asp; Cys 101→ Phe; Leu 105→ Gly; His 106→ Glu; Lys 108 →Thr; Val 1 10→ Gly; Gly 1 12→ Val; Lys 1 14→ Ala; Lys 121→ Thr;

(p) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Asp; Phe 28→ Asp; Asn 32→ Thr;

Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Tyr; Ala 86→ Asp; Cys 101→ Phe; Leu 105→ Gly; His 106→ Gin; Lys 108→ Thr; Val 1 10→ Gly; Gly 1 12→ Leu; Lys 1 14→ Ala; Lys 121→ Thr;

(q) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Ser; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84 → Tyr; Ala 86→ Asp; Cys 101→ Phe; Leu 105→ Gly; His 106→ Gin; Lys 108 →Thr; Val 1 10→ Gly; Gly 1 12→ Met; Lys 1 14→ Ala; Lys 121→ Thr;

(r) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Ala; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84 → Tyr; Ala 86→ Asp; Cys 101→ Phe; Leu 105→ Gly; His 106→ Lys; Lys 108 →Thr; Val 1 10→ Gly; Gly 1 12→ Met; Lys 1 14→ Ala; Lys 121→ Thr;

(s) Ser 14→ Pro; Asp 25→ Ser; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; Ala 86→ Asp; Cys 101 → Phe; Leu 105→ Gly; His 106→ Gin; Lys 108→ Thr; Val 1 10→ Asn; Gly 1 12→ Met; Val 1 13→ Ala; Lys 1 14→ Ala; Lys 121→Thr;

(t) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Gly; Phe 28→ Asp; Met 31 → Leu;

Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Tyr; Ala 86→ Asp; Cys 101 → Phe; Leu 105→ Gly; His 106→ Pro; Lys 108→ Thr; Val 1 10→ Gly; Gly 1 12→ Met; Lys 1 14→ Ala; Lys 121→Thr; (u) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Asp; Phe 28→ Asp; Asn 32→ Thr; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; His 84→ Leu; Ala 86→ Asp; Cys 101→ Phe; Leu 105→ Gly; His 106→ Gin; Lys 108→ Thr; Val 1 10→ Gly; Gly 1 12→ Met; Val 1 13→ Leu; Lys 1 14→ Ala; Lys 121→Thr;

(v) Ser 14→ Pro; Asp 25→ Ser; Arg 26→ Gly; Phe 28→ Asp; Asn 32→ Met; Lys 52→ Arg; Met 55→ Val; Ser 58→ Asp; Ala 66→ Asn; Ala 79→ Glu; Ala 86 → Asp; Cys 101 → Phe; Leu 105→ Gly; His 106→ Gin; Lys 108→ Thr; Val 1 10→ Gly; Gly 1 12→ Met; Lys 1 14→ Ala; Lys 121→ Thr.

Item 18. The fusion polypeptide of any one of items 1-12, wherein the second subunit is a LAG-3-specific lipocalin mutein comprising one of the following sets of amino acid residue mutations in comparison with the linear polypeptide sequence of the human tear lipocalin (SEQ ID NO: 1 ):

(a) Ala 5→ Thr; Glu 8→ Gin; Lys 65→ Glu; Glu 69→ Gly; Val 85→ Ala; Ser 154 → Ala; insertion of Pro between positions 156 and 157;

(b) Ala 5→ Thr; Gly 46→ Asp; Lys 65→ Glu; Val 85→ Ala; Ser 150→ Gly; insertion of Pro between positions 156 and 157;

(c) Asp 7→ Gly; Val 85→ Asp; Arg 148→ Trp; Thr 152→ Pro; insertion of Pro between positions 156 and 157;

(d) Ala 5 → Thr; Val 53 → Ala; Lys 65 → Glu; Ala 79 → Thr; Tyr 97 → His; insertion of Pro between positions 156 and 157;

(e) Glu 63→ Asp; Val 85→ Asp; Arg 90→ Ser; His 96→ Asn; Leu 124→ Gin;

(f) Thr 16→ Met; Leu 44→ His; Lys 65→ Glu; lie 89→ Ser;

(g) Glu 63 → Asp; Lys 65 → Glu; Gin 149 → Leu; insertion of Pro between positions 156 and 157;

(h) Lys 65→ Glu; Lys 70→ Arg;

(i) Ala 5→ Thr; Lys 65→ Glu; Asp 80→ Gly; lie 89→ Asn; lie 98→ Val; insertion of Pro between positions 156 and 157;

G) Me 10→ Phe; Lys 65→ Glu; Glu 73→ Ala; lie 89→ Asn; Val 93→ Glu;

(k) Arg 26→ Asp; Asn 32→ Thr; His 84→ Tyr; Val 1 10→ Gly; Gly 1 12→ Met; (I) Arg 26→ Glu; Met 31→ Leu; Asn 32→ Thr; His 84→ Tyr; His 106→ Gin; Val

1 10→ Gly; Gly 1 12→ Met;

(m) Arg 26→ Glu; Asn 32→ Thr; His 84→ Tyr; His 106→ Glu; Gly 1 12→ Val; (n) Arg 26→ Asp; Asn 32→ Thr; His 84→ Tyr; His 106→ Gin; Val 1 10→ Gly; Gly 1 12→ Leu;

(o) Arg 26→ Ser; Asn 32→ Thr; His 84→ Tyr; His 106→ Gin; Val 1 10→ Gly; Gly 1 12→ Met;

(p) Arg 26→ Ala; Asn 32→ Thr; His 84→ Tyr; His 106→ Lys; Val 1 10→ Gly; Gly 1 12→ Met;

(q) Asn 32→ Thr; His 106→ Gin; Val 1 10→ Asn; Gly 1 12→ Met; Val 1 13→ Ala; (r) Arg 26→ Gly; Met 31→ Leu; Asn 32→ Thr; His 84→ Tyr; His 106→ Pro; Val

1 10→ Gly; Gly 1 12→ Met; or

(s) Arg 26→ Asp; Asn 32→ Thr; His 84→ Leu; His 106→ Gin; Val 1 10→ Gly;

Gly 1 12→ Met; Val 1 13→ Leu.

Item 19. The fusion polypeptide of any one of items 1-18, wherein the LAG-3-specific lipocalin mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-17 and 19-27 or of a fragment or variant thereof.

Item 20. The fusion polypeptide of any one of items 1-18, wherein the LAG-3-specific lipocalin mutein has at least 85%, at least 90%, at least 95%, at least 97.5% or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-17 and 19-27.

Item 21 . The fusion polypeptide of any one of items 1-20, wherein one subunit can be linked to another subunit as essentially described in Figure 1 via a linker.

Item 22. The fusion polypeptide of item 21 , wherein the peptide bond is an unstructured (Gly- Gly-Gly-Gly-Ser) ₃ linker (SEQ ID NO: 54).

Item 23. The fusion polypeptide of any one of items 1-22, wherein the first subunit is a monoclonal antibody.

Item 24. The fusion polypeptide of item 23, wherein the monoclonal antibody comprises an antigen-binding region which cross-blocks or binds to the same epitope as a PD-1- binding antibody comprising the VH and VL regions of antibodies nivolumab, pembrolizumab, PDR001 , MEDI0680, pidilizumab, ENUM-388D4, or ENUM-244C8.

Item 25. The fusion polypeptide of item 23, wherein the monoclonal antibody comprises the same three heavy chain CDRs and three light chain CDRs (LCDR1 , LCDR2 and LCDR3) as an antibody selected from the group consisting of nivolumab, pembrolizumab, PDR001 , MEDI0680, pidilizumab, ENUM-388D4, and ENUM- 244C8. Item 26. The fusion polypeptide of item 23, wherein the monoclonal antibody has an antigen- binding region which cross-blocks or binds to any one of the sequences selected from the group consisting of SEQ ID NOs: 146-176.

Item 27. The fusion polypeptide of item 23, wherein the monoclonal antibody comprises the amino acid sequence of SEQ ID NOs: 65 and 62, SEQ ID NOs: 61 and 66, SEQ ID NOs: 57 and 58, or SEQ ID NOs: 59 and 60.

Item 28. The fusion polypeptide of item 23, wherein the variable region of the heavy chain of the monoclonal antibody is selected from a group consisting of SEQ ID NOs: 104, 106, or 108, and wherein the variable region of the light chain of the monoclonal antibody is selected from a group consisting of SEQ ID NOs: 105, 107, or 109.

Item 29. The fusion polypeptide of item 23, wherein the monoclonal antibody comprises a heavy chain comprising that is any one of SEQ ID NOs: 57, 59, 61 , 63, and 65 and a light chain that is any one of SEQ ID NOs: 58, 60, 62, 64, and 66.

Item 30. The fusion polypeptide of item 23, wherein the monoclonal antibody comprises a HCVR and LCVR, respectively, as follows: SEQ ID NOs: 61 and 66, or SEQ ID NOs: 65 and 62.

Item 31 . The fusion polypeptide of item 23, wherein the variable region of the heavy chain of the monoclonal antibody has at least 70% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 104, 106, or 108 and wherein the variable region of the light chain of the monoclonal antibody has at least 70% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 105, 107, or 109.

Item 32. The fusion polypeptide of item 23, wherein the heavy chain of the monoclonal antibody has at least 70 % sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 59, 61 , 63, and 65 and wherein the light chain of the monoclonal antibody has at least 70% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 60, 62, 64, and 66.

Item 33. The fusion polypeptide of item 23, wherein the heavy chain variable region of the monoclonal antibody has following CDR sequences:

(a) HCDR1 : GYTFTDYE (SEQ ID NO: 1 16), HCDR2: IDPGTGGT (SEQ ID NO:

1 17), HCDR3: TSEKFGSNYYFDY (SEQ ID NO: 1 18);

(b) HCDR1 : GYTFTSYW (HCDR1 , SEQ ID NO: 1 19), HCDR2: IDPSNSET (SEQ ID NO: 120), HCDR3: ARSRGNYAYEMDY (SEQ ID NO: 121 ); (c) HCDR1 : GYTFTDYW (SEQ ID NO: 122), HCDR2: IDTSDSYT (SEQ ID NO: 123), HCDR3: ARRDYGGFGY (SEQ ID NO: 124);

(d) HCDR1 : GYTFTDYN (SEQ ID NO: 125), HCDR2: IDPNNGDT (SEQ ID NO:

126), HCDR3: ARWRSSMDY (SEQ ID NO: 127);

(e) HCDR1 : GYSITSDYA (SEQ ID NO: 128), HCDR2: ITYSGSP (SEQ ID NO:

129), HCDR3: ARGLGGHYFDY (SEQ ID NO: 130); or

(f) HCDR1 : GFSLTSYG (SEQ ID NO: 131 ), HCDR2: IWRGGNT (SEQ ID NO:

132), HCDR3: AASMIGGY (SEQ ID NO: 133).

Item 34. The fusion polypeptide of item 23, wherein the light chain variable region of the monoclonal antibody has following CDR sequences:

(a) LCDR1 : QTIVHSDGNTY (SEQ ID NO: 134), LCDR2: KVS, LCDR3:

FQGSHVPLT (SEQ ID NO: 135);

(b) LCDR1 : SSVSSNY (SEQ ID NO: 136), LCDR2: STS, LCDR3: HQWSSYPP (SEQ ID NO: 137);

(c) LCDR1 : QDISSY (SEQ ID NO: 138), LCDR2: YTS, LCDR3: QQYSELPW (SEQ ID NO: 139);

(d) LCDR1 : QGISNY (SEQ ID NO: 140), LCDR2: YTS, LCDR3: QQYSNLPW (SEQ ID NO: 141 );

(e) LCDR1 : QSISDY (SEQ ID NO: 142), LCDR2: YAS, LCDR3: QNGRSYPY (SEQ ID NO: 143); or

(f) LCDR1 : QSIVHSNGNTY (SEQ ID NO: 144), LCDR2: KVS, LCDR3:

FQGSHVPL (SEQ ID NO: 145).

Item 35. The fusion polypeptide of item 23, wherein the monoclonal antibody has following CDR sequences:

(a) HCDR1 : GYTFTDYE (SEQ ID NO: 1 16), HCDR2: IDPGTGGT (SEQ ID NO:

1 17), HCDR3: TSEKFGSNYYFDY (SEQ ID NO: 1 18), LCDR1 : QTIVHSDGNTY (SEQ ID NO: 134), LCDR2: KVS, LCDR3: FQGSHVPLT (SEQ ID NO: 135);

(b) HCDR1 : GYTFTSYW (SEQ ID NO: 1 19), HCDR2: IDPSNSET (SEQ ID NO:

120), HCDR3: ARSRGNYAYEMDY (SEQ ID NO: 121 ), LCDR1 : SSVSSNY (SEQ ID NO: 136), LCDR2: STS, LCDR3: HQWSSYPP (SEQ ID NO: 137);

(c) HCDR1 : GYTFTDYW (SEQ ID NO: 122), HCDR2: IDTSDSYT (SEQ ID NO:

123), HCDR3: ARRDYGGFGY (SEQ ID NO: 124), LCDR1 : QDISSY (SEQ ID NO: 138), LCDR2: YTS, LCDR3: QQYSELPW (SEQ ID NO: 139); (d) HCDR1 : GYTFTDYN (SEQ ID NO: 125), HCDR2: IDPNNGDT (SEQ ID NO: 126), HCDR3: ARWRSSMDY (SEQ ID NO: 127), LCDR1 : QGISNY (SEQ ID NO: 140), LCDR2: YTS, LCDR3: QQYSNLPW (SEQ ID NO: 141 );

(e) HCDR1 : GYSITSDYA (SEQ ID NO: 128), HCDR2: ITYSGSP (SEQ ID NO:

129), HCDR3: ARGLGGHYFDY (SEQ ID NO: 130), LCDR1 : QSISDY (SEQ ID NO: 142), LCDR2: YAS, LCDR3: QNGRSYPY (SEQ ID NO: 143); or

(f) HCDR1 : GFSLTSYG (SEQ ID NO: 131 ), HCDR2: IWRGGNT (SEQ ID NO:

132), HCDR3: AASMIGGY (SEQ ID NO: 133), LCDR1 : QSIVHSNGNTY (SEQ ID NO: 144), LCDR2: KVS, LCDR3: FQGSHVPL (SEQ ID NO: 145).

Item 36. The fusion polypeptide of item 23, wherein the monoclonal antibody is selected from the group consisting of nivolumab, pembrolizumab, PDR001 , MEDI0680, pidilizumab, ENUM-388D4, and ENUM-244C8.

Item 37. The fusion polypeptide of item 23, wherein the monoclonal antibody has an lgG1 , lgG2, lgG3 or lgG4 backbone.

Item 38. The fusion polypeptide of item 37, wherein the lgG4 backbone has any one of the following mutations selected from the group consisting of S228P, N297A, F234A and L235A.

Item 39. The fusion polypeptide of item 23, wherein the monoclonal antibody has an lgG1 backbone.

Item 40. The fusion polypeptide of item 39, wherein the lgG1 backbone has any one of the following mutations selected from the group consisting of N297A, L234A and L235A.

Item 41 . The fusion polypeptide of any one of items 1-23, wherein the fusion polypeptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 74-88 and 177-181 .

Item 42. The fusion polypeptide of any one of items 1-23, wherein the fusion polypeptide comprises the amino acids shown in SEQ ID NOs: 74 and 66, or the amino acids shown in SEQ ID NOs: 61 and 75, or the amino acids shown in SEQ ID NOs: 76 and 66, or the amino acids shown in SEQ ID NOs: 61 and 77, or the amino acids shown in SEQ ID NOs: 78 and 62, or the amino acids shown in SEQ ID NOs: 65 and 79, or the amino acids shown in SEQ ID NOs: 80 and 62, or the amino acids shown in SEQ ID NOs: 65 and 81 , or the amino acids shown in SEQ ID NOs: 78 and 79, or the amino acids shown in SEQ ID NOs: 57 and 84, or the amino acids shown in SEQ ID NOs: 85 and 66, or the amino acids shown in SEQ ID NOs: 61 and 86, or the amino acids shown in SEQ ID NOs: 59 and 87, or the amino acids shown in SEQ ID NOs: 88 and 60, or the amino acids shown in SEQ ID NOs: 57 and 181 ; or the amino acids shown in SEQ ID NOs: 185 and 66, or the amino acids shown in SEQ ID NOs: 61 and 187, or the amino acids shown in SEQ ID NOs: 189 and 62, or the amino acids shown in SEQ ID NOs: 65 and 191 , or the amino acids shown in SEQ ID NOs: 193 and 62, or the amino acids shown in SEQ ID NOs: 65 and 194.

Item 43. The fusion polypeptide of any one of items 1 -42, wherein the fusion polypeptide is able to bind PD-1 with an EC ₅₀ value of at most about 1 nM.

Item 44. The fusion polypeptide of any one of items item 1 -42, wherein the fusion polypeptide is able to bind PD-1 with and EC ₅₀ value of at most about 0.3 nM.

Item 45. The fusion polypeptide of any one of items 2-42, wherein the fusion polypeptide is able to bind PD-1 with an EC ₅₀ value at least as good as or superior to the EC ₅₀ value of the antibody specific for PD-1 as included in such fusion polypeptide.

Item 46. The fusion polypeptide of any one of items 2-42, wherein the fusion polypeptide is able to bind PD-1 with a lower EC ₅₀ than the EC ₅₀ value of the antibody specific for

PD-1 as included in such fusion polypeptide.

Item 47. The fusion polypeptide of any one of items 1 -42, wherein the fusion polypeptide is able to bind LAG-3 with an EC ₅₀ value of at most about 1 nM.

Item 48. The fusion polypeptide of any one of items 1 -42, wherein the fusion polypeptide is able to bind LAG-3 with an EC ₅₀ value of at most about 0.5 nM.

Item 49. The fusion polypeptide of any one of items 2-42, wherein the fusion polypeptide is able to bind LAG-3 with an EC ₅₀ value comparable to or lower than the EC ₅₀ value of the lipocalin mutein specific for LAG-3 as included in such fusion polypeptide.

Item 50. The fusion polypeptide of any one of items 1 -42, wherein the fusion polypeptide is capable of simultaneously binding of PD-1 and LAG-3.

Item 51 . The fusion polypeptide of any one of items 1 -42, wherein the fusion polypeptide is capable of simultaneously binding of PD-1 and LAG-3, with an EC ₅₀ value of at most about 10 nM.

Item 52. The fusion polypeptide of any one of items 1 -42, wherein the fusion polypeptide is capable of simultaneously binding of PD-1 and LAG-3, with an EC ₅₀ value of at most about 0.5 nM.

Item 53. The fusion polypeptide of any one of items 43-52, wherein the EC ₅₀ value is determined by enzyme-linked immunosorbent assay (ELISA).

Item 54. The fusion polypeptide of any one of items 1-53, wherein the fusion polypeptide competitively inhibits the binding of LAG-3 to major histocompatibility complex

(MHC) class II. Item 55. The fusion polypeptide of item 54, wherein the ability of the fusion polypeptide to competitively inhibit the binding of LAG-3 to major histocompatibility (MHC) class II is analyzed by fluorescence-activated cell sorting (FACS).

Item 56. The fusion polypeptide of any one of items 1-55, wherein the fusion polypeptide is capable of co-stimulating T cell responses.

Item 57. The fusion polypeptide of item 56, wherein the capability of co-stimulating T cell responses is measured in a functional T cell activation assay.

Item 58. The fusion polypeptide of any one of items 1-57, wherein the fusion polypeptide is able to induce IL-2 or IFN-γ production in the presence of stimulation of the T cells. Item 59. The fusion polypeptide of item 58, wherein the ability to induce IL-2 or IFN-γ production is measured in a functional T cell activation or killing assay.

Item 60. The fusion polypeptide of any one of items 1-59, wherein the fusion polypeptide is able to compete with PD-L1 and/or PD-L2 for binding to PD-1 .

Item 61 . The fusion polypeptide of item 60, fusion polypeptide is able to compete with PD-L1 and/or PD-L2 for binding to PD-1 with an IC ₅₀ value of at most about 100 nM.

Item 62. The fusion polypeptide of item 60 or 61 , fusion polypeptide is able to compete with

PD-L1 and/or PD-L2 for binding with PD-1 to an IC ₅₀ value of at most about 10 nM. Item 63. The fusion polypeptide of any one of items 60-62, fusion polypeptide is able to compete with PD-L1 and/or PD-L2 for binding to PD-1 with an IC ₅₀ value of at most about 7 nM.

Item 64. The fusion polypeptide of any one of items, wherein the ability to compete with PD-

L1 and/or PD-L2 measured by enzyme-linked immunosorbent assay (ELISA).

Item 65. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of any one of items 1-64.

Item 66. The nucleic acid molecule of item 65, wherein the nucleic acid molecule is operably linked to a regulatory sequence to allow expression of said nucleic acid molecule. Item 67. The nucleic acid molecule of items 65 or 66, wherein the nucleic acid molecule is comprised in a vector or in a phagemid vector.

Item 68. A host cell containing a nucleic acid molecule of any one of items 65-67.

Item 69. A method of producing the fusion polypeptide according to any one of items 1-64, wherein the fusion polypeptide is produced starting from the nucleic acid coding for the mutein by means of genetic engineering methods.

Item 70. The method of item 69, wherein the fusion polypeptide is produced in bacterium or eukaryotic host organism and is isolated from this host organism or its culture. Item 71 . A use of the fusion polypeptide according to any one of items 1-64 or a composition comprising such fusion polypeptide for simultaneously inhibiting immune checkpoints PD-1 and LAG-3.

Item 72. A use of the fusion polypeptide according to any one of items 1-64 or a composition comprising such fusion polypeptide for increasing anti-tumor lymphocyte cell activity. Item 73. A method of simultaneously inhibiting immune checkpoints PD-1 and LAG-3, comprising applying the fusion polypeptides according to any one of items 1-64 or a composition comprising such fusion polypeptide.

Item 74. A method of increasing anti-tumor lymphocyte cell activity, comprising applying the fusion polypeptides according to any one of items 1-64 or a composition comprising such fusion polypeptide.

Item 75. A method of interfering with the binding of human LAG-3 to major histocompatibility complex (MHC) class II in a subject, comprising applying one or more fusion polypeptides of any one of items 1-59 or one or more compositions comprising such fusion polypeptides.

V. EXAMPLES

[00185] Example 1 : Expression and analysis of representative fusion polypeptides

[00186] To engage PD-1 and LAG-3 at the same time, we generated several representative antibody-lipocalin mutein fusion polypeptides, fusing together a PD-1 specific antibody having the heavy chain provided by SEQ ID NO: 57, 59, 61 , or 65, or comprising the CDRs GYTFTDYE (HCDR1 , SEQ ID NO: 1 16), IDPGTGGT (HCDR2, SEQ ID NO: 1 17), and TSEKFGSNYYFDY (HCDR3; SEQ ID NO: 1 18), and light chains provided by SEQ ID NO: 58, 60, 62, or 66, or comprising the CDRs QTIVHSDGNTY (LCDR1 , SEQ I D NO: 134), KVS (LCDR2), and FQGSHVPLT (LCDR3, SEQ ID NO: 135), and the LAG-3-specific lipocalin muteins of SEQ ID NO: 8, 240, 14, 246, 21 , 21 1 or 247 via a linker, such as an unstructured (G ₄ S) ₃ linker, a polyproline linker, or a proline-alanine-serine polymer. The different formats that were generated are depicted in Figure 1A-1 F. Such fusion polypeptides, e.g., SEQ ID NOs: 74 and 66; SEQ ID NOs: 61 and 75; SEQ ID NOs:76 and 66; SEQ ID NOs: 61 and 77; SEQ ID NOs: 78 and 62; SEQ ID NOs: 65 and 79; SEQ ID NOs:80 and 62; SEQ ID NOs: 65 and 81 ; SEQ ID NOs: 78 and 79; SEQ ID NOs: 57 and 84; SEQ ID NOs: 85 and 66; SEQ ID NOs: 61 and 86; SEQ ID NOs: 59 and 87; SEQ ID NOs: 88 and 60; SEQ ID NOs: 57 and 181 SEQ ID NOs: 185 and 66; SEQ ID NOs: 61 and 187; SEQ ID NOs: 189 and 62; SEQ ID NOs: 65 and 191 ; SEQ ID NOs: 193 and 62; SEQ ID NOs: 65 and 194, and SEQ ID NOs: 236 and 66 were generated via fusion of the lipocalin mutein of SEQ ID NO: 8, 240, 14, 246, 21 , 21 1 or 247 to either one of the four termini of an antibody comprising of the heavy chain provided by SEQ ID NO: 57, 59, 61 , or 65 or comprising the heavy chain CDRs GYTFTDYE (HCDR1 , SEQ ID NO: 1 16), IDPGTGGT (HCDR2, SEQ ID NO: 1 17), and TSEKFGSNYYFDY (HCDR3; SEQ ID NO: 1 18), and the light chain provided by SEQ ID NO: 58, 60, 62, or 66 or comprising the light CDRs QTIVHSDGNTY (LCDR1 , SEQ ID NO: 134), KVS (LCDR2), and FQGSHVPLT (LCDR3, SEQ ID NO: 135).The PD-1 specific antibodies comprising of the heavy chain of SEQ ID NO: 57 and the light chain of SEQ ID NO: 58, or comprising of the heavy chain of SEQ ID NO: 59 and the light chain of SEQ ID NO: 60, or comprising of the heavy chain of SEQ ID NO: 65 and the light chain of SEQ ID NO: 62, or comprising of the heavy chain of SEQ ID NO: 61 and the light chain of SEQ ID NO: 66, as well as all antibody lipocalin mutein fusion polypeptides described here had an engineered lgG4 backbone, which contained a S228P mutation to minimize lgG4 half-antibody exchange in-vitro and in-vivo (Silva et al.,J Biol Chem, 2015). Additional mutations in the lgG4 backbones may also exist in all antibodies and fusion polypeptides described here, including any oneor more of mutations F234A, L235A, M428L, N434S, M252Y, S254T, and T256E. F234A and L235A mutations may be introduced to decrease ADCC and ADCP (Glaesner et a\., Diabetes Metab Res Rev, 2010). M428L and N434S mutations or M252Y, S254T, and T256E mutations may be introduced for extended serum half-life (Dall'Acqua et al.,J Biol Chem, 2006, Zalevsky et a\.,Nat Biotechnol, 2010).

[00187] All antibodies were expressed without the carboxy-terminal lysine to avoid heterogeneity. In addition, a lipocalin mutein Fc fusion was generated by fusing the LAG-3 specific lipocalin mutein of SEQ ID NO: 8 via an unstructured (G ₄ S) ₃ linker (SEQ ID NO: 54) to the C-terminus of the Fc region of an antibody provided in SEQ ID NO: 73. The resulting construct is provided in SEQ ID NO: 83. The present invention also embodies other antibody- lipocalin mutein fusion formats, such as those depicted in Figure 1 G-1 I. Such fusion polypeptides may be made in the same manner described above. The present invention also embodies asymmetrical antibody-lipocalin mutein fusion formats where, for example, one light chain of the antibody may be fused with a lipocalin mutein while the other is not.

[00188] The constructs of the fusion polypeptides were generated by gene synthesis and cloned into a mammalian expression vector. They were then transiently expressed in Expi293F™ cells (Life Technologies). The concentration of fusion polypeptides in the cell culture medium was measured by HPLC (Agilent Technologies) employing a POROS® protein A affinity column (Applied Biosystems).

[00189] The fusion polypeptides were purified using Protein A chromatography followed by size-exclusion chromatography (SEC) in phosphate-buffered saline (PBS). After SEC purification, the fractions containing monomeric protein are pooled and analyzed again using analytical SEC.

[00190] Example 2: Binding of fusion polypeptides towards PD-1 in enzyme- linked immunosorbent assay (ELISA)

[00191] We employed an enzyme-linked immunosorbent assay (ELISA) to determine the binding potency of the fusion polypeptides to recombinant human PD-1 -His (PD-1 with a C-terminal polyhistidine tag, ACROBiosystems). PD-1 -His at the concentration of 1 μg mL in PBS was coated overnight on microtiter plates at 4°C. After washing with PBS-0.05%T (PBS supplemented with 0.05% (v/v) Tween 20), the plates were blocked with 2% BSA (w/v) in PBS-0.1 %T (PBS supplemented with 0.1 % (v/v) Tween 20) for 1 h at room temperature. After washing with 100 μί PBS-0.05%T five times, PD-1 specific antibodies (SEQ ID NOs: 65 and 62; SEQ ID NOs: 61 and 66) or the fusion polypeptides at different concentrations were added to the wells and incubated for 1 h at room temperature, followed by another wash step. Bound antibodies/fusion polypeptides under study were detected by incubation with 1 :5000 diluted anti-human IgG Fc-HRP (Jackson Laboratory) in PBS-0.1 %T-2%BSA. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescence microplate reader.

[00192] The result of the experiment is depicted in Figure 2A and 2B, together with the fit curves resulting from a 1 :1 binding sigmoidal fit, where the EC ₅₀ value and the maximum signal were free parameters, and the slope was fixed to unity. The resulting EC ₅₀ values are provided in Table 1. The observed EC ₅₀ values for all tested molecules were very similar and were comparable to the PD-1 -specific antibodies (SEQ ID NOs: 65 and 62; SEQ ID NOs: 61 and 66) included in the fusion polypeptides. The experiment shows that when included in fusion polypeptides the described PD-1 -specific antibodies can be fused with the lipocalin mutein at either one of the four termini of the antibody and still binds to PD-1 .

[00193] Table 1 : ELISA data for PD-1 binding

Clone Name ECso PD-1 [nM]

SEQ ID NOs: 78 and 62 0.3

SEQ ID NOs: 65 and 79 0.24

SEQ ID NOs: 80 and 62 0.25

SEQ ID NOs: 65 and 81 0.38

SEQ ID NOs: 78 and 79 0.31

SEQ ID NOs: 74 and 66 0.19

SEQ ID NOs: 61 and 75 0.19

SEQ ID NOs: 76 and 66 0.22

SEQ ID NOs: 61 and 77 0.27

SEQ ID NOs: 85 and 66 0.21

SEQ ID NOs: 61 and 86 0.18

SEQ ID NOs: 65 and 62 0.15

SEQ ID NOs: 61 and 66 0.14

[00194] Example 3: Binding of fusion polypeptides towards LAG -3 in ELISA

[00195] We employed an ELISA assay to determine the binding potency of the antibody-lipocalin mutein fusion polypeptides to recombinant LAG-3-His (ACROBiosystems). LAG-3-His at the concentration of 1 μg mL in PBS was coated overnight on microtiter plates at 4°C. After washing with PBS-0.05%T (PBS supplemented with 0.05% (v/v) Tween 20), the plates were blocked with 2% BSA (w/v) in PBS-0.1 %T (PBS supplemented with 0.1 % (v/v) Tween 20) for 1 h at room temperature and subsequently washed. Different concentrations of the antibody-lipocalin mutein fusion polypeptides were added to the wells and incubated for 1 h at room temperature, followed by another wash step. A 1 :5000 diluted anti-human IgG Fc-HRP (Jackson Laboratory) in PBS-0.1 %T-2%BSA was added for 1 h at room temperature. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well, and the fluorescence intensity was detected using a fluorescence microplate reader.

[00196] The results of the experiments are depicted in Figure 3A and 3B, together with the fit curves resulting from a 1 :1 sigmoidal binding fit, where the EC ₅₀ value and the maximum signal were free parameters, and the slope was fixed to unity. The resulting EC ₅₀ values are shown in Table 2. The binding potencies to LAG-3 for the different fusion formats when the same lipocalin mutein was included in the polypeptides were comparable with each other.

[00197] Table 2: ELISA data for LAG-3 binding Clone Name ECso LAG -3 [nM]

SEQ ID NOs: 78 and 62 0.06

SEQ ID NOs: 65 and 79 0.06

SEQ ID NOs: 80 and 62 0.06

SEQ ID NOs: 65 and 81 0.06

SEQ ID NOs: 78 and 79 0.08

SEQ ID NOs: 74 and 66 0.07

SEQ ID NOs: 61 and 75 0.05

SEQ ID NOs: 76 and 66 0.06

SEQ ID NOs: 61 and 77 0.08

SEQ ID NOs: 85 and 66 0.77

SEQ ID NOs: 61 and 86 0.87

[00198] Example 4: Fluorescence-activated cell sorting (FACS) analysis of fusion polypeptides binding to cells expressing PD-1 or LAG -3

[00199] We employed fluorescence-activated cell sorting (FACS) studies in order to assess the specific binding of fusion polypeptides versus negative controls to Chinese hamster ovary (CHO) cells stably transfected with human PD-1 (CHO-huPD-1 ) or human LAG-3 (CHO-huLAG-3), respectively. The cell lines were generated using the Flp-ln system (Invitrogen) according to the manufacturer ^' s instructions. Mock-transfected Flp-ln CHO cells served as the negative control.

[00200] Transfected CHO cells were maintained in Ham ^' s F12 medium (Invitrogen) supplemented with 10% Fetal Calf Serum (FCS, Biochrom) and 500 μg ml Hygromycin B (Roth). Cells were cultured in cell culture flasks under standard conditions according to manufacturer's instruction (37°C, 5% C0 ₂ atmosphere). In order to dissociate the adherent cells for subculture or FACS experiments, Accutase (PAA Laboratories) was employed according to the manufacturer ^' s instructions.

[00201] To perform the experiment, PD-1 -positive cells, LAG-3 positive cells and negative control Flp-ln CHO cells were incubated with fusion polypeptides, and bound fusion polypeptides were detected by using a fluorescently labeled anti-human IgG antibody in FACS analysis as described in the following.

[00202] 2.5 x 10 ⁴ cells per well were pre-incubated for 1 h in ice-cold PBS containing 5% fetal calf serum (PBS-FCS). Subsequently, a dilution series of the fusion polypeptides, control antibodies and negative controls typically ranging from 100 to 0.001 nM was added to the cells and incubated on ice for 1 h. Cells were washed twice in ice-cold PBS using centrifugation at 300 xg and then incubated with a rabbit anti-hlgG antibody labeled with the fluorescent dye Alexa488 (Pieris) for 30 min on ice. Cells were subsequently washed and analyzed using iQue Flow cytometer (Intellicyte). Fluorescent data generated by the fusion polypeptides binding to PD-1 or LAG-3 expressing cells were analyzed using Forecyt® software and resulted geometric fluorescent means were plotted and fitted using Graphpad software.

[00203] The fusion polypeptides show similar binding abilities to PD-1 expressing cells (single digit nanomolar range) and LAG3 expressing cells (picomolar range). Exemplary data for selected fusions showing binding with high affinity to PD-1 are shown in Figure 4A-4D and LAG-3 are shown in Figure 4C-4H. Selected data are also shown in Table 3. Each of fusion 1 through fusion 3, when used in the present disclosure, refers to fusion polypeptides comprising humanized antibodies that have the CDR sequences of the 388D4 antibody, namely the heavy chain CDRs GYTFTDYE (HCDR1 , SEQ ID NO: 1 16), IDPGTGGT (HCDR2, SEQ ID NO: 1 17), and TSEKFGSNYYFDY (HCDR3; SEQ ID NO: 1 18), and the light chain CDRs QTIVHSDGNTY (LCDR1 , SEQ ID NO: 134), KVS (LCDR2), and FQGSHVPLT (LCDR3, SEQ ID NO: 135). Negative controls did not bind to human PD-1 or human LAG-3 expressed on cells (data not shown) as expected. Similarly, no binding of the fusion polypeptides was detected on Mock-transfected Flp-ln CHO (data not shown).

[00204] Table 3: FACS data for binding to huPD-1 and hu LAG-3

SEQ ID NOs: 57 and 84 0.68 0.053

SEQ ID NOs: 85 and 66 0.25 0.38

SEQ ID NOs: 61 and 86 0.32 0.56

SEQ ID NOs: 88 and 60 0.26 0.033

SEQ ID NO: 246-fusion 1 3.42 0.18

SEQ ID NO: 21 1 -fusion 1 5.19 0.24

SEQ ID NO: 213-fusion 1 3.85 0.31

SEQ ID NO: 214-fusion 1 4.57 0.30

SEQ ID NO: 246-fusion 2 4.14 0.48

SEQ ID NO: 21 1 -fusion 3 4.19 0.29

SEQ ID NO: 213-fusion 3 4.82 0.22

SEQ ID NO: 214-fusion 3 2.92 0.27

SEQ ID NO: 246-fusion 3 4.25 0.39

[00205] Example 5: Demonstration of simultaneous target binding in an ELISA- based setting

[00206] In order to demonstrate the simultaneous binding of the fusion polypeptides to PD-1 and LAG-3, a dual-binding ELISA format was used. Recombinant PD-1 -His (ACROBiosystems) in PBS (1 g/mL) was coated overnight on microtiter plates at 4°C. The plates were washed five times after each incubation step with 100 μΙ_ PBS-0.05%T. The plates were blocked with 2% BSA (w/v) in PBS-0.1 %T for 1 h at room temperature and subsequently washed again. Different concentrations of the fusion polypeptides were added to the wells and incubated for 1 h at room temperature, followed by a wash step. Subsequently, biotinylated human LAG-3-Fc (R&D Systems) was added at a constant concentration of 2 μg/mL in PBS-0.1 %T-2%BSA for 1 h. After washing, a 1 :5000 dilution of Extravidin-HRP (Sigma-Aldrich) in PBS-0.1 %T-2%BSA was added to the wells and incubated for 1 h. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well, and the fluorescence intensity was detected using a fluorescence microplate reader.

[00207] Dual binding data of the fusion polypeptides are shown in Figure 5, together with the fit curves resulting from a 1 :1 sigmoidal binding fit, where the EC ₅₀ value and the maximum signal were free parameters, and the slope was fixed to unity. The EC ₅₀ values are summarized in Table 4. Each of fusion 1 through fusion 1 1 , when used in the present disclosure, refers to fusion polypeptides comprising humanized antibodies that have the CDR sequences of the 388D4 antibody, namely the heavy chain CDRs GYTFTDYE (HCDR1 , SEQ ID NO: 1 16), IDPGTGGT (HCDR2, SEQ ID NO: 1 17), and TSEKFGSNYYFDY (HCDR3; SEQ ID NO: 1 18), and the light chain CDRs QTIVHSDGNTY (LCDR1 , SEQ ID NO: 134), KVS (LCDR2), and FQGSHVPLT (LCDR3, SEQ ID NO: 135). All fusion polypeptides showed clear binding signals, demonstrating that the fusion polypeptides are able to engage PD-1 and LAG-3 simultaneously.

[00208] Table 4: ELISA data for simultaneous target binding of both PD-1 and LAG-3

[00209] Example 6: FACS analysis of competitive binding of fusion polypeptides with major histocompatibility complex (MHC) class II expressing cells for human LAG- 3. [00210] To assess whether a given fusion polypeptide interferes with LAG-3 binding to major histocompatibility complex (MHC) class II on MHC class ll-positive cells, a competition FACS experiment was utilized. In this experiment, a constant concentration of human LAG-3- Fc fusion (huLAG-3-Fc, R&D system) and a dilution series of the fusion polypeptides were incubated with the MHC class II positive human cell line A375, and cell-bound huLAG-3-Fc was detected using a fluorescently labeled anti-IgG Fc antibody. In this assay, competitive fusion polypeptides interfering with the binding of LAG-3 with its ligand MHC class II lead to a reduction of huLAG-3-Fc binding to the MHC class II positive cell line A375.

[00211] The melanoma cell line A375 was maintained in DMEM medium (Invitrogen) supplemented with 10% Fetal Calf Serum (FCS, Biochrom). Cells were cultured in cell culture flasks under standard conditions according to manufacturer's instruction (37°C, 5% C0 ₂ atmosphere). In order to dissociate the adherent cells for subculture or FACS experiments, Accutase (PAA Laboratories) was employed according to the manufacturer ^' s instructions.

[00212] For FACS assay, 5 10 ⁴ A375 cells per well were incubated for 1 h in PBS- FCS, followed by addition of 3 nM huLAG-3-Fc and varying concentrations of the fusion polypeptides. Cells were washed twice in ice-cold PBS, re-suspended in PBS-FCS and incubated 30 min on ice with phycoerythrin labeled anti-human IgG Fc antibody (Jackson Immunologics). Cells were subsequently washed and analyzed using an Intellicyt IQue Flow cytometer (Intellicyt). Fluorescent data generated by huLAG-3-Fc binding to A375 cells were analyzed using Forecyt software and resulted geometric fluorescent means were normalized to huLAG-3-Fc maximal binding. Percent of huLAG-3-Fc binding were plotted and fitted using the Graphpad software. Selected competition binding curves are provided in Figure 6. The data show that the tested antibody-lipocalin mutein fusion polypeptides and the Fc-lipocalin mutein fusion polypeptides compete with binding of LAG-3 to its ligand MHC class II on human MHC class II expressing cells. The inhibitory effect on LAG-3/MHC class II molecules binding of the fusion polypeptides appeared at concentrations comparable to the reference LAG-3 monoclonal antibody (SEQ ID NOs: 5 and 6). The antibodies (SEQ ID NOs: 65 and 62; SEQ ID NOs: 61 and 66 and the SEQ ID NOs: 57 and 58) which did not bind to LAG-3, did not show any competition, see Figure 6.

[00213] Example 7: Assessment of T cell activation using human peripheral blood mononuclear cells (PBMCs) [00214] We employed a T cell assay to assess the ability of the selected fusion polypeptides to revert the inhibitory signaling of the negative checkpoint molecules LAG-3 and PD-1 by blocking the interaction between LAG-3 and PD-1 and their respective ligands. For this purpose, fusion polypeptides at different concentrations were added to staphylococcal enterotoxin B (SEB) stimulated human peripheral blood mononuclear cells (PBMCs) and incubated for 4 days at 37°C. As a readout, secreted IL-2 levels in the supernatants were assessed.

[00215] PBMCs from healthy volunteer donors were isolated from buffy coats by centrifugation through a polysucrose density gradient (Biocoll, 1 .077 g/mL, Biochrom), following Biochrom ^' s protocols. The purified PBMCs were resuspended in a buffer consisting of 90% FCS and 10% DMSO, immediately frozen down using liquid nitrogen and stored in liquid nitrogen until further use. For the assay, PBMCs were thawed and rested for 16 h in culture media (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1 % Penicillin-Streptomycin (Life Technologies).

[00216] The following procedure was performed using triplicates for each experimental condition.

[00217] 2.5x10 ⁴ PBMCs were incubated in each well of a 384 well flat-bottom tissue culture plates in culture media supplemented or not with SEB at different concentrations. Subsequently, a dilution series of the fusion polypeptides, control antibodies, cocktails of antibody and the lipocalin Fc fusion proteins, and negative controls typically ranging from 100 to 0.001 nM were added to the cells. Plates were covered with a gas permeable seal (4titude) and incubated at 37°C in a humidified 5% C0 ₂ atmosphere for four days. Subsequently, IL-2 levels in the supernatant were assessed.

[00218] Human IL-2 in the cell culture supernatants were quantified using the IL-2 DuoSet kit from R&D Systems.

[00219] The following procedure describes the IL-2 quantification.

[00220] In the first step, a 384 well plate was coated at room temperature for 2 h with 1 μg mL "Human IL-2 Capture Antibody" (R&D Systems) in PBS. Subsequently, wells were washed 5 times with 80 μΙ PBS-0.05%T. After 1 h blocking in PBS-0.05%T additionally containing 1 % casein (w/w), pooled supernatants and a concentration series of an IL-2 standard diluted in culture medium was incubated in the 384-well plate overnight at 4°C. To allow for detection and quantitation of captured IL-2, a mixture of 100 ng/mL goat anti-hlL-2- Bio detection antibody (R&D Systems) and ^g mL Sulfotag-labelled streptavidin (Mesoscale Discovery) in PBS-T containing 0.5% casein were added and incubated at room temperature for 1 h. After washing, 25 μΙ_ reading buffer was added to each well, and the electrochemiluminescence (ECL) signal of every well was read using a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.

[00221] The result of a representative experiment is depicted in Figure 7. It shows the increased IL-2 secretion level induced by the fusion polypeptides (SEQ ID NOs: 74 and 66; SEQ ID NOs: 61 and 75, SEQ ID NO: 61 and 86, SEQ ID NOs: 57 and 84, SEQ ID NOs: 57 and 181 , SEQ ID NOs: 21 1 -fusion 1 , SEQ ID NO: 213-fusion 1 , SEQ ID NO: 214-fusion 1 , SEQ ID NO: 246-fusion 1 , SEQ ID NO: 246-fusion 2, SEQ ID NO: 21 1 -fusion 3, SEQ ID NO: 213-fusion 3, SEQ ID NO: 214-fusion 3, SEQ ID NO: 246-fusion 3, SEQ ID NOs: 236 and 66). Each of fusion 1 through fusion 3, when used in the present disclosure, refers to fusion polypeptides comprising humanized antibodies that have the CDR sequences of the 388D4 antibody, namely the heavy chain CDRs GYTFTDYE (HCDR1 , SEQ ID NO: 1 16), IDPGTGGT (HCDR2, SEQ ID NO: 1 17), and TSEKFGSNYYFDY (HCDR3; SEQ ID NO: 1 18), and the light chain CDRs QTIVHSDGNTY (LCDR1 , SEQ ID NO: 134), KVS (LCDR2), and FQGSHVPLT (LCDR3, SEQ ID NO: 135). The fusion polypeptides show increased cytokine secretion compared to the building block antibodies (SEQ ID NOs: 61 and 66 and SEQ ID NOs: 57 and 58) and the lipocalin-Fc mutein (SEQ ID NO: 83 and SEQ ID NO: 182) alone or an anti-LAG-3 antibody (SEQ ID NOs: 238 and 239) alone and compared to the cocktail of the building block antibody (SEQ ID NOs: 61 and 66 and SEQ ID NOs: 57 and 58) and the lipocalin-Fc mutein (SEQ ID NO: 83 and SEQ ID NO: 182) or anti-LAG-3 antibody (SEQ ID NOs: 238 and 239). hlgG4 (Sigma) used as negative control do not induce increased IL-2 production by T cells compared to basal activity.

[00222] Example 8: Stability assessment of the fusion polypeptides

[00223] To determine melting temperatures (T _m s) as a general indicator for overall stability, the fusion polypeptides at a protein concentration of 1 mg/mL in PBS (Gibco) were scanned (25-100°C) at 1 °C/min using a capillary nanoDSC instrument (CSC 6300, TA Instruments). The T _m s were calculated from the displayed thermogram using the integrated Nano Analyze software.

[00224] The resulting T _m s as well as the onset of melting for the fusion polypeptides are listed in Table 5 below. All fusion polypeptides have T _m s as well as the onset of melting in the same range as the PD-1 antibodies. [00225] Table 5: Melting temperature (T _m ) and the onset of melting of fusion polypeptides as determined by nanoDSC. Three distinct peaks of the melting curve have been identified and correspond to the domains/regions of the molecule.

[00226] To assess storage stability, the fusion polypeptides were incubated at a concentration of 1 mg/mL in PBS for 1 week at 37°C and a concentration of 5 mg/ml in PBS for 4 weeks at 40°C. Monomeric protein content was measured in an analytical size exclusion chromatography. Data for selected polypeptides are shown in Table 6.

[00227] Analytical size exclusion chromatography was performed on an Agilent HPLC system with two Superdex 200, 3.2/300lncrease (GE Healthcare) in a row with PBS (Gibco) as an eluent at a flow rate of 0.3 mL/min. The percentage recovery of monomer was determined by the monomer peak area for each sample referencing against non-stressed reference sample frozen at -20°C.

[00228] To further assess the storage stability in plasma, fusion polypeptides at the concentration of 0.5 mg/mL were incubated for 1 week at 37°C in human plasma. Active fusion polypeptide was measured in a quantitative ELISA setting.

[00229] For assaying protein activity, the simultaneous binding ELISA as described in Example 5 was applied.

[00230] A calibration curve with standard protein dilutions was prepared. Three different, independent dilutions within the linear range of the calibration curve were prepared for each sample. PBS-0.1 %T-2%BSA supplemented with 1 % human plasma was used for the dilutions. The percentage recovery of activity for each sample was calculated from the calibration curve, referencing against an unstressed sample stored at -20 °C at the same concentration and in the same matrix.

[00231] Table 6: Stability after 1 -week storage in PBS at 37°C assessed by recovery of monomer content in analytical SEC or human plasma (HPL) at 37°C assessed by recovery of activity in qELISA and Stability after 4-week storage in PBS at 40°C assessed by recovery of monomer content in analytical SEC and by recovery of activity in qELISA: stable in qELISA = 100 +/- 15 %; stable in aSEC = 100 +/- 5%; for all samples including references a monomer content of at least 99 area percent has been detected.

[00232] Example 9: ELISA analysis of competitive binding of fusion polypeptides with PD-L1 and PD-L2.

[00233] We employed an ELISA-based competition assay to analyze the inhibition of the interaction of PD-1 with PD-L1 and PD-L2. Recombinant human PD-L1 -Fc (R&D) or recombinant human PD-L2-Fc (R&D) at a concentration of 1 μς in PBS was coated overnight on microtiter plates at 4°C. After washing with PBS-0.05%T (PBS supplemented with 0.05% (v/v) Tween 20), the plates were blocked with 2% BSA (w/v) in PBS-0.1 %T (PBS supplemented with 0.1 % (v/v) Tween 20) for 1 h at room temperature. After washing with 100 μΙ_ PBS-0.05%T five times, a mixture of biotinylated human PD-1 -Fc (R&D, in-house biotinylation via primary amines) at a constant concentration of 20 nM and PD-1 specific antibodies (SEQ ID NOs: 65 and 62; SEQ ID NOs: 61 and 66) or the fusion polypeptides at different concentrations were added to the wells and incubated for 20 min at room temperature. This mixture of human PD-1 and PD-1 specific antibodies or fusion polypeptides had previously been incubated at room temperature for 1 h. Another wash step followed. Biotinylated PD-1 that still bound to PD-L1/PD-L2 after previous incubation with antibody or fusion polypeptide, "free PD-1 ", was detected by incubation with 1 :5000 diluted Extravidin-HRP (Sigma-Aldrich) in PBS-0.1 %T-2%BSA for 1 h. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescence microplate reader.

[00234] In addition, calibration curves for the quantification of free PD-1 were prepared by applying different concentrations of biotinylated human PD-1 -Fc alone to the same assays. The concentration of free (non-neutralized) PD-1 after incubation with PD-1 antibodies or fusion polypeptides was calculated from these calibration curves.

[00235] The data were analyzed by using a 1 :1 sigmoidal binding fit, where the IC ₅₀ value and the maximum signal were free parameters, and the slope was fixed to one. The resulting IC ₅₀ values are provided in Table 7. The PD-1 specific antibodies as well as the fusion polypeptides all clearly inhibited the interaction between PD-1 and PD-L1 and PD-L2. The observed IC ₅₀ values for all tested molecules were similar and were comparable to the PD-1 -specific antibodies (SEQ ID NOs: 65 and 62; SEQ ID NOs: 61 and 66) included in the fusion polypeptides. A commercially available non-competitive mouse anti-human PD-1 antibody was used as negative control (not shown).

[00236] Table 7 shows that all of the exemplary PD-1 antibodies and fusion polypeptides tested, as indicated below, compete with PD-L1 and PDL-2 for binding to PD-1 :

s: 5 an . .

[00237] Example 10: Functional T cell activation assay using A375 tumor cells expressing LAG-3 and PD-1 ligands

[00238] We employed a further T cell assay to assess the ability of the fusion polypeptides revert the inhibitory signaling of the negative checkpoint molecules LAG-3 and PD-1 by blocking the interaction of LAG-3 and PD-1 with their respective ligands. We applied fusion polypeptides at different concentrations to SEB stimulated T cells, in the presence of the melanoma cell line A375 which expresses MHC II, the ligand of LAG-3, and PD-L1 , the ligand of PD-1 , followed by 3-day incubation at 37°C. As readouts, we assessed secreted IL- 2 and IFN-γ levels in the supernatants.

[00239] Human peripheral blood mononuclear cells (PBMC) from healthy volunteer donors were isolated from buffy coats by centrifugation through a Polysucrose density gradient (Biocoll 1 .077 g/mL from Biochrom), following Biochrom ^' s protocols. The T lymphocytes were isolated from the resulting PBMC using a Pan T cell purification Kit (Miltenyi Biotec GmbH) and the manufacturer ^' s protocols. Purified T cells were resuspended in a buffer consisting of 90% FCS and 10% DMSO, immediately frozen down using liquid nitrogen and stored in liquid nitrogen until further use.

[00240] For the assay, T cells were thawed for 16 h and cultivated in culture media (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1 % Penicillin- Streptomycin (Life Technologies). [00241] Melanoma cell line A375 were treated 1 hour at 37°C with mitomycin C (Sigma Aldrich) at a concentration of 3C^g/ml in order to block their proliferation. Mitomycin treated A375 were then washed twice in culture medium and plated at 6.25 x 10 ³ cells per well and allowed to adhere overnight at 37°C in a humidified 5% C0 ₂ atmosphere. The target cells had before been grown under standard conditions, detached using Accutase (PAA Laboratories), and resuspended in culture media. The following procedure was performed using triplicates for each experimental condition.

[00242] The following procedure was performed using triplicates for each experimental condition.

[00243] On the next days, plates were washed twice with PBS, and 25μΙ_ of T cell suspension (corresponding to 1 .25 x 10 ⁴ T cells), the selected fusion polypeptides (SEQ ID NOs: 74 and 66 and SEQ ID NOs: 61 and 75), PD-1 -specific benchmark antibody (SEQ ID NOs: 61 and 66), Fc-lipocalin mutein (SEQ ID NO: 83) or LAG-3 benchmark antibody (SEQ ID NOs: 5 and 6) the negative controls, at concentrations ranging from .0001 nM to 10nM, were added to each well. Finally, 5 μΙ_ of SEB, corresponding to a final concentration of 0.05 ng/mL of SEB diluted in culture media, were added to each well. Plates were covered with a gas permeable seal (4titude) and incubated at 37°C in a humidified 5% C0 ₂ atmosphere for 3 days.

[00244] Subsequently, IL-2 and IFN-γ levels in the supernatant were assessed as described in Example 7 for IL-2 secretion (data on IFN-γ secretion are not shown).

[00245] Exemplary data shown in Figure 8. These data indicate a clear increase of IL- 2 secretion levels with the treatment of the PD-1 and LAG-3 bispecific fusion polypeptides.

[00246] Example 11 : Tumor growth inhibition by fusion polypeptides in humanized mouse tumor model

[00247] In order to investigate the activity of the exemplary fusion polypeptide SEQ ID NO: 21 1 -fusion 1 (lipocalin mutein of SEQ ID NO: 21 1 specific for LAG-3 fused to a humanized antibody specific for PD-1 comprising the heavy chain CDRs GYTFTDYE, IDPGTGGT, and TSEKFGSNYYFDY and the light chain CDRs QTIVHSDGNTY, KVS, and FQGSHVPLT). in an in-vivo mouse model, we employed immune deficient NOG mice engrafted with human HCC827 tumor cells and human PBMC.

[00248] 4 to 6 week-old NOG mice were subcutaneously (s.c.) injected with 5 x 10 ⁶ HCC827 cells in a matrigel/PBS (1 :1 ) solution. Tumors were allowed to grow for 10 days and on day 0 of the experiment mice were randomized into treatment (or control) groups according to tumor size and animal weight. Mice were given 5 x 10 ⁶ fresh human PBMC intravenously (i.v.) via tail vein injection. Mice received treatment or control (PBS) via intraperitoneal (i.p.) injection after the i.v. PBMC injection on day 1 , and again received treatment or control on day 4, day 8, and day 12. The molecules under study were the fusion polypeptide SEQ ID NO: 21 1 -fusion 1 (25mg/kg) and the PD-1 -specific antibody included in SEQ ID NO: 21 1 -fusion 1 (20 mg/mk). Tumor growth was recorded every 3-4 days.

[00249] Figure 9 reflects, for each of the treatment and control groups, the change in tumor volume as measured on day 4, day 7, day 1 1 , and day 14 of the study. Tumor growth inhibition was achieved by both the fusion polypeptide SEQ ID NO: 21 1 -fusion 1 and the PD- 1 -specific antibody included in SEQ ID NO: 21 1 -fusion 1 . The fusion polypeptide SEQ ID NO: 21 1 -fusion 1 almost completely inhibited the growth of tumor over the course of the study, showing much enhanced antitumor activity than the PD-1 -specific antibody alone.

[00250] Embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present embodiments have been specifically disclosed by preferred embodiments and optional features, modification and variations thereof may be resorted to by those skilled in the art and that such modifications and variations are considered to be within the scope of this invention. All patents, patent applications, textbooks, and peer-reviewed publications described herein are hereby incorporated by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised 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 become apparent from the following claims.

[00251] Equivalents: Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

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