Novel proteins with high binding affinity for programmed death ligand 1 (PD-L1)

Abstract

The present invention relates to novel proteins specific for programmed (cell) death ligand 1 (PD-L1). The PD-L1 specific protein of the present invention binds to human and mouse PD-L1 with high affinity. The present invention also relates to a PD-L1 specific protein further comprising a diagnostic or therapeutic active ingredient. Further aspects of the invention encompass the use of these PD-L1-specific binding proteins in medical treatment, for example in the diagnosis and treatment of PD-L1-related cancers.

Description

Technical Field

[0001] The present invention relates to novel proteins specific for programmed (cell) death ligand 1 (PD-L1). The PD-L1-specific proteins of the present invention have high affinity binding to human and mouse PD-L1. The present invention further relates to PD-L1-specific proteins further comprising diagnostic or therapeutic active components. Further aspects of the present invention encompass the use of these PD-L1-specific binding proteins in medicine, such as in the diagnosis and treatment of PD-L1-related cancers. Background Art

[0002] The transmembrane protein programmed (cell) death ligand 1 (PD-L1; also known as B7-H, B7H1, CD274) belongs to the B7 family of immunomodulatory molecules and is involved in regulating cellular and humoral immune responses. The PD-L1 ligand interacts with PD-1 (programmed cell death 1). PD-L1 is expressed on a variety of normal cells and immune cells, and tumor cells express PD-L1 as a mechanism of immune evasion.

[0003] Both PD-1 and PD-L1 belong to the family of immune checkpoint proteins, which act as co-inhibitors. They can prevent or limit the development of T cell responses, such as inhibiting T cell activation and proliferation and inducing apoptosis of activated T cells. The PD-1 / PD-L1 interaction ensures that the immune system is activated only at the appropriate time to minimize the possibility of chronic autoimmune inflammation.

[0004] PD-L1 expressed on tumor cells binds to the PD-1 receptor on activated T cells, which results in the inhibition of cytotoxic T cells and, in turn, in reduced cytokine production and T cell proliferation. By upregulating PD-L1 expression, tumor cells evade detection and destruction by the immune system. In cancer, PD-L1 provides resistance to T cell-mediated cytotoxicity. By blocking the immune checkpoint receptor PD-1 or its ligand PD-L1, the immune system can overcome the ability of cancer to resist immune responses and stimulate the body's own mechanisms to maintain its effective defense against cancer.

[0005] A variety of immune checkpoint humanized IgG1 antibodies have been developed as PD-1 / PD-L1 inhibitors for cancer immunotherapy, such as atezolizumab, durvalumab, avelumab, BMS-936559, and MEDI4736.

[0006] Existing options often fail to adequately address the diagnosis and treatment of PD-L1-associated cancers, and thus many patients fail to fully benefit from current strategies.

[0007] Undoubtedly, new strategies are urgently needed for the diagnosis and treatment of PD-L1-overexpressing tumors as well as other PD-L1-associated diseases and conditions, including (viral) infections.

[0008] An object of the present invention is to provide proteins that specifically target PD-L1 to allow targeted diagnostic and therapeutic options, including detection of PD-L1 positive tumors (e.g., by radiodiagnostic methods). Targeting this tumor-associated protein can benefit patients who have not yet met the needs of new diagnostic and therapeutic approaches. Due to the low and limited distribution of PD-L1 in normal tissues, targeting PD-L1 indicates a potential non-toxic diagnostic and therapeutic method. Therefore, binding proteins with specificity for PD-L1 can provide effective medical options for cancer treatment and ultimately improve the quality of life of patients.

[0009] The present invention provides novel PD-L1 binding molecules, novel strategies and improved strategies for the diagnosis and treatment of PD-L1 positive tumors. In addition, the novel PD-L1 binding molecules of the present invention provide improved strategies for the diagnosis and treatment of infectious diseases (e.g., chronic viral infections).

[0010] The above objects and advantages are achieved by the subject matter of the appended claims. The present invention meets the above needs by providing new high affinity PD-L1 binding proteins. The above summary does not necessarily describe all problems solved by the present invention. Summary of the invention

[0011] The present disclosure provides the following items 1 to 15, but is not particularly limited thereto:

[0012] 1. A protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, wherein the protein: (i) has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at the position corresponding to position 6 of SEQ ID NO: 1, and has an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at the position corresponding to position 8 of SEQ ID NO: 1; and (ii) exhibits a binding affinity of less than 100 nM for human programmed (cell) death ligand 1 (PD-L1); a preferred aspect relates to a dimeric PD-L1 binding protein comprising an amino acid sequence of SEQ ID NO: 4 and an amino acid sequence having at least 96 ... NO: 1 has an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at position 8; and (ii) exhibits a binding affinity for human programmed death ligand 1 (PD-L1) of less than 15 nM as determined by surface plasmon resonance.

[0013] 2. The protein according to item 1, wherein the protein has phenylalanine (F) at the position corresponding to position 6 of SEQ ID NO: 1, and has aspartic acid (D) at the position corresponding to position 8 of SEQ ID NO: 1.

[0014] 3. The protein according to item 1 or 2, wherein the protein is stable in serum after incubation at 37°C for 24h.

[0015] 4. The protein according to any one of items 1 to 3, wherein the protein exhibits a binding affinity to human PD-L1 of less than 15 nM.

[0016] 5. A multimer comprising the protein according to any one of items 1 to 4, wherein the multimer is a dimer, trimer, tetramer, pentamer or hexamer.

[0017] 6. A multimer according to item 5, comprising a protein according to any one of items 1 to 4 and a protein comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 4, wherein the multimer is preferably a dimer.

[0018] 7. The protein according to any one of items 1 to 6, further comprising one or more coupling sites for coupling of a chemical moiety, wherein the chemical moiety is preferably selected from any one of a chelator, a drug, a toxin, a dye and a small molecule.

[0019] 8. The protein according to any one of items 1 to 7, further comprising at least one diagnostically active part, or at least one therapeutically active part.

[0020] 9. The protein according to any one of items 1 to 8, further comprising at least one pharmacokinetics-regulating portion, wherein the at least one pharmacokinetics-regulating portion is preferably selected from any one of the following: serum albumin; albumin binding protein; immunoglobulin binding protein; immunoglobulin or immunoglobulin fragment; polysaccharide; unstructured amino acid sequence comprising amino acids alanine, glycine, serine, proline; polyethylene glycol; sialic acid; transferrin; and transferrin receptor binding protein; or any combination thereof.

[0021] 10. The protein according to any one of items 1 to 9, for use in the diagnosis or treatment of PD-L1 positive tumors or for use in the diagnosis or treatment of infectious diseases such as chronic viral infections.

[0022] 11. A composition comprising the protein according to any one of items 1 to 10.

[0023] 12. The composition of item 11 for use in medical treatment.

[0024] 13. The composition for use in medical treatment according to item 12, which is for use in the diagnosis or treatment of PD-L1 positive tumors, or for use in the diagnosis or treatment of infectious diseases such as chronic viral infections.

[0025] 14. A method for producing a protein according to any one of items 1 to 10, comprising the steps of: a) culturing a host cell under conditions suitable for obtaining said protein and b) isolating said protein produced.

[0026] 15. A method for detecting PD-L1 in a sample, the method comprising contacting the sample with the protein according to any one of items 1 to 10, and detecting the binding of PD-L1 in the sample with the protein according to any one of items 1 to 10.

[0027] This summary does not necessarily describe all features of the present invention. Other embodiments will become apparent upon reading the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings show:

[0029] Figure 1: shows the binding affinity of dimeric PD-L1 binding protein after long-term incubation in human serum. Binding affinity (K) for PD-L1 expressed on cells after 24h incubation in serum DFigure 1A shows the binding affinity of 224058 (comprising SEQ ID NO: 2) to PD-L1 after incubation in human serum. Figure 1B shows the binding affinity of 224180 (comprising SEQ ID NO: 3) to PD-L1 after incubation in human serum. The results confirm the stability of the PD-L1 binding protein. Even after long-term incubation in human serum, the PD-L1 binding protein still binds to PD-L1 expressed on cells with high affinity. DETAILED DESCRIPTION

[0030] The inventors have developed a solution to meet the continued strong demand in the art for expanding medical options for diagnosis and treatment of cancer and infectious diseases by providing new PD-L1 binding proteins. The PD-L1-specific proteins defined herein are functionally characterized by a high specific affinity for (human) PD-L1 of less than 100 nM, preferably less than 15 nM. In addition, they show a high level of stability in serum and at high temperatures. The PD-L1 binding proteins of the present invention (including their multimeric forms disclosed herein) can inhibit the interaction of PD-L1 with PD-1. This inhibition is believed to provide blocking of PD-1 inhibitory signaling. Therefore, the PD-L1 binding proteins described herein provide a molecular form with good physicochemical properties, high-level expression in bacteria, and allow a simple production method. The new proteins described herein can broaden the medical strategies for diagnosis and therapy of PD-L1-related cancers and infectious diseases that have not been met so far. In particular, the proteins described herein can be used for imaging purposes, such as for the presence of tumor cells expressing PD-L1, and for radiotherapy treatment of tumors expressing PD-L1, or for immuno-oncology treatment options.

[0031] Before describing the present invention in more detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only used to describe specific aspects and embodiments, and are not intended to limit the scope of the present invention as reflected by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. This includes technicians working in the field of protein engineering and purification, as well as technicians working in the field of developing new target-specific binding molecules for use in technical applications and in treatment and diagnosis.

[0032] Preferably, according to "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland) defines the terms used herein.

[0033] Throughout the specification and in the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. The term "comprises" or "comprising" may encompass limitations to "consists of" or "consisting of" if such limitations are necessary for any reason and to any extent.

[0034] Multiple documents (e.g., patents, patent applications, scientific publications, manufacturer specifications, instructions, GenBank accession number sequence submissions, etc.) may be cited in this specification. Nothing herein should be construed as an admission that the present invention is not entitled to preempt such disclosures by virtue of prior inventions. Some documents cited herein may be characterized as "incorporated by reference." If the definitions or teachings of these incorporated references conflict with the definitions or teachings in this specification, the text of this specification shall prevail.

[0035] All sequences mentioned herein are disclosed in the attached sequence listing, the entire content and disclosure of which constitute part of the disclosure of this specification.

[0036] General definitions of important terms used in this application

[0037] The term "PD-L1" as used herein refers to Uniprot accession number Q9NZQ7 (programmed cell death ligand 1 or programmed death ligand 1). The term "PD-L1" includes all polypeptides that show at least 70%, 75%, 80%, 85%, 90%, 95%, 96% or 97% or more, or 100% sequence identity with the PD-L1 (human) of Uniprot accession number Q9NZQ7. Human PD-L1 has 73% identity to mouse PD-L1 (accession number Q9EP73) and 74% identity to rat PD-L1. The term "PD-L1" includes the extracellular domain (residues 19-238) of PD-L1. Human PD-L1 has three domains: an extracellular domain (aa 19-238), a transmembrane domain (aa239-259), and a cytoplasmic domain (aa 260-290). The extracellular domain of human PD-L1 is shown in SEQ ID NO:11.

[0038] The term "PD-L1 binding protein" refers to a protein of the invention that binds to PD-L1 (programmed cell death ligand 1, or programmed death ligand 1) with high affinity, including its multimeric forms disclosed herein. Thus, the proteins of the present disclosure are PD-L1 binding proteins. As described elsewhere herein, the proteins of the present disclosure exhibit specific binding affinity to human PD-L1, which is a binding affinity in the nanomolar range.

[0039] The terms "protein" and "polypeptide" refer to any chain of two or more amino acids linked by peptide bonds, and do not refer to a specific length of the product. Thus, "peptide," "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included in the definition of "polypeptide," and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the products of post-translational modifications of a polypeptide, as is well known in the art.

[0040] The term "modification" or "amino acid modification" refers to the substitution, deletion or insertion of a reference amino acid by another amino acid at a specific position in the parent polypeptide sequence. Given the known genetic code and recombinant and synthetic DNA technology, skilled scientists can easily construct DNA encoding amino acid variants. The term "amino acid substitution" is understood as the exchange of one amino acid by another.

[0041] The term "ubiquitin" refers to the amino acid sequence given in SEQ ID NO:10.

[0042] The terms "binding affinity" and "binding activity" are used interchangeably herein and refer to the ability of a polypeptide to bind to another protein, peptide, or fragment or domain thereof. Binding affinity is typically measured by the equilibrium dissociation constant (K D ) to measure and report, which is used to assess and rank the strength of biomolecular interactions.

[0043] The term "fusion protein" relates to a protein comprising at least one first protein genetically linked to at least one second protein. A fusion protein is produced by linking two or more genes that originally encode different proteins. The fusion protein may further comprise additional domains that are not involved in target binding, such as, but not limited to, for example, a multimerization moiety, a polypeptide tag, a polypeptide linker, or a moiety that binds to a target different from PD-L1.

[0044] The term "amino acid sequence identity" refers to a quantitative comparison of the identity (or difference) of the amino acid sequences of two or more proteins. "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the sequence that are identical to the amino acid residues in the reference polypeptide sequence, after the sequences are aligned and gaps are introduced, if necessary, to achieve maximum percent sequence identity. To determine sequence identity, the sequence of the query protein is aligned with the sequence of a reference protein or polypeptide. Methods for sequence alignment are well known in the art. For example, in order to determine the degree of amino acid sequence identity of any polypeptide relative to another amino acid sequence, the SIM local similarity program known in the art is preferably used. For multiple alignment analysis, Clustal Omega, known to those skilled in the art, is preferably used.

[0045] Detailed description of embodiments of the present invention

[0046] Structural characterization of programmed death ligand 1 (PD-L1) binding proteins. The proteins described herein comprise an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, wherein the protein has: (a) an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at a position corresponding to position 6 of SEQ ID NO: 1, and (b) an acidic amino acid selected from aspartic acid (D) or glutamic acid (E), or a basic amino acid such as lysine (K) or arginine (R) at a position corresponding to position 8 of SEQ ID NO: 1.

[0047] In various embodiments, the protein is selected from an amino acid sequence having at least 90%, 92%, 93%, 94%, 96%, 97% or 98% identity to the amino acid sequence of SEQ ID NO: 1. The PD-L1 binding protein described herein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, wherein in a preferred embodiment, the protein has: (a) a phenylalanine (F) at a position corresponding to position 6 of SEQ ID NO: 1, and (b) an aspartic acid (D) at a position corresponding to position 8 of SEQ ID NO: 1, and has a binding affinity of at least (or less than) 100 nM for human PD-L1. Specific examples of proteins described herein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 are provided in SEQ ID NO: 2 and SEQ ID NO: 3.

[0048] As described herein, ubiquitin modifications that result in high affinity binding to PD-L1 are located in a region comprising amino acid positions 6 and 8 of SEQ ID NO: 10, and may further comprise modifications in a region comprising amino acid position 62 of ubiquitin (SEQ ID NO: 10).

[0049] As a general concept, the present invention provides a protein comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, wherein the protein: (i) has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at the position corresponding to position 6 of SEQ ID NO: 1, and has a charged amino acid selected from any one of aspartic acid (D), glutamic acid (E), lysine (K), arginine (R) and / or histidine (H) at the position corresponding to position 8 of SEQ ID NO: 1; and (ii) exhibits a binding affinity of less than 100 nM for PD-L1.

[0050] Aspects and embodiments involving an acidic amino acid at the position corresponding to position 8 of SEQ ID NO: 1:

[0051] More specifically, the present invention provides a protein comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, wherein the protein: (i) has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at a position corresponding to position 6 of SEQ ID NO: 1, and has an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at a position corresponding to position 8 of SEQ ID NO: 1; and (ii) exhibits a binding affinity of less than 100 nM for PD-L1. In various embodiments, the acidic amino acid is glutamic acid (E). In preferred embodiments, the acidic amino acid is aspartic acid (D).

[0052] In various other preferred embodiments, the aromatic amino acid is phenylalanine (F), and the acidic amino acid is aspartic acid (D) or glutamic acid (E). More preferably, the aromatic amino acid is phenylalanine (F), and the acidic amino acid is aspartic acid (D).

[0053] In various other preferred embodiments, the aromatic amino acid is tyrosine (Y), and the acidic amino acid is aspartic acid (D) or glutamic acid (E). More preferably, the aromatic amino acid is tyrosine (Y), and the acidic amino acid is aspartic acid (D).

[0054] In various other preferred embodiments, the aromatic amino acid is tryptophan (W), and the acidic amino acid is aspartic acid (D) or glutamic acid (E). More preferably, the aromatic amino acid is tryptophan (W), and the acidic amino acid is aspartic acid (D).

[0055] In various other embodiments, the protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at the position corresponding to position 6 of SEQ ID NO: 1, an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at the position corresponding to position 8 of SEQ ID NO: 1, and an amino acid selected from tryptophan (W), glutamine (Q) or alanine (A) at the position corresponding to position 62 of SEQ ID NO: 1. In various embodiments, the acidic amino acid is glutamic acid (E). In various preferred embodiments, the acidic amino acid is aspartic acid (D).

[0056] In various other preferred embodiments, the aromatic amino acid is phenylalanine (F), the acidic amino acid is aspartic acid (D) or glutamic acid (E), and the amino acid at the position corresponding to position 62 of SEQ ID NO: 1 is tryptophan (W). More preferably, the aromatic amino acid is phenylalanine (F), the acidic amino acid is aspartic acid (D), and the amino acid at the position corresponding to position 62 of SEQ ID NO: 1 is tryptophan (W).

[0057] In various other preferred embodiments, the aromatic amino acid is phenylalanine (F), the acidic amino acid is aspartic acid (D) or glutamic acid (E), and the amino acid at the position corresponding to position 62 of SEQ ID NO: 1 is glutamine (Q). More preferably, the aromatic amino acid is phenylalanine (F), the acidic amino acid is aspartic acid (D), and the amino acid at the position corresponding to position 62 of SEQ ID NO: 1 is glutamine (Q).

[0058] In various other preferred embodiments, the aromatic amino acid is phenylalanine (F), the acidic amino acid is aspartic acid (D) or glutamic acid (E), and the amino acid at the position corresponding to position 62 of SEQ ID NO: 1 is alanine (A). More preferably, the aromatic amino acid is phenylalanine (F), the acidic amino acid is aspartic acid (D), and the amino acid at the position corresponding to position 62 of SEQ ID NO: 1 is alanine (A).

[0059] In the aspects and embodiments described herein relating to SEQ ID NO: 1, particularly including in the aspects and embodiments described herein relating to an acidic amino acid at a position corresponding to position 8 of SEQ ID NO: 1, the protein may comprise an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 1. Preferably, the protein may comprise an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 1. More preferably, the protein may comprise an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 1. Still more preferably, the protein may comprise an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 1. Even more preferably, the protein may comprise an amino acid sequence having at least 97% or at least 98% identity with the amino acid sequence of SEQ ID NO: 1.

[0060] As further described herein, a protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 may in various embodiments describe a variant amino acid sequence, which is (only) based on amino acid substitutions compared to a reference sequence disclosed herein (particularly the sequence of SEQ ID NO: 1). Such a protein (or protein monomer) of the invention has a length of about 76 amino acids, or comprises about 76 amino acids, as described elsewhere herein. In other embodiments, a protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 may in various embodiments describe a variant amino acid sequence, which is (only) based on amino acid substitutions compared to a reference sequence disclosed herein (particularly the sequence of SEQ ID NO: 1) and any one of 1, 2, 3, 4 or 5 amino acid residues deleted at the (N- or C-) terminal. Such a protein (or protein monomer) of the invention has a length of about 71, 72, 73, 74 or 75 amino acids, or comprises about 71, 72, 73, 74 or 75 amino acids.

[0061] The aspects and embodiments described herein relating to SEQ ID NO:1, particularly including the aspects and embodiments described herein relating to the acidic amino acid at the position corresponding to position 8 of SEQ ID NO:1, also apply to the multimeric forms disclosed herein, which multimeric forms contain the protein described herein comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1.

[0062] Aspects and embodiments relating to a basic amino acid at the position corresponding to position 8 of SEQ ID NO: 1:

[0063] Further, the present invention also provides a protein comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, wherein the protein: (i) has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at a position corresponding to position 6 of SEQ ID NO: 1, and has a basic amino acid selected from lysine (K), arginine (R) or histidine (H) at a position corresponding to position 8 of SEQ ID NO: 1; and (ii) exhibits a binding affinity of less than 100 nM for PD-L1. In various embodiments, the basic amino acid is histidine (H). In various preferred embodiments, the basic amino acid is lysine (K) or arginine (R).

[0064] Therefore, in various preferred embodiments, the aromatic amino acid is phenylalanine (F), and the basic amino acid is lysine (K) or arginine (R). More preferably, the aromatic amino acid is phenylalanine (F), and the basic amino acid is lysine (K).

[0065] In various other embodiments, the protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at the position corresponding to position 6 of SEQ ID NO: 1, a basic amino acid selected from lysine (K), arginine (R) or histidine (H) at the position corresponding to position 8 of SEQ ID NO: 1; and an amino acid selected from tryptophan (W), glutamine (Q) or alanine (A) at the position corresponding to position 62 of SEQ ID NO: 1. In various preferred embodiments, the basic amino acid is lysine (K) or arginine (R).

[0066] Therefore, in various preferred embodiments, the aromatic amino acid is phenylalanine (F), the basic amino acid is lysine (K) or arginine (R), and the amino acid at the position corresponding to position 62 of SEQ ID NO: 1 is tryptophan (W). More preferably, the aromatic amino acid is phenylalanine (F), the basic amino acid is lysine (K), and the amino acid at the position corresponding to position 62 of SEQ ID NO: 1 is tryptophan (W).

[0067] In all aspects and embodiments described herein involving SEQ ID NO: 1, particularly including aspects and embodiments described herein involving a basic amino acid at a position corresponding to position 8 of SEQ ID NO: 1, the protein may comprise an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 1. Preferably, the protein may comprise an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 1. More preferably, the protein may comprise an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 1. Still more preferably, the protein may comprise an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 1. Even more preferably, the protein may comprise an amino acid sequence having at least 97% or at least 98% identity with the amino acid sequence of SEQ ID NO: 1.

[0068] The aspects and embodiments described herein relating to SEQ ID NO:1, particularly including the aspects and embodiments described herein relating to a basic amino acid at a position corresponding to position 8 of SEQ ID NO:1, also apply to the multimeric forms disclosed herein, which multimeric forms contain a protein described herein comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:1.

[0069] The proteins of the present invention (including multimeric forms thereof disclosed herein) are believed to bind to the same or overlapping epitopes of PD-L1. In various embodiments, the proteins of the present invention (including multimeric forms thereof disclosed herein) bind or are believed to bind to the extracellular domain of PD-L1. In various embodiments, the proteins of the present invention (including multimeric forms thereof disclosed herein) bind or are believed to bind to the N-terminal region of the extracellular domain of PD-L1. More specifically, the PD-L1 binding proteins of the present invention (including multimeric forms thereof disclosed herein) bind or are believed to bind to the N-terminal region of the extracellular domain of PD-L1, which N-terminal region comprises amino acid residues 1 to about 50 of SEQ ID NO: 11 (corresponding to amino acid residues 19 to about 68 of the amino acid sequence of PD-L1). The PD-L1 binding proteins disclosed herein (including multimeric forms thereof) can bind to an epitope in the N-terminal region of the PD-L1 extracellular domain as described above, wherein the epitope comprises amino acid residues 9 to 15 of SEQ ID NO: 11 (corresponding to amino acid residues 27 to 33 of the amino acid sequence of PD-L1), or consists of the same.

[0070] Functional characterization. The PD-L1 binding proteins described herein (including their multimeric forms disclosed herein) have a binding affinity (KD) of less than (at least) 100 nM for PD-L1. In various embodiments, the protein binds to PD-L1 with a measurable binding affinity of any of the following: less than 100 nM, less than 50 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 2 nM and / or even less than 1 nM.

[0071] As described elsewhere in this article, K D The lower the value, the greater the binding affinity of the biomolecule for its binding partner. D The higher the value, the weaker the binding partners bind to each other (see Examples). Therefore, as described herein, the terms "less than 100 nM" and "at least 100 nM" can be used interchangeably herein, as they both refer to the high binding affinity of the protein of the present invention to PD-L1. More specifically, in the context of high binding affinity, the terms "less than 100 nM" and "at least 100 nM" each refer to an indication of a K DThe value range is ≤ 100 nM (= including 100 nM), because K D The lower the value, the greater the binding affinity. The same considerations regarding the interchangeable use of the terms "less than" and "at least" apply to all preferred binding affinities for PD-L1 disclosed throughout the specification, e.g., "less than 15 nM" and "at least 15 nM", etc. Again, the terms "less than 15 nM" and "at least 15 nM" each indicate an indication of K D The value range is ≤ 15 nM (= including 15 nM), because K D The lower the value, the greater the binding affinity.

[0072] In a preferred embodiment, the PD-L1 binding protein (including its multimeric form disclosed herein) binds to PD-L1 with a measurable binding affinity of less than 15 nM. In various embodiments of the present invention, as described above and throughout the specification, a binding affinity of less than 100 nM for PD-L1 means a binding affinity of less than (at least) 100 nM for human PD-L1 (hPD-L1). Preferred binding affinities (less than 50 nM, etc.) are described above and elsewhere herein and are applicable to embodiments related to binding of proteins and multimers of the present invention to hPD-L1.

[0073] Suitable methods are known to those skilled in the art or described in the literature. The methods for determining binding affinity are known per se and can be selected from, for example, the following methods known in the art: enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), kinetic exclusion analysis (KinExA assay), biolayer interferometry (BLI), flow cytometry, fluorescence spectroscopy, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA) and enhanced chemiluminescence (ECL). Some of these methods are described in the examples below. Typically, the dissociation constant K is determined at 20° C., 25° C. or 30° C. D Unless otherwise specified, the K D The values ​​were determined by SPR at 25°C. D The lower the value, the greater the binding affinity of the biomolecule for its binding partner. D The higher the value, the weaker the binding partners bind to each other (see Examples).

[0074] As disclosed herein, in the above aspects and embodiments, the protein may comprise an amino acid sequence having at least 92% identity, at least 93% identity, or at least 94% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity of less than 100 nM to PD-L1. As further disclosed herein, in the above aspects and embodiments, the protein may comprise an amino acid sequence having at least 92% identity, at least 93% identity, or at least 94% identity to the amino acid sequence of SEQ ID NO: 1, and preferably exhibits a binding affinity of less than 50 nM to PD-L1. More preferably, the protein may comprise an amino acid sequence having at least 92% identity, at least 93% identity, or at least 94% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity of less than 20 nM to PD-L1. Even more preferably, the protein may comprise an amino acid sequence having at least 92% identity, or at least 93% identity, or at least 94% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity of less than 15 nM or less than 10 nM to PD-L1. Still more preferably, in the above aspects and embodiments involving an acidic / basic amino acid at a position corresponding to position 8 of SEQ ID NO: 1, the protein may comprise an amino acid sequence having at least 92% identity, or at least 93% identity, or at least 94% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity of less than 5 nM to PD-L1. In particularly preferred embodiments, the protein may comprise an amino acid sequence having at least 92% identity, or at least 93% identity, or at least 94% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity of less than 2 nM, or even less than 1 nM to PD-L1.

[0075] As further disclosed herein, in the above aspects and embodiments, the protein may comprise an amino acid sequence having at least 96% identity, or at least 97% identity, or at least 98% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity of less than 100 nM to PD-L1. In the above aspects and embodiments, the protein may comprise an amino acid sequence having at least 96% identity, or at least 97% identity, or at least 98% identity to the amino acid sequence of SEQ ID NO: 1, and preferably exhibits a binding affinity of less than 50 nM to PD-L1. More preferably, the protein may comprise an amino acid sequence having at least 96% identity, or at least 97% identity, or at least 98% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity of less than 20 nM to PD-L1. Even more preferably, the protein may comprise an amino acid sequence having at least 97% identity, or at least 98% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity of less than 15 nM or less than 10 nM to PD-L1. Still more preferably, in the above aspects and embodiments, the protein may comprise an amino acid sequence having at least 97% identity or at least 98% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity of less than 5 nM to PD-L1. In particularly preferred embodiments, the protein may comprise an amino acid sequence having at least 97% identity or at least 98% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity of less than 2 nM, or even less than 1 nM to PD-L1.

[0076] As further disclosed herein, in the above aspects and embodiments involving SEQ ID NO: 1 (see "Structural Characterization of PD-L1 Binding Proteins"), the protein (including its multimeric form disclosed herein) preferably exhibits a binding affinity of less than 50 nM for PD-L1. More preferably, the protein disclosed herein (including its multimeric form) exhibits a binding affinity of less than 20 nM for PD-L1. Even more preferably, the protein disclosed herein (including its multimeric form) exhibits a binding affinity of less than 15 nM or less than 10 nM for PD-L1. Still more preferably, the protein disclosed herein (including its multimeric form) exhibits a binding affinity of less than 5 nM for PD-L1. It is particularly preferred that the protein disclosed herein (including its multimeric form) exhibits a binding affinity of less than 2 nM, or even less than 1 nM for PD-L1.

[0077] The aspects and embodiments described herein relating to the functional characterization of the PD-L1 binding proteins of the invention also apply to the multimeric forms disclosed herein, which contain the proteins described herein comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.

[0078] Cellular PD-L1 binding of the proteins described herein can be determined by standard methods, including immunofluorescence microscopy and flow cytometric analysis. In some embodiments, the specific PD-L1 binding of the proteins of the present invention is determined by cellular PD-L1 binding analysis. In the above aspects and embodiments involving SEQ ID NO: 1 (see "Structural Characterization of PD-L1 Binding Proteins"), in various embodiments, the protein preferably exhibits a binding affinity of less than 5nM, preferably less than 3nM, to PD-L1 on PD-L1 expressing cells (see Examples). More specifically, in various embodiments, the protein preferably exhibits a binding affinity of less than 5nM, preferably less than 3nM, to PD-L1 on PD-L1 expressing cells as determined by flow cytometric analysis (see Examples).

[0079] In some embodiments, the proteins described herein inhibit the interaction of PD-1 binding to PD-L1. In preferred embodiments, the proteins described herein (including their multimeric forms disclosed herein) specifically bind to (human) PD-L1 with high affinity (i.e., less than 100nM, preferably less than 50nM, etc., as described elsewhere herein) and inhibit the interaction of PD-L1 with PD-1. As further described herein, in various embodiments, inhibiting the interaction of PD-L1 with PD-1 means (or provides) blocking PD-1 inhibitory signaling.

[0080] In some embodiments, the proteins described herein are particularly stable under different conditions. In a preferred embodiment, the protein is stable at least 24 h at 37 ° C in the presence of (human or mouse or rat) serum. In some embodiments, the proteins described herein are stable at least 24 h at 37 ° C in the presence of human serum, as described in more detail in Example 6 and Figure 1. In some embodiments, the proteins described herein are stable at least 24 h at 37 ° C in the presence of mouse serum. For example, the binding affinity (K) can be measured by using the standard methods described herein above and in the Examples after incubation in serum for a long period of time at a temperature of up to 37 ° C. D) to determine the stability of the PD-L1 binding protein. In the above aspects and embodiments involving SEQ ID NO: 1 (see "Structural Characterization of PD-L1 Binding Protein"), in various embodiments, even after incubation in (human) serum at 37°C for 24h, the protein preferably exhibits a binding affinity for PD-L1 on PD-L1 expressing cells of less than 5nM, preferably less than 3nM. More specifically, in various embodiments, even after incubation in (human) serum at 37°C for 24h, the protein preferably exhibits a binding affinity for PD-L1 on PD-L1 expressing cells of less than 5nM, preferably less than 3nM as determined by flow cytometry analysis.

[0081] In some embodiments, the PD-L1 binding proteins described herein are stable at high temperatures (preferably at least 60° C.). For stability analysis, spectroscopic or fluorescence-based methods, such as those associated with chemical or physical unfolding, are known to those skilled in the art. For example, thermal melting (T m ) temperature to determine the stability of the molecule, thermal melting (T m ) is the temperature at which half of the molecules unfold, in degrees Celsius (℃). Usually, T m The higher the value, the more stable the molecule. Temperature stability was determined by differential scanning fluorimetry (DSF), as described in further detail in Example 3 and Table 1.

[0082] In addition, the above-mentioned further aspects and embodiments related to the functional characterization of the PD-L1 binding protein of the present invention are applicable to the multimeric form disclosed herein, which contains the protein described throughout the present invention comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.

[0083] Multimer. The present invention provides a multimer comprising a protein of the present invention as described above and throughout the present specification, that is, as a general concept, the present invention provides a multimer comprising a protein comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, wherein the protein: (i) has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at the position corresponding to position 6 of SEQ ID NO: 1, and has a charged amino acid selected from any one of aspartic acid (D), glutamic acid (E), lysine (K), arginine (R) and / or histidine (H) at the position corresponding to position 8 of SEQ ID NO: 1; and (ii) exhibits a binding affinity of less than 100 nM for programmed (cell) death ligand 1 (PD-L1).

[0084] More specifically, the present invention provides a multimer comprising a protein described elsewhere herein regarding the “aspects and embodiments involving an acidic amino acid at a position corresponding to position 8 of SEQ ID NO:1”, and also provides a multimer comprising a protein described elsewhere herein regarding the “aspects and embodiments involving a basic amino acid at a position corresponding to position 8 of SEQ ID NO:1”.

[0085] In the present invention, the multimer can be any one of a dimer, a trimer, a tetramer, a pentamer, or a hexamer. In the present invention, the multimer is preferably a dimer or a trimer, more preferably a dimer. Therefore, the multimer of the present invention comprises at least two domains or at least two monomers (= dimer). Therefore, the protein of the present invention as described above throughout this specification can be regarded as a protein domain or protein monomer of the multimer provided by the present invention. Such a protein domain or protein monomer can have a length of about 76 amino acids, or contain about 76 amino acids.

[0086] In some embodiments, the multimer may comprise one, two, three, four or more proteins described herein. In one embodiment, the protein comprises 2, 3, 4 or more proteins connected to each other, that is, the protein may be a dimer, trimer or tetramer as described above, etc., wherein at least one protein of the multimer comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, wherein the protein has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at a position corresponding to position 6 of SEQ ID NO: 1, and has an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at a position corresponding to position 8 of SEQ ID NO: 1, and wherein the multimeric protein has a binding affinity of less than 100 nM to human PD-L1.

[0087] In a preferred embodiment, the PD-L1 binding protein comprises two proteins linked to each other, wherein one protein in the multimer comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, wherein the protein has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at the position corresponding to position 6 of SEQ ID NO: 1, and has an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at the position corresponding to position 8 of SEQ ID NO: 1, and wherein the dimeric PD-L1 binding protein has a binding affinity for human PD-L1 of less than 100 nM. In a preferred embodiment, the PD-L1 binding protein comprises two interconnected proteins, one of which has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, wherein the protein has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at the position corresponding to position 6 of SEQ ID NO: 1, and has an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at the position corresponding to position 8 of SEQ ID NO: 1, and the second protein has an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, and wherein the dimeric PD-L1 binding protein has a binding affinity for human PD-L1 of less than 100 nM.

[0088] In various preferred embodiments of the present invention, the multimer comprises: the protein of the present invention described above and throughout this specification, and a protein comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 4. In this case, the multimer of the present invention comprises at least two protein domains. The at least two protein domains of the multimer of the present invention are located at the N-terminus and the C-terminus, respectively.

[0089] In a preferred embodiment, the PD-L1 binding protein comprises two proteins linked to each other, wherein the protein comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, wherein the protein has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at the position corresponding to position 6 of SEQ ID NO: 1, and has an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at the position corresponding to position 8 of SEQ ID NO: 1, the acidic amino acid is located at the N-terminus or C-terminus, preferably the C-terminus, and wherein the dimeric PD-L1 binding protein has a binding affinity for human PD-L1 of less than 100 nM. In a preferred embodiment, the PD-L1 binding protein comprises 2 proteins linked to each other, wherein the protein having an amino acid sequence with at least 90% identity has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at the position corresponding to position 6 of SEQ ID NO: 1, and has an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at the position corresponding to position 8 of SEQ ID NO: 1, which is located at the N-terminus or C-terminus, preferably the C-terminus, and wherein the second protein having an amino acid sequence with at least 90% identity to SEQ ID NO: 4 is located at the N-terminus or C-terminus, preferably the N-terminus, and wherein the dimeric PD-L1 binding protein has a binding affinity for human PD-L1 of less than 100 nM.

[0090] In various embodiments, the multimer comprises: a protein of the present invention as described above and throughout this specification, and a protein comprising an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID NO: 4. In various preferred embodiments, the multimer comprises: a protein of the present invention as described above and throughout this specification, and a protein comprising an amino acid sequence having at least 93% identity with the amino acid sequence of SEQ ID NO: 4. More preferably, the multimer comprises: a protein of the present invention as described above and throughout this specification, and a protein comprising an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 4. Still more preferably, the multimer comprises: a protein of the present invention as described above and throughout this specification, and a protein comprising an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 4. Even more preferably, the multimer comprises: a protein of the present invention as described above and throughout this specification, and a protein comprising an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 4. In a particularly preferred embodiment, the multimer comprises: a protein of the invention as described above and throughout this specification, and a protein comprising an amino acid sequence that is at least 98% or even 100% identical to the amino acid sequence of SEQ ID NO:4.

[0091] Preferably, the multimer is a dimer or trimer, comprising: a protein of the present invention as described above and throughout this specification, and a protein comprising an amino acid sequence as described above that has at least 90% identity with the amino acid sequence of SEQ ID NO: 4. More preferably, the multimer is a dimer, comprising: a protein of the present invention as described above and throughout this specification, and a protein comprising an amino acid sequence as described above that has at least 90% identity with the amino acid sequence of SEQ ID NO: 4.

[0092] A preferred embodiment relates to a dimeric PD-L1 binding protein comprising an amino acid sequence of SEQ ID NO:4 and an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO:1, wherein the protein: (i) has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at a position corresponding to position 6 of SEQ ID NO:1, and has an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at a position corresponding to position 8 of SEQ ID NO:1; and (ii) exhibits a binding affinity for human programmed death ligand 1 (PD-L1) of less than 15 nM as determined by surface plasmon resonance.

[0093] Preferably, in the multimer of the present invention, the multimer comprises a protein of the present invention as described above and throughout this specification and a protein comprising an amino acid sequence as described above that has at least 90% identity with the amino acid sequence of SEQ ID NO: 4, the protein of the present invention (domain / monomer) as described above and throughout this specification is located at the C-terminus, and the protein (domain / monomer) as described above that comprises an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 4 is located at the N-terminus.

[0094] In one embodiment, the multimer is a dimer comprising two proteins, wherein the second (C-terminal) protein corresponds to SEQ ID NO: 1 and the first (N-terminal) protein corresponds to SEQ ID NO: 4. The sequence of the dimer is SEQ ID NO: 5 (224039). In another embodiment, the multimer is a dimer comprising two proteins, wherein the second (C-terminal) protein corresponds to SEQ ID NO: 2 and the first (N-terminal) protein corresponds to SEQ ID NO: 4. The sequence of the dimer is SEQ ID NO: 6 (224058). In another embodiment, the multimer is a dimer comprising two proteins, wherein the second (C-terminal) protein corresponds to SEQ ID NO: 3 and the first (N-terminal) protein corresponds to SEQ ID NO: 4. The sequence of the dimer is SEQ ID NO: 7 (224180).

[0095] In some embodiments, the PD-L1 binding protein has at least 90% identity to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In a preferred embodiment, the PD-L1 binding protein has at least 90% identity to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, provided that the aromatic amino acid at the position corresponding to position 82 of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 and the acidic amino acid at position 84 of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 remain unchanged.

[0096] In some embodiments, two or more proteins are directly linked. In some embodiments, two or more proteins as described herein are linked by a peptide linker. In various embodiments, two or more proteins as described herein are linked by a peptide linker of up to 30 amino acids. In other embodiments, two or more proteins as described herein are linked by a peptide linker of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids.

[0097] The multimer as described above can be considered as a chimeric polypeptide comprising a first part and a second part, wherein the first part comprises the amino acid sequence of the protein of the present invention as described above and throughout this specification, and wherein the second part comprises the amino acid sequence of the protein as described above comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:4.

[0098] The functional properties of the proteins of the present invention as described above and throughout the specification are fully applicable to any multimers, fusion proteins and chimeric polypeptides provided by the present invention.

[0099] Coupling site. In some embodiments, the PD-L1 binding protein described herein further comprises one or more coupling sites for coupling of a chemical moiety. The coupling site is capable of reacting with other chemical groups to couple the PD-L1 binding protein to the chemical moiety. The limited number and limited position of the coupling site enable the chemical moiety to be site-specifically coupled to the PD-L1 binding protein described herein. Therefore, if necessary, a large number of chemical moieties can be bound to the PD-L1 binding protein. Those skilled in the art can adjust the number of coupling sites to the optimal number for a particular application, thereby adjusting the amount of the chemical moiety accordingly. In a selected embodiment, the coupling site can be selected from one or more groups of amino acids that can be labeled with a specific chemical substance, such as one or more cysteine ​​residues, one or more lysine residues, one or more tyrosines, one or more tryptophans, or one or more histidine residues. The PD-L1 binding protein may comprise 1 to 20 coupling sites, preferably 1 to 6 coupling sites, preferably 2 coupling sites, or preferably 1 coupling site.

[0100] Coupling domain. One embodiment provides a PD-L1 binding protein comprising at least one coupling domain of 1 to 80 amino acids, the coupling domain comprising one or more coupling sites. In some embodiments, the coupling domain of 1 to 80 amino acids may comprise alanine, proline or serine and cysteine ​​as a coupling site. In other embodiments, the coupling domain of 5 to 80 amino acids may consist of alanine, proline, serine and cysteine ​​as a coupling site. In one embodiment, the coupling domain consists of 20-60% alanine, 20-40% proline, 10-60% serine and one or more cysteines as coupling sites at the C-terminus or N-terminus of the PD-L1 binding protein described herein. In some embodiments, the amino acids alanine, proline and serine are randomly distributed throughout the coupling domain amino acid sequence, so that the adjacent identical amino acid residues are no more than 2, 3, 4 or 5, preferably up to 3 amino acids. The composition of 1 to 20 coupling domains may be different or the same.

[0101] Chemical moieties. In some embodiments, the chemical moieties are selected from any one of chelators, drugs, toxins, dyes, and small molecules. In some embodiments, at least one of the chemical moieties is a chelator, which is designed as a complexing agent for coupling one or more other moieties to a target compound of a PD-L1 binding protein disclosed herein. One embodiment relates to a PD-L1 binding protein, wherein the chelator is a complexing agent for coupling one or more radioisotopes or other detectable labels.

[0102] Diagnostic moiety. In some embodiments, a protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 further comprises at least one diagnostically active moiety, wherein the protein has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at a position corresponding to position 6 of SEQ ID NO: 1, and has an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at a position corresponding to position 8 of SEQ ID NO: 1. In various embodiments, the PD-L1 binding protein further comprises a diagnostic moiety. In other embodiments, the PD-L1 binding protein further comprises more than one diagnostic moiety. In some embodiments, such diagnostic moieties may be selected from radionuclides, fluorescent proteins, photosensitizers, dyes, fluorophores, enzymes, magnetic beads, metal beads, colloidal particles, electron-dense reagents, biotin, digoxin, haptens, or any combination thereof. In some embodiments, a PD-L1 binding protein comprising at least one diagnostic moiety may be used, for example, as an imaging agent, for example to assess the presence of tumor cells or metastases, tumor distribution, and / or tumor recurrence. The method for detecting or monitoring cancer cells relates to an imaging method. Such methods involve imaging PD-L1 associated cancer cells by, for example, radioimaging or photoluminescence or fluorescence.

[0103] Therapeutic moiety. In some embodiments, a protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 further comprises at least one therapeutically active moiety, wherein the protein has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at a position corresponding to position 6 of SEQ ID NO: 1, and has an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at a position corresponding to position 8 of SEQ ID NO: 1. In other embodiments, the PD-L1 binding protein further comprises more than one therapeutically active moiety. In some embodiments, such therapeutically active moieties may be selected from monoclonal antibodies or fragments thereof, extracellular domains of receptors or fragments thereof, radionuclides, cytotoxic compounds, cytokines, chemokines, enzymes, or derivatives thereof, or any combination thereof. In some embodiments, the PD-L1 binding protein comprising a therapeutically active ingredient can be used to target and accumulate any of the components listed above to tumor cells expressing PD-L1, thereby producing low levels of toxicity to normal cells.

[0104] Radionuclides. Radionuclides suitable for use in imaging (e.g., in vitro) or radiotherapy include, for example, but not limited to, a group of gamma-emitting isotopes, a group of positron emitters, a group of beta emitters, and a group of alpha emitters. In some embodiments, suitable conjugation partners include chelating agents such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA) or their activated derivatives, nanoparticles, and liposomes. In various embodiments, DOTA may be suitable as a complexing agent for radioisotopes and other agents used for imaging.

[0105] Moieties that regulate pharmacokinetics. In some embodiments, a protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 further comprises at least one moiety that regulates pharmacokinetics, wherein the protein has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at a position corresponding to position 6 of SEQ ID NO: 1, and an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at a position corresponding to position 8 of SEQ ID NO: 1. In some embodiments, the moiety that regulates pharmacokinetics is selected from polyethylene glycol, human serum albumin, an albumin-binding peptide, an immunoglobulin binding peptide or an immunoglobulin or an immunoglobulin fragment, sialic acid, or transferrin, transferrin receptor binding protein, a polysaccharide (e.g., hydroxyethyl starch), or an unstructured amino acid sequence that increases the hydrodynamic radius (e.g., a polymer comprising amino acids alanine, glycine, serine, proline). In various embodiments, the moiety increases the half-life of the PD-L1 binding protein by at least 1.5 times. A variety of techniques for producing PD-L1 binding proteins with extended half-life are known in the art, for example, directly fusing or chemically coupling a pharmacokinetic modulating moiety to a PD-L1 binding protein as described above. As described above, a pharmacokinetic modulating moiety can be, for example, linked to one or more sites of a PD-L1 binding protein via a peptide linker sequence or via a coupling site.

[0106] The protein or non-protein moiety can be conjugated to the PD-L1 binding protein using chemical methods well known in the art. In some embodiments, coupling chemistry dedicated to the derivatization of cysteine ​​or lysine residues may be applicable. Chemical coupling can be performed by chemical methods well known to those skilled in the art, including but not limited to substitution, addition, or cycloaddition or oxidation chemistry (e.g., disulfide bond formation).

[0107] Molecules for purification / detection. In some embodiments, additional amino acids may be extended at the N-terminus or C-terminus or both of the PD-L1 binding protein. Additional sequences may include, for example, sequences introduced for purification or detection. In one embodiment, the additional amino acid sequence includes one or more peptide sequences that confer affinity for certain chromatography column materials. Typical examples of such sequences include, but are not limited to, Strep-tags, oligohistidine-tags, glutathione S-transferases, maltose binding proteins, inteins, intein fragments, or the albumin binding domain of protein G.

[0108] Composition. Various embodiments relate to a composition comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, wherein the protein has an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at a position corresponding to position 6 of SEQ ID NO: 1, and an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at a position corresponding to position 8 of SEQ ID NO: 1, and wherein the protein has a binding affinity of less than 100 nM for human PD-L1. Various embodiments relate to a composition comprising a PD-L1 binding protein as defined above, for use in medical treatment. Various preferred embodiments relate to a composition comprising a PD-L1 binding protein as defined above, for use in the diagnosis or treatment of various PD-L1-related (PD-L1-positive) tumors or sensor diseases such as chronic viral infections. The composition comprising the above-mentioned PD-L1 binding protein can be used in clinical applications for diagnostic and therapeutic purposes. In particular, the composition comprising the above-mentioned PD-L1 binding protein can be used for clinical applications of imaging, monitoring, and eliminating or inactivating pathological cells expressing PD-L1.

[0109] Various embodiments relate to a diagnostic composition for diagnosing PD-L1-related cancers, comprising a PD-L1 binding protein as defined herein and a diagnostically acceptable carrier and / or diluent. These include, for example, but not limited to, stabilizers, surfactants, salts, buffers, colorants, etc. The composition may be in the form of a liquid preparation, a lyophilisate, a granulate, an emulsion or a liposomal preparation.

[0110] The diagnostic compositions comprising the PD-L1 binding proteins described herein can be used to diagnose PD-L1-related cancers, as described above.

[0111] Various embodiments relate to a pharmaceutical (e.g., therapeutic) composition for treating a disease, comprising a PD-L1 binding protein as disclosed herein and a pharmaceutically (e.g., therapeutically) acceptable carrier and / or diluent. The pharmaceutical (e.g., therapeutic) composition may optionally contain other adjuvants and excipients known per se. These include, for example, but not limited to, stabilizers, surfactants, salts, buffers, colorants, and the like.

[0112] Pharmaceutical compositions comprising a PD-L1 binding protein as defined herein may be used to treat a disease, as described above.

[0113] The composition contains an effective dose of a PD-L1 binding protein as defined herein. The amount of protein administered depends on the organism, the type of disease, the age and weight of the patient and other factors known per se. According to the galenic formulation, these compositions can be administered parenterally, systemically, intraperitoneally, intramuscularly, subcutaneously, transdermally, or by other commonly used application methods by injection or infusion.

[0114] The composition can be in the form of a liquid preparation, a lyophilisate, an ointment, a lotion for topical application, an aerosol, a powder, a granule, an emulsion or a liposome preparation. The type of preparation depends on the type of disease, the route of administration, the severity of the disease, the patient and other factors known to those skilled in the medical field.

[0115] The various components of the composition may be packaged into a kit together with instructions for use.

[0116] Used in medical treatment. Various embodiments relate to the PD-L1 binding proteins disclosed herein for use in medical treatment. In one embodiment, the PD-L1 binding protein is used in medical treatment to diagnose or treat cancer associated with PD-L1 expression. Therefore, a method for diagnosing or treating PD-L1-related cancer or infectious disease is disclosed herein. The PD-L1 binding protein disclosed herein allows for the selective diagnosis and treatment of PD-L1-related cancer cells or cancer tissues, such as cancer cells or cancer tissues from melanoma, NSCLC, head and neck cancer, bladder cancer, breast cancer, etc. It is known that PD-L1 is upregulated in tumor cells, which may lead to the uncontrolled growth of tumor cells and the formation of metastases. The PD-L1 binding protein disclosed herein further allows for the selective diagnosis and treatment of PD-L1-related infectious diseases, such as chronic viral infections (HIV, HBV, HCV). In one embodiment, PD-L1 binding protein is used to diagnose PD-L1-related cancer or infectious diseases by applying an in vitro method.

[0117] One embodiment is a method for diagnosing (including monitoring) a subject with a PD-L1-related cancer or infectious disease (e.g., chronic viral infection), the diagnostic (monitoring) method comprising administering to the subject a PD-L1 binding protein described herein, the protein optionally conjugated to a radioactive molecule. In various embodiments, the PD-L1 binding protein disclosed herein can be used to diagnose PD-L1-related cancers or infectious diseases, such as chronic viral infections, optionally wherein the PD-L1 binding protein is conjugated to a radioactive molecule. In some embodiments, the PD-L1 binding protein is used for (in vitro) imaging methods using labels such as radioactive articles or fluorescence, and can be used to visualize PD-L1 on specific tissues or cells, for example, to assess the presence of PD-L1-related tumor cells (PD-L1-positive tumor cells), PD-L1-related tumor distribution, recurrence of PD-L1-related tumors (PD-L1-positive tumors), and / or assess the patient's response to therapeutic treatment.

[0118] One embodiment is a method for treating a subject with a PD-L1-related (positive) cancer (tumor) or infectious disease (e.g., chronic viral infection), the method comprising administering to the subject a PD-L1-specific binding protein described herein, the protein optionally conjugated to a radioactive molecule and / or a cytotoxic agent, or as an immuno-oncology agent. In various embodiments, the PD-L1 binding protein disclosed herein can be used to treat PD-L1-related cancers or infectious diseases such as chronic viral infections, optionally wherein the PD-L1 binding protein is conjugated to a cytotoxic agent and / or a radioactive molecule or expressed on the surface of a target-specific CarT cell. Some embodiments relate to the use of PD-L1 binding proteins labeled with suitable radioisotopes or cytotoxic compounds for the treatment of PD-L1-related (positive) tumor cells, particularly controlling or killing PD-L1-related (positive) tumor cells, such as malignant cells. In one embodiment, a therapeutic dose of radiation is selectively delivered to PD-L1-related (positive) tumor cells, but not to normal cells.

[0119] As further described herein, in various embodiments, PD-L1-associated cancers are characterized by cancer cells that express or overexpress PD-L1. In various embodiments, PD-L1-associated cancers are solid tumors that express or overexpress PD-L1. Use as an immuno-oncology agent refers to the use of the PD-L1 binding protein disclosed herein as a therapeutic composition for treating a disease, i.e., promoting an immune response against cancer. Certain cancer cells suppress the immune response against the cell, such as by suppressing a T cell response against the cell. The PD-L1 binding protein disclosed herein as part of a medical composition used as a therapeutic agent can counteract the suppression of the immune response.

[0120] Production of PD-L1 binding proteins. The PD-L1 binding proteins described herein can be prepared by any of a variety of conventional and well-known techniques, such as common organic synthesis strategies, solid phase-assisted synthesis techniques, fragment ligation techniques, or by commercially available automatic synthesizers. On the other hand, they can also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques. In addition, they can also be prepared by cell-free in vitro transcription / translation.

[0121] Various embodiments relate to a polynucleotide encoding a PD-L1 binding protein disclosed herein. One embodiment further provides an expression vector comprising the polynucleotide, and a host cell comprising the isolated polynucleotide or expression vector.

[0122] Various embodiments relate to a method of producing a PD-L1 binding protein disclosed herein, comprising the steps of a) culturing a host cell under appropriate conditions allowing expression of the protein and b) isolating the protein.

[0123] For example, one or more polynucleotides encoding PD-L1 binding proteins can be expressed in a suitable host, and the produced PD-L1 binding proteins can be isolated. The host cell comprises the nucleic acid molecule or vector. Suitable host cells include prokaryotic cells or eukaryotic cells. A vector refers to any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) that can be used to transfer protein coding information to a host cell. Various cell culture systems (e.g., but not limited to mammals, yeast, plants, or insects) can also be used to express recombinant proteins. Appropriate conditions for culturing prokaryotic or eukaryotic host cells are well known to those skilled in the art. Cell culture and protein expression for protein production purposes can be performed on any scale from small-volume shake flasks to large fermenters using techniques well known to those skilled in the art.

[0124] One embodiment relates to a method for producing a protein as detailed above, the method comprising the following steps: (a) preparing a nucleic acid encoding a PD-L1 binding protein as defined herein; (b) introducing the nucleic acid into an expression vector; (c) introducing the expression vector into a host cell; (d) culturing the host cell; (e) subjecting the host cell to culture conditions for expressing the PD-L1 binding protein, thereby producing the PD-L1 binding protein as defined herein; (f) optionally isolating the PD-L1 binding protein produced in step (e); and (g) optionally conjugating the PD-L1 binding protein to other functional parts as defined herein.

[0125] In general, the purified PD-L1 binding protein can be isolated from the culture mixture using conventional methods and techniques well known in the art, such as centrifugation, precipitation, flocculation, different embodiments of chromatography, filtration, dialysis, concentration, and combinations thereof. Chromatography is well known in the art and includes, but is not limited to, ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), hydrophobic interaction chromatography, or affinity chromatography.

[0126] To simplify purification, the PD-L1 binding protein can be fused to other peptide sequences with increased affinity for the separation material. Preferably, such fusion is selected so that it does not have an adverse effect on the function of the PD-L1 binding protein, or can be separated after purification due to the introduction of a specific protease cleavage site. Such methods are also known to those skilled in the art.

[0127] Method for detecting PD-L1 in a sample. Some embodiments relate to detecting PD-L1 in a sample, the method comprising contacting the sample with the above-mentioned PD-L1 binding protein, detecting the binding of PD-L1 in the sample to the above-mentioned PD-L1 binding protein. In some embodiments, the protein described herein is used in a method to determine the presence of PD-L1. Some embodiments relate to a method for analyzing the presence of PD-L1 in a sample, the method comprising the following steps: (i) providing a sample containing PD-L1, (ii) providing a binding protein for PD-L1, (iii) contacting the sample containing PD-L1 with the binding protein for PD-L1 described herein under conditions that allow at least one protein to bind to PD-L1, (iv) separating (eluting) the complex of PD-L1 and the binding protein for PD-L1, and (v) determining the amount of the binding protein for PD-L1, which indicates the amount of PD-L1 in the sample (i). In some embodiments, the sample can be a liquid sample, such as a blood sample or a urine sample or a tumor sample in a liquid.

[0128] Example

[0129] The following examples are provided to further illustrate the present invention. However, the present invention is not limited thereto, and the following examples merely show the practicality of the present invention based on the above description.

[0130] Example 1. Expression and purification of PD-L1 binding protein

[0131] The genes for proteins 224039, 224058, 224180, and 224121 (SEQ ID NOs: 5-8) were cloned into expression vectors using standard methods known to those skilled in the art, purified, and analyzed as described below. All proteins were expressed and highly purified by affinity chromatography and gel filtration. After affinity chromatography purification, the proteins were purified using System and Superdex TM Size exclusion chromatography (SEC) was performed on a 200 HiLoad 16 / 600 column (GE Healthcare). The column had a volume of 120 ml and was balanced using 2CV. The sample was applied at a flow rate of 1 ml / min using PBS as a running buffer. Fraction collection was started when the signal intensity reached 10 mAU. After SDS-PAGE analysis, positive fractions were pooled and their protein concentrations were measured.

[0132] Purity was confirmed by SDS-PAGE, SE-HPLC and RP-HPLC. Purity determined by RP-HPLC was at least 98%. Protein concentration was determined by absorbance measurement at 280 nm using the specific molar absorption coefficient. The protein was purified using an Ultimate 3000 HPLC system (Thermo Fisher Scientific) and PLRP-S (5 μm, ) column (Agilent) for RP chromatography (RP HPLC).

[0133] Example 2: Mammalian Expression of Human and Mouse PD-L1

[0134] Expi293-F- cells were cultured at 0.5-1 million cells / ml in Expi293-F expression medium (Fisher scientific, 13489756) at 135rpm, 37°C, 8% CO2 and 95% humidity in a shake flask. One day before transfection, cells were inoculated at a density of 2.0 million cells / ml. On the day of transfection, cells were inoculated at a density of 2.5 million cells / ml. 1 μg of the plasmid DNA of the extracellular domain of hPD-L1-Fc or mPD-L1-Fc was diluted in Opti-MEM I reduced serum medium (Life Technologies, 31985-062) per ml culture volume. According to the manufacturer's information, ExpiFectamine was diluted in Opti-MEM I reduced serum medium and incubated at room temperature for 5 min. Subsequently, the DNA-solution was added to the ExpiFectamine-mixture, incubated at room temperature for 20 min and mixed with the cells. After 16 h, the enhancer was added to the cells. After 96-120 h, the supernatant was collected, centrifuged and filtered through a 0.45 μm membrane.

[0135] Example 3. Stability at high temperature (above 60°C)

[0136] The thermal stability of proteins was determined by differential scanning fluorimetry (DSF). Each probe was transferred to a MicroAmp Optical 96-well plate at a concentration of 0.1 μg / μL, and SYPRO orange dye was added with an appropriate dilution. The temperature was programmed to 95°C from 25°C at a heating rate of 1°C / min. Fluorescence was continuously measured at 520nm for excitation and 623nm for emission (ViiA7 real-time PCR system (Thermo Scientific)). The midpoint of the transition (Tm, melting point) of thermal unfolding was determined, and as shown in Table 1.

[0137] Example 4. Biochemical binding analysis (surface plasmon resonance, SPR)

[0138] After NHS / EDC activation, recombinant protein A was immobilized on a high-capacity amine sensor chip (Bruker) using a Sierra SPR-32 system (Bruker) to generate approximately 1000RU. The chip was balanced with SPR running buffer (PBS 0.05%, Tween pH7.3). Ethanolamine was injected after ligand immobilization to block unreacted NHS groups. The Fc-tagged extracellular domains of hPD-L1 and mPD-L1 were injected at 30nM, followed by PD-L1 binding protein at a flow rate of 30μl / min. A flow cell without ligand was used as a reference. Once the ligand binds, the protein analyte accumulates on the surface, increasing the refractive index. This change in refractive index is measured in real time and plotted as a response or resonance unit (RU) versus time. The analyte was applied to the chip in serial dilutions. Association was performed for 120 seconds and dissociation was performed for 180 seconds. After each run, the chip surface was regenerated with 30 μl regeneration buffer (10 mM glycine pH 2.0) and equilibrated with running buffer.Binding studies were performed by using a Sierra SPR-32 system (Bruker) and data evaluation was performed by the Sierra Analyser software provided by the manufacturer.

[0139] The binding affinity to PD-L1 is shown in Table 1. Binding of 211828 to mouse PD-L1 was detectable, but an effective KD could not be determined using SPR.

[0140] Table 1. Binding affinity (K D ) and temperature stability.

[0141]

[0142] Example 5. Functional characterization: specific binding to hPD-L1 and mPD-L1 expressed on the cell surface (flow cytometry)

[0143] Flow cytometry was used to analyze the specific interaction of PD-L1 binding proteins with surface-exposed human PD-L1 (hPD-L1) or mouse PD-L1 (mPD-L1). Transfected HEK293-hPD-L1 cells, HEK293-mPD-L1 cells, empty vector control HEK293-pEntry cells, and native hPD-L1-expressing H460 cell lines were trypsinized and resuspended in medium containing FCS and washed in pre-cooled FACS blocking buffer. A cell concentration of 1 million cells / ml was prepared for cell staining and loaded into 96-well plates (Greiner) at 100 μl / / well in triplicate for each cell line. Dilution series of proteins or 1 μg / ml Avelumab (Merck) were added as positive controls to PD-L1-expressing cells and control cells. After 45 min, the supernatant was removed and 100 μl / well rabbit anti-Strep-tag antibody (GenScript; A00626) (diluted 1:300 in FACS blocking buffer) was added to the wells with diluted bound protein. Avelumab was detected using anti-human-IgG-Alexa 488 (Invitrogen; A-11013) at a dilution of 1:1000. After removal of the anti-Strep-tag antibody, goat anti-rabbit IgG Alexa Fluor 488 antibody (Invitrogen; A11008) was applied to the other wells at a dilution of 1:1000. Flow cytometry measurements were performed on a Guava easyCyte 5HT device (Merck-Millipore) at an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

[0144] 224039, 224058, 224180 (SEQ ID NOs: 5-7) showed specific binding on human PD-L1 or mouse PD-L1 overexpressing HEK293-cells. No binding was detected on HEK293-pEntry-cells. 224039, 224058, 224180 also showed significant binding to native human PD-L1-expressing cells, while 224121 and 211828 showed only weak binding. In Table 2, Pos 6 and Pos 8 refer to the positions in the second (C-terminal) monomer of the protein.

[0145] Table 2: Cell binding analysis (flow cytometry)

[0146]

[0147] Example 6. Binding affinity to PD-L1 in human serum after long-term incubation (cell binding assay - flow cytometry)

[0148] The amount of 224058 (SEQ ID NO: 6) was increased from 3.3 x 10 -6 M to 7x 10 -13 M and 224180 (SEQ ID NO: 7) or 224039 (SEQ ID NO: 5) from 1 x 10 -5 M to 7x 10 -13 A dilution series of 1 μM was incubated in human serum at 37°C for 0 h and 24 h. PD-L1 overexpressing HEK293 cells were trypsinized, washed with FACS blocking buffer, seeded in 96-well round-bottom plates at a density of 0.1 million cells / 100 μl, and the protein dilution series was added to the cells. After 45 min, the supernatant was removed and the cells were washed. In the first step, binding was demonstrated with 100 ul / well rabbit anti-Strep-tag antibody diluted 1:300 in FACS blocking buffer, and in the second step, binding was demonstrated with goat anti-rabbit IgG Alexa Fluor 488 antibody diluted 1:1000. The readouts were as described above. The protein was stable in human serum for at least 24 h (Figure 1), with only a slight loss of binding affinity to PD-L1 expressed on cells (224058: K for human PD-1 after 24 h). D 2.4nM; 224180: K of human PD-1 after 24h D 224039 was stable in human serum even after 24 h incubation, with a K of 1.2 nM for human PD-L1. D is 1.3nM.

[0149] Example 7. Serum stability of PD-L1 binding protein (ELISA)

[0150] High binding plates (Greiner, 781061) were immobilized with 2.5 μg / ml recombinant human PD-L1-Fc overnight at 4°C. 1 μM to 7x10 -8A μM dilution series of 224058 (SEQ ID NO: 6), 224180 (SEQ ID NO: 7) and 224039 (SEQ ID NO: 5) was incubated for 24 h. The ELISA plate was washed 3 times with PBST (PBS, 0.1% Tween) and blocked with 3% BSA / 0.5% Tween / PBS for 2 h at room temperature. The dilution series after incubation for 0 h or 24 h in the presence of serum was incubated on the ELISA plate for 1 h at room temperature. After washing with PBST, the wells were incubated with biotinylated anti-ubiquitin antibody (1:300) for 1 h at room temperature. The binding was visualized using streptavidin-HRP (1:5000). PD-L1 binding proteins are stable in rat and mouse serum. After 24 h serum incubation, they did not show K D significant changes (Table 3).

[0151] Table 3. Stability of bound proteins in serum.

[0152]

[0153] Appendix - Sequence

[0154] SEQ ID NO: 1 (224039-2)

[0155]

[0156] SEQ ID NO: 2 (224058-2)

[0157]

[0158] SEQ ID NO: 3 (224180-2)

[0159]

[0160] SEQ ID NO: 4(224039-1 / 224058-1 / 224180-1 / 224121-1 / 211828-1)

[0161]

[0162] SEQ ID NO: 5 (224039)

[0163]

[0164] SEQ ID NO:6(224058)

[0165]

[0166] SEQ ID NO:7(224180)

[0167]

[0168] SEQ ID NO:8(224121)

[0169]

[0170] SEQ ID NO:9(211828)

[0171]

[0172] SEQ ID NO: 10 (ubiquitin)

[0173]

[0174] SEQ ID NO: 11 (extracellular domain of hPD-L1)

[0175]

[0176] SEQ ID NO:12(224121-2)

[0177]

[0178] SEQ ID NO: 13 (211828-2)

[0179]

Claims

1. A dimeric PD-L1 binding protein comprising an amino acid sequence of SEQ ID NO: 4 and an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 1, wherein: The protein: (i) having an aromatic amino acid selected from phenylalanine (F), tyrosine (Y) or tryptophan (W) at the position corresponding to position 6 of SEQ ID NO: 1, and having an acidic amino acid selected from aspartic acid (D) or glutamic acid (E) at the position corresponding to position 8 of SEQ ID NO: 1; and (ii) exhibits a binding affinity of less than 15 nM for human programmed death ligand 1 (PD-L1) as determined by surface plasmon resonance.

2. The dimeric PD-L1 binding protein according to claim 1, wherein The protein has phenylalanine (F) at the position corresponding to position 6 of SEQ ID NO:1, and has aspartic acid (D) at the position corresponding to position 8 of SEQ ID NO:

1.

3. The dimeric PD-L1 binding protein according to claim 1 or 2, wherein The protein was stable in serum after incubation at 37°C for 24 h.

4. The dimeric PD-L1 binding protein according to any one of claims 1 to 3, further comprising one or more coupling sites for coupling of a chemical moiety, wherein The chemical moiety is preferably selected from any one of a chelator, a drug, a toxin, a dye and a small molecule. 5 . The dimeric PD-L1 binding protein according to claim 1 , further comprising at least one diagnostically active portion or at least one therapeutically active portion.

6. The dimeric PD-L1 binding protein according to any one of claims 1 to 7, further comprising at least one pharmacokinetics-regulating portion, wherein The at least one pharmacokinetic-modulating moiety is preferably selected from any one of the following: serum albumin; albumin binding protein; immunoglobulin binding protein; immunoglobulin or immunoglobulin fragment; polysaccharide; unstructured amino acid sequence comprising the amino acids alanine, glycine, serine, proline; polyethylene glycol; sialic acid; transferrin; and transferrin receptor binding protein; or any combination thereof.

7. The dimeric PD-L1 binding protein according to any one of claims 1 to 6, for use in the diagnosis or treatment of PD-L1 positive tumors, or for use in the diagnosis or treatment of infectious diseases such as chronic viral infections.

8. A composition comprising the dimeric PD-L1 binding protein according to any one of claims 1 to 7.

9. The composition of claim 8 for use in medicine.

10. The composition for use in medical treatment according to claim 9, which is for use in the diagnosis or treatment of PD-L1 positive tumors, or for use in the diagnosis or treatment of infectious diseases such as chronic viral infections.

11. A method for producing a dimeric PD-L1 binding protein according to any one of claims 1 to 7, comprising the following steps: a) culturing the host cell under conditions suitable for obtaining the dimeric PD-L1 binding protein and b) isolating the produced dimeric PD-L1 binding protein.

12. A method for detecting PD-L1 in a sample, the method comprising contacting the sample with the dimeric PD-L1 binding protein according to any one of claims 1 to 7, and detecting the binding of PD-L1 in the sample to the dimeric PD-L1 binding protein according to any one of claims 1 to 7.

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