A novel protein with high binding affinity to programmed death-ligand 1 (PD-L1)

Abstract

The present invention relates to novel proteins specific for programmed death-ligand 1 (PD-L1). The PD-L1-specific proteins of the invention bind to human and mouse PD-L1 with high affinity. The invention further relates to PD-L1-specific proteins which further comprise a diagnostic or therapeutic active component. Further aspects of the invention include the use of these PD-L1-specific binding proteins in medicine, for example in the diagnosis and treatment of PD-L1-associated cancers.

Description

[Technical Field]

[0001] The present invention relates to novel proteins specific for programmed death-ligand 1 (PD-L1). The PD-L1-specific proteins of the invention bind to human and mouse PD-L1 with high affinity. The invention further relates to PD-L1-specific proteins which further comprise a diagnostic or therapeutic active moiety. Further aspects of the invention include the use of these PD-L1-specific binding proteins in medicine, for example in the diagnosis and treatment of PD-L1-associated cancers. [Background technology]

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

[0003] Both PD-1 and PD-L1 belong to a family of immune checkpoint proteins that act as co-inhibitors. They can halt or limit the development of T cell responses, for example, by suppressing T cell activation and proliferation and inducing apoptosis of activated T cells. PD-1 / PD-L1 interaction ensures that the immune system is activated only at the appropriate time to minimize the potential for chronic autoimmune inflammation.

[0004] PD-L1 expressed on tumor cells binds to the PD-1 receptor on activated T cells, thereby inhibiting cytotoxic T cells and reducing cytokine production and T-cell proliferation. Upregulation of PD-L1 expression allows tumor cells to evade detection and destruction by the immune system. In cancer, PD-L1 confers resistance to T-cell-mediated cytotoxicity. By inhibiting the immune checkpoint receptor PD-1 or its ligand PD-L1, the immune system can overcome the cancer's ability to resist the immune response and stimulate the body's own mechanisms to effectively maintain defense against cancer.

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

[0006] Diagnosis and treatment of PD-L1-associated cancers are often inadequately addressed by existing options, and as a result, many patients do not fully benefit from current strategies.

[0007] Needless to say, there is a strong need for new strategies for the diagnosis and treatment of PD-L1-overexpressing tumors and other PD-L1-associated diseases and disorders, including (viral) infections.

[0008] One objective of the present invention is to provide proteins for specifically targeting PD-L1 to enable targeted diagnostic and therapeutic options, including the detection of PD-L1-positive tumors (e.g., by radiodiagnostic methods). Targeting this tumor-associated protein may benefit patients with an unmet need for new diagnostic and therapeutic pathways. Targeting PD-L1 suggests potentially non-toxic diagnostic and therapeutic approaches due to the low and restricted distribution of PD-L1 in normal tissues. Thus, binding proteins with specificity for PD-L1 may enable effective medical options for cancer treatment, ultimately improving patients' quality of life.

[0009] The present invention provides novel PD-L1-binding molecules for new and improved strategies for the diagnosis and treatment of PD-L1-positive tumors. Furthermore, the novel PD-L1-binding molecules of the present invention provide improved strategies in the diagnosis and treatment of infectious diseases, such as chronic viral infections.

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

[0011] The present invention provides the following items 1 to 15, but is not particularly limited thereto: 1. A protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, wherein (i) it 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 (ii) it exhibits a binding affinity for human programmed death-ligand 1 (PD-L1) of less than 100 nM. A preferred embodiment relates to a dimeric PD-L1-binding protein comprising an amino acid sequence set forth in SEQ ID NO:4 and an amino acid sequence which has at least 96% identity to the amino acid sequence of SEQ ID NO:1, wherein: (i) the dimeric PD-L1-binding 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 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) the dimeric PD-L1-binding protein exhibits a binding affinity for human programmed death-ligand 1 (PD-L1) of less than 15 nM, as measured by surface plasmon resonance.

[0012] 2. The protein according to item 1, which has a phenylalanine (F) at the position corresponding to position 6 of SEQ ID NO: 1 and an aspartic acid (D) at the position corresponding to position 8 of SEQ ID NO: 1.

[0013] 3. The protein according to item 1 or 2, which is stable in serum after incubation at 37°C for 24 hours.

[0014] 4. The protein of any one of items 1 to 3, which exhibits a binding affinity to human PD-L1 of less than 4.15 nM.

[0015] 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.

[0016] 6. The multimer according to item 5, which comprises 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, and is preferably a dimer.

[0017] 7. The protein according to any one of items 1 to 6, further comprising one or more binding sites for attachment of a chemical moiety, the chemical moiety preferably being selected from any one of a chelator, a drug, a toxin, a dye, and a small molecule.

[0018] 8. The protein according to any one of items 1 to 7, further comprising at least one diagnostically active moiety or at least one therapeutically active moiety.

[0019] 9. The protein according to any one of items 1 to 8, further comprising at least one pharmacokinetic-modulating moiety, wherein the at least one pharmacokinetic-modulating moiety is preferably selected from any one of 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.

[0020] 10. The protein according to any one of items 1 to 9 for use in the diagnosis or treatment of a PD-L1 positive tumor, or for use in the diagnosis or treatment of an infectious disease, such as a chronic viral infection.

[0021] 11. A composition comprising a protein according to any one of 1 to 10.

[0022] 12. The composition according to item 11 for use in medicine.

[0023] 13. A composition for use in a medicine according to item 12, for use in the diagnosis or treatment of a PD-L1 positive tumor, or for use in the diagnosis or treatment of an infectious disease, such as a chronic viral infection.

[0024] 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 the protein, and b) isolating the produced protein.

[0025] 15. A method for detecting PD-L1 in a sample, the method comprising contacting the sample with a protein according to any one of items 1 to 10, and detecting binding between PD-L1 in the sample and the protein according to any one of items 1 to 10.

[0026] This summary does not necessarily describe all features of the invention, and other embodiments will become apparent upon consideration of the following detailed description.

[0027] The drawings show: [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 shows the binding affinity of dimeric PD-L1 binding proteins after long-term incubation in human serum. After 24 hours of incubation in serum, the binding affinity (KD value) to PD-L1 expressed on cells showed only slight changes. The binding affinity of 224058 (comprising SEQ ID NO: 2) to PD-L1 after incubation in human serum is shown in Figure 1A. The binding affinity of 224180 (comprising SEQ ID NO: 3) to PD-L1 after incubation in human serum is shown in Figure 1B. The results confirmed the stability of the PD-L1 binding proteins. Even after long-term incubation in human serum, the PD-L1 binding proteins bind with high affinity to PD-L1 expressed on cells. DETAILED DESCRIPTION OF THE INVENTION

[0029] The inventors have developed a solution that satisfies a strong and continuing need in the art to expand medical options for the diagnosis and treatment of cancer and infectious diseases by providing novel 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. Furthermore, they exhibit a high level of stability both in serum and at elevated temperatures. The PD-L1-binding proteins of the present invention (including their multimeric forms disclosed herein) can inhibit the interaction between PD-L1 and PD-1. This inhibition is believed to prevent PD-1 inhibitory signaling. The PD-L1-binding proteins described herein thereby provide a molecular format with favorable physicochemical properties and high-level expression in bacteria, allowing for facile manufacturing methods. The novel proteins described herein may expand previously unmet medical strategies for the diagnosis and treatment of PD-L1-associated cancer and infectious diseases. In particular, the proteins described herein may be used for imaging purposes, such as for the presence of tumor cells that express PD-L1, and for the treatment of tumors that express PD-L1 by radiotherapy or as an immuno-oncology treatment option.

[0030] Before describing the present invention in further detail below, it should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular aspects and embodiments only, and is not intended to limit the scope of the present invention as reflected in the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. This includes those skilled in the art of protein engineering and purification, as well as those skilled in the art of technical applications and the development of new target-specific binding molecules for use in therapy and diagnosis.

[0031] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W., Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0032] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" 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 terms "comprise(s)" or "comprising" may include the limitation "consists of" or "consisting of," regardless of why and to what extent such a limitation is necessary.

[0033] Several documents (e.g., patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank accession number sequence submissions, etc.) may be cited throughout this specification. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein may be characterized as "incorporated by reference." In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching set forth herein, the body of this specification shall control.

[0034] All sequences referred to herein are disclosed in the accompanying sequence listing, the entire contents and disclosure of which are incorporated herein by reference.

[0035] General definitions of important terms used in this application As used herein, the term "PD-L1" refers to Uniprot Accession No. Q9NZQ7 (programmed cell death ligand 1 or programmed death ligand 1). The term "PD-L1" includes all polypeptides that exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97% or more, or 100% sequence identity to PD-L1 in Uniprot Accession No. Q9NZQ7 (human). Human PD-L1 is 73% identical to mouse PD-L1 (Accession No. Q9EP73) and 74% identical to rat PD-L1. The term "PD-L1" includes the extracellular domain of PD-L1 (residues 19-238). Human PD-L1 has three domains: the extracellular domain (aa 19-238), the transmembrane domain (aa 239-259), and the cytoplasmic domain (aa 260-290). The extracellular domain of human PD-L1 is shown in SEQ ID NO:11.

[0036] The term "PD-L1 binding protein" refers to proteins of the invention (including multimeric forms thereof as disclosed herein) that have high affinity for PD-L1 (programmed cell death ligand 1 or programmed death ligand 1). Thus, the proteins of the disclosure are PD-L1 binding proteins. As described elsewhere herein, the proteins of the disclosure exhibit specific binding affinity for human PD-L1, with binding affinity in the nanomolar range.

[0037] The terms "protein" and "polypeptide" refer to any chain of two or more amino acids joined 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 within 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 products of post-translational modifications of polypeptides, which are well known in the art.

[0038] The term "modification" or "amino acid modification" refers to the substitution, deletion, or insertion of a reference amino acid at a particular position in a parent polypeptide sequence with another amino acid. Given the known genetic code and recombinant and synthetic DNA techniques, skilled scientists can readily construct DNA encoding amino acid variants. The term "amino acid substitution" is understood as the replacement of one amino acid with another.

[0039] The term "ubiquitin" refers to the amino acid sequence shown in SEQ ID NO:10.

[0040] The terms "binding affinity" and "binding activity" may be 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 ), which is used to assess and rank the strength of biomolecular interactions.

[0041] The term "fusion protein" refers to a protein comprising at least a first protein joined genetically to at least a second protein. Fusion proteins are created by joining two or more genes that originally encoded separate proteins. Fusion proteins may further comprise additional domains that are not involved in target binding, such as, but not limited to, a multimerization moiety, a polypeptide tag, a polypeptide linker, or a moiety that binds to a target other than PD-L1.

[0042] 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" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. To determine sequence identity, the sequence of the query protein is aligned to the sequence of the reference protein or polypeptide. Methods for sequence alignment are well known in the art. For example, to determine the degree of identity of the amino acid sequence of any polypeptide to another amino acid sequence, the SIM Local similarity program, known in the art, is preferably used. For multiple alignment analysis, Clustal Omega is preferably used, as known to those skilled in the art.

[0043] Detailed Description of Embodiments of the Invention Structural features of the programmed death-ligand 1 (PD-L1) binding protein 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 proteins have (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.

[0044] 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.

[0045] The PD-L1 binding proteins described herein comprise an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO:1, and in a preferred embodiment, the protein has (a) a phenylalanine (F) at the position corresponding to position 6 of SEQ ID NO:1, and (b) an aspartic acid (D) at the position corresponding to position 8 of SEQ ID NO:1, and has a binding affinity for human PD-L1 of at least 100 nM (or less).

[0046] Specific examples of proteins described herein that include amino acid sequences that have 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.

[0047] As described herein, modifications of ubiquitin that result in high affinity binding to PD-L1 are located in the region comprising amino acids 6 and 8 of SEQ ID NO: 10, and may also include modifications in the region comprising amino acid 62 of ubiquitin (SEQ ID NO: 10).

[0048] In general terms, the present invention provides a protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, wherein (i) it 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 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) it exhibits a binding affinity to PD-L1 of less than 100 nM.

[0049] Aspects and embodiments relating to an acidic amino acid at a position corresponding to position 8 of SEQ ID NO: 1: More specifically, the present invention provides a protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, wherein (i) 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 (ii) the protein exhibits a binding affinity to PD-L1 of less than 100 nM. In various embodiments, the acidic amino acid is glutamic acid (E). In various preferred embodiments, the acidic amino acid is aspartic acid (D).

[0050] 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).

[0051] 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).

[0052] 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).

[0053] In various other embodiments, a 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 a position corresponding to position 6 of SEQ ID NO:1, 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 an amino acid selected from tryptophan (W), glutamine (Q), or alanine (A) at a 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).

[0054] 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).

[0055] 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).

[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 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).

[0057] In aspects and embodiments described herein relating to SEQ ID NO:1 (including particularly aspects and embodiments described herein relating to the acidic amino acid at the position corresponding to position 8 of SEQ ID NO:1), the protein may comprise an amino acid sequence having at least 92% identity to the amino acid sequence of SEQ ID NO:1. Preferably, the protein may comprise an amino acid sequence having at least 93% identity to the amino acid sequence of SEQ ID NO:1. More preferably, the protein may comprise an amino acid sequence having at least 94% identity to the amino acid sequence of SEQ ID NO:1. Even more preferably, the protein may comprise an amino acid sequence having at least 96% identity to 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 to the amino acid sequence of SEQ ID NO:1.

[0058] 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, exhibit (only) a variant amino acid sequence 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 present 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, exhibit (only) a variant amino acid sequence based on amino acid substitutions compared to a reference sequence disclosed herein, particularly the sequence of SEQ ID NO: 1, as well as (only) a variant amino acid sequence based on (N- or C-) terminal deletion of any one of 1, 2, 3, 4, or 5 amino acid residues. Such a protein (or protein monomer) of the present invention may have a length of about 71, 72, 73, 74, or 75 amino acids or comprises about 71, 72, 73, 74, or 75 amino acids.

[0059] The aspects and embodiments described herein with respect to SEQ ID NO: 1 (including in particular the aspects and embodiments described herein with respect 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, including proteins comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 described herein.

[0060] Aspects and embodiments relating to the basic amino acid at the position corresponding to position 8 of SEQ ID NO: 1: Additionally, the present invention also provides a protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, wherein (i) 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 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) the protein has a binding affinity for PD-L1 of less than 100 nM. In various embodiments, the basic amino acid is histidine (H). In various preferred embodiments, the basic amino acid is lysine (K) or arginine (R).

[0061] Thus, 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).

[0062] In various other embodiments, a 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 a position corresponding to position 6 of SEQ ID NO: 1, 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 an amino acid selected from tryptophan (W), glutamine (Q), or alanine (A) at a position corresponding to position 62 of SEQ ID NO: 1. In various preferred embodiments, the basic amino acid is lysine (K) or arginine (R).

[0063] Thus, 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).

[0064] In all aspects and embodiments described herein relating to SEQ ID NO:1 (including in particular aspects and embodiments described herein relating to the basic amino acid at the position corresponding to position 8 of SEQ ID NO:1), the protein may comprise an amino acid sequence having at least 92% identity to the amino acid sequence of SEQ ID NO:1. Preferably, the protein may comprise an amino acid sequence having at least 93% identity to the amino acid sequence of SEQ ID NO:1. More preferably, the protein may comprise an amino acid sequence having at least 94% identity to the amino acid sequence of SEQ ID NO:1. Even more preferably, the protein may comprise an amino acid sequence having at least 96% identity to 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 to the amino acid sequence of SEQ ID NO:1.

[0065] The aspects and embodiments described herein with respect to SEQ ID NO: 1 (including in particular the aspects and embodiments described herein with respect to the basic amino acid at the position corresponding to position 8 of SEQ ID NO: 1) also apply to the multimeric forms disclosed herein, including proteins comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 described herein.

[0066] Proteins of the invention (including multimeric forms thereof disclosed herein) are believed to bind to the same or overlapping epitopes of PD-L1. In various embodiments, proteins of the invention (including multimeric forms thereof disclosed herein) bind, or are believed to bind, to the extracellular domain of PD-L1. In various embodiments, proteins of the 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, PD-L1-binding proteins of the 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 comprising 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). PD-L1 binding proteins (including multimeric forms thereof disclosed herein) may bind to an epitope in the N-terminal region of the extracellular domain of PD-L1 as described above, which epitope comprises or consists of 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).

[0067] Functional Characteristics. The PD-L1 binding proteins described herein (including multimeric forms thereof disclosed herein) have a binding affinity (KD) for PD-L1 of (at least) less than 100 nM. In various embodiments, the proteins bind to PD-L1 with a measurable binding affinity of any of 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 less than 1 nM.

[0068] As described elsewhere herein, K D The lower the value, the greater the binding affinity of the biomolecule for its binding partner. DThe higher the value, the weaker the binding of the binding partners to each other (see Examples). Thus, as described herein, the terms "less than 100 nM" and "at least 100 nM" may be used interchangeably herein, as they both refer to a high binding affinity of the proteins of the invention to PD-L1. More specifically, K D In the context of high binding affinity, the terms "less than 100 nM" and "at least 100 nM" respectively indicate K D " refers to a range of values ​​indicating ≦100 nM (= 100 nM inclusive). The same considerations regarding the interchangeable use of the terms "less than" and "at least" apply with respect to all preferred binding affinities to PD-L1 disclosed throughout this specification (e.g., "less than 15 nM" and "at least 15 nM"). Here again, K D The terms "less than 15 nM" and "at least 15 nM" respectively refer to K D It refers to a range of values ​​indicating a value ≦15 nM (= 15 nM inclusive).

[0069] In preferred embodiments, the PD-L1 binding proteins (including multimeric forms thereof disclosed herein) bind to PD-L1 with a measurable binding affinity of less than 15 nM. In various embodiments of the invention, a binding affinity of less than 100 nM to PD-L1 as described above and throughout this specification means a binding affinity of less than 100 nM (at least 100 nM) to human PD-L1 (hPD-L1). Preferred binding affinities (such as less than 50 nM) are described above and elsewhere herein and apply to embodiments relating to binding of proteins and multimers of the invention to hPD-L1.

[0070] Suitable methods are known to those skilled in the art or described in the literature.Methods for determining binding affinity are known per se and can be selected, for example, from the following methods known in the art: enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), kinetic exclusion (KinExA assay), biolayer interferometry (BLI), flow cytometry, fluorescence spectroscopy techniques, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL).Some of the methods are described in the examples below.Typically, the dissociation constant K D is determined at 20°C, 25°C, or 30°C. Unless otherwise specified, the K D Values ​​are determined by SPR at 25°C.

[0071] 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).

[0072] 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 to PD-L1 of less than 100 nM. 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 to PD-L1 of less than 50 nM. 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 to PD-L1 of less than 20 nM. Even 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 to PD-L1 of less than 15 nM or less than 10 nM. Even more preferably, in the above aspects and embodiments relating to the acidic / basic amino acid at the position corresponding to position 8 of SEQ ID NO: 1, 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 to PD-L1 of less than 5 nM. In particularly preferred embodiments, the protein comprises 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 to PD-L1 of less than 2 nM or less than 1 nM.

[0073] As further disclosed herein, in the above aspects and embodiments, the protein may comprise an amino acid sequence having at least 96% identity, at least 97% identity, or at least 98% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity to PD-L1 of less than 100 nM. In the above aspects and embodiments, the protein may comprise an amino acid sequence having at least 96% identity, 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 to PD-L1 of less than 50 nM. More preferably, the protein may comprise an amino acid sequence having at least 96% identity, at least 97% identity, or at least 98% identity to the amino acid sequence of SEQ ID NO: 1, and exhibits a binding affinity to PD-L1 of less than 20 nM. 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 to PD-L1 of less than 15 nM or less than 10 nM. Even 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 to PD-L1 of less than 5 nM. 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 to PD-L1 of less than 2 nM or less than 1 nM.

[0074] As further disclosed herein, in the above aspects and embodiments relating to SEQ ID NO:1 (see "Structural Features of PD-L1-Binding Proteins"), the protein (including multimeric forms thereof as disclosed herein) preferably exhibits a binding affinity to PD-L1 of less than 50 nM. More preferably, the protein (including multimeric forms thereof as disclosed herein) exhibits a binding affinity to PD-L1 of less than 20 nM. Even more preferably, the protein (including multimeric forms thereof as disclosed herein) exhibits a binding affinity to PD-L1 of less than 15 nM or less than 10 nM. Even more preferably, the protein (including multimeric forms thereof) disclosed herein exhibits a binding affinity to PD-L1 of less than 5 nM. Particularly preferably, the protein (including multimeric forms thereof as disclosed herein) exhibits a binding affinity to PD-L1 of less than 2 nM or less than 1 nM.

[0075] The aspects and embodiments described herein relating to the functional characteristics of the PD-L1 binding proteins of the invention also apply to the multimeric forms disclosed herein, including proteins comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1 described herein.

[0076] The binding of the proteins described herein to cellular PD-L1 can be determined by standard methods, including immunofluorescence microscopy and flow cytometry analysis. In some embodiments, the specific PD-L1 binding of the proteins of the invention is determined by cellular PD-L1 binding analysis. In aspects and embodiments related to SEQ ID NO: 1 above (see "Structural Features of PD-L1-Binding Proteins"), the protein preferably, in various embodiments, exhibits a binding affinity for PD-L1 on PD-L1-expressing cells of less than 5 nM, preferably less than 3 nM (see Examples). More specifically, in various embodiments, the protein preferably exhibits a binding affinity for PD-L1 on PD-L1-expressing cells of less than 5 nM, preferably less than 3 nM, as determined by flow cytometry analysis (see Examples).

[0077] In some embodiments, the proteins described herein inhibit the binding interaction between PD-1 and PD-L1. In preferred embodiments, the proteins described herein (including multimeric forms thereof as disclosed herein) specifically bind to (human) PD-L1 with high affinity (i.e., less than 100 nM, preferably less than 50 nM, 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 preventing (or providing prevention of) PD-1 inhibitory signaling.

[0078] In some embodiments, the proteins described herein are particularly stable under different conditions. In preferred embodiments, the proteins are stable in the presence of (human, mouse, or rat) serum at 37°C for at least 24 hours. In some embodiments, the proteins described herein are stable in the presence of human serum at 37°C for at least 24 hours, as described in more detail in Example 6 and Figure 1. In some embodiments, the proteins described herein are stable in the presence of mouse serum at 37°C for at least 24 hours. For example, the stability of a PD-L1-binding protein can be determined by measuring the binding affinity (KD) after extended incubation in serum at the elevated temperature of 37°C, using standard methods as described herein above and in the Examples. In aspects and embodiments relating to SEQ ID NO: 1 above (see the section entitled "Structural Features of PD-L1-Binding Proteins"), the protein preferably exhibits a binding affinity for PD-L1 on PD-L1-expressing cells of less than 5 nM, preferably less than 3 nM, even after 24 hours of incubation in (human) serum at 37°C. More specifically, the proteins of various embodiments preferably exhibit a binding affinity for PD-L1 of less than 5 nM, preferably less than 3 nM, on PD-L1-expressing cells, as measured by flow cytometry analysis, even after incubation in (human) serum at 37°C for 24 hours.

[0079] In some embodiments, the PD-L1 binding proteins described herein are stable at elevated temperatures, preferably at elevated temperatures of at least 60°C. Stability analysis includes, for example, spectroscopic or fluorescence-based methods related to chemical or physical unfolding known to those skilled in the art. For example, the stability of a molecule can be assessed using standard methods to measure the thermal melting temperature (T), which is the temperature (in degrees Celsius (°C)) at which half of the molecule unfolds. m ) can be determined by measuring the T m The higher the σ, the more stable the molecule. Temperature stability was determined by differential scanning fluorimetry (DSF), as described in more detail in Example 3 and Table 1.

[0080] The above-described further aspects and embodiments relating to the functional characteristics of the PD-L1-binding proteins of the invention also apply to the multimeric forms disclosed herein, including proteins comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1 as described throughout this specification.

[0081] Multimers. The present invention provides multimers comprising the proteins of the invention as described above and throughout this specification, i.e., in general terms, the present invention provides multimers comprising 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 a charged amino acid selected from 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 for programmed (cell) death-ligand 1 (PD-L1) of less than 100 nM.

[0082] More specifically, the present invention provides multimers comprising proteins described elsewhere herein with respect to "Aspects and embodiments relating to an acidic amino acid at a position corresponding to position 8 of SEQ ID NO:1," and also provides multimers comprising proteins described elsewhere herein with respect to "Aspects and embodiments relating to a basic amino acid at a position corresponding to position 8 of SEQ ID NO:1."

[0083] In the present invention, a multimer may be any of a dimer, trimer, tetramer, pentamer, or hexamer. In the present invention, a multimer is preferably a dimer or trimer, more preferably a dimer. Thus, a multimer of the present invention comprises at least two domains or at least two monomers (=dimer). Thus, the proteins of the present invention described above throughout this specification can be considered as multimeric protein domains or protein monomers provided by the present invention. Such protein domains or protein monomers have a length of about 76 amino acids or consist of about 76 amino acids.

[0084] In some embodiments, a multimer may comprise one, two, three, four, or more proteins described herein. In one embodiment, the proteins comprise two, three, four, or more proteins linked to one another, i.e., the proteins may be dimers, trimers, tetramers, etc., as described above, wherein at least one protein of the multimer comprises an amino acid sequence having at least 90% identity 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 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 multimeric protein exhibits a binding affinity to human PD-L1 of less than 100 nM.

[0085] In a preferred embodiment, the PD-L1-binding protein comprises two proteins bound to each other, wherein one protein of the multimer comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and 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 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 proteins linked to each other, one protein having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1, which 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 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 at least 90% identical to SEQ ID NO: 4, and the dimeric PD-L1-binding protein has a binding affinity for human PD-L1 of less than 100 nM.

[0086] In various preferred embodiments of the present invention, the multimers include the proteins of the present invention described above and throughout the specification, as well as proteins comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4. In such cases, the multimers of the present invention comprise at least two protein domains, which are located at the N-terminus and C-terminus, respectively.

[0087] In a preferred embodiment, the PD-L1-binding protein comprises two proteins bound to each other, wherein the proteins comprise an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and wherein the proteins have 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 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, located at their N- 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 two proteins bound to each other, wherein the protein with at least 90% amino acid sequence 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 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 is located at the N-terminus or C-terminus, preferably the C-terminus, and the second protein with an amino acid sequence at least 90% identical to SEQ ID NO: 4 is located at the N-terminus or C-terminus, preferably the N-terminus, and the dimeric PD-L1-binding protein has a binding affinity for human PD-L1 of less than 100 nM.

[0088] In various embodiments, the multimers comprise proteins of the invention as described above and throughout the specification, as well as proteins comprising an amino acid sequence at least 92% identical to the amino acid sequence of SEQ ID NO:4. In various preferred embodiments, the multimers comprise proteins of the invention as described above and throughout the specification, as well as proteins comprising an amino acid sequence at least 93% identical to the amino acid sequence of SEQ ID NO:4. More preferably, the multimers comprise proteins of the invention as described above and throughout the specification, as well as proteins comprising an amino acid sequence at least 94% identical to the amino acid sequence of SEQ ID NO:4. Even more preferably, the multimers comprise proteins of the invention as described above and throughout the specification, as well as proteins comprising an amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:4. Even more preferably, the multimers comprise proteins of the invention as described above and throughout the specification, as well as proteins comprising an amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO:4. In particularly preferred embodiments, the multimers comprise proteins of the invention as described above and throughout the specification, as well as proteins comprising an amino acid sequence at least 98% or 100% identical to the amino acid sequence of SEQ ID NO:4.

[0089] Preferably, the proteins of the present invention described above and throughout this specification, and multimers comprising proteins comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 4, are dimers or trimers. More preferably, the proteins of the present invention described above and throughout this specification, and multimers comprising proteins comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 4, are dimers.

[0090] A preferred embodiment relates to a dimeric PD-L1 binding protein comprising the amino acid sequence of SEQ ID NO:4 and an amino acid sequence which has at least 96% identity to the amino acid sequence of SEQ ID NO:1, wherein: (i) it 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 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) it exhibits a binding affinity for human programmed death-ligand 1 (PD-L1) of less than 15 nM, as measured by surface plasmon resonance.

[0091] Preferably, in the proteins of the present invention described above and throughout this specification, and in the multimers of the present invention comprising a protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4, the proteins of the present invention (domains / monomers) described above and throughout this specification are located on the C-terminal side, and the proteins (domains / monomers) comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4 are located on the N-terminal side.

[0092] In one embodiment, the multimer is a dimer of two proteins, where 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 of two proteins, where 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 of two proteins, where 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).

[0093] 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 preferred 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, with the exception of the aromatic amino acid at a 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, remaining unchanged.

[0094] In some embodiments, two or more proteins are directly linked. In some embodiments, two or more proteins described herein are linked by a peptide linker. In various embodiments, two or more proteins described herein are linked via a peptide linker of up to 30 amino acids. In other embodiments, two or more proteins described herein are linked via a peptide linker of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0095] The multimer may be considered a chimeric polypeptide comprising a first portion and a second portion, wherein the first portion comprises the amino acid sequence of a protein of the invention as described above and throughout this specification, and the second portion comprises the amino acid sequence of a protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4 as described above.

[0096] The functional characteristics of the proteins of the invention described above and throughout this specification apply fully to any of the multimeric, fusion proteins, and chimeric polypeptides provided by the present invention.

[0097] Binding Sites. In some embodiments, the PD-L1-binding proteins described herein further comprise one or more binding sites for attachment of a chemical moiety. The binding sites can react with other chemical groups to attach the PD-L1-binding protein to the chemical moiety. The defined number and defined locations of the binding sites allow for site-specific attachment of chemical moieties to the PD-L1-binding proteins described herein. Thus, multiple chemical moieties can be attached to the PD-L1-binding protein as desired. The number of binding sites can be adjusted by one of skill in the art to the optimal number for a particular application, and the amount of chemical moieties can be adjusted accordingly. In selected embodiments, the binding sites may be selected from a group of one or more amino acids that can be labeled with a specific chemistry, such as one or more cysteine ​​residues, one or more lysine residues, one or more tyrosine residues, one or more tryptophans, or one or more histidine residues. The PD-L1-binding protein may contain 1 to 20 binding sites, preferably 1 to 6 binding sites, preferably 2 binding sites, or preferably 1 binding site.

[0098] Binding Domain. One embodiment provides a PD-L1-binding protein comprising at least one binding domain of 1-80 amino acids that comprises one or more binding sites. In some embodiments, the 1-80 amino acid binding domain may comprise alanine, proline, or serine, and a cysteine ​​as a binding site. In other embodiments, the 5-80 amino acid binding domain may consist of alanine, proline, serine, and a cysteine ​​as a binding site. In one embodiment, the binding domain consists of 20-60% alanine, 20-40% proline, 10-60% serine, and one or more cysteines as binding sites at the C-terminus or N-terminus of a PD-L1-binding protein described herein. In some embodiments, the alanine, proline, and serine amino acids are randomly distributed throughout the amino acid sequence of the binding domain and are flanked by up to 2, 3, 4, or 5 identical amino acid residues, preferably up to 3 amino acids. The composition of the 1-20 binding domains may be different or identical.

[0099] Chemical moieties. In some embodiments, the chemical moieties are selected from any of a chelator, a drug, a toxin, a dye, and a small molecule. In some embodiments, at least one of the chemical moieties is a chelator designed as a complexing agent for attaching one or more additional moieties to targeting compounds for the PD-L1 binding proteins disclosed herein. One embodiment relates to a PD-L1 binding protein in which the chelator is a complexing agent for attaching one or more radioisotopes or other detectable labels.

[0100] Diagnostic moiety. In some embodiments, the protein comprises an amino acid sequence having at least 90% identity 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 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 further comprises at least one diagnostically active moiety. In various embodiments, the PD-L1-binding protein further comprises a diagnostic moiety. In other embodiments, the PD-L1-binding protein further comprises two or more diagnostic moieties. 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, digoxigenin, haptens, or any combination of the above. In some embodiments, PD-L1-binding proteins comprising at least one diagnostic moiety can be used, for example, as imaging agents to assess the presence of tumor cells or metastases, tumor distribution, and / or tumor recurrence. Methods for detecting or monitoring cancer cells include imaging methods. Such methods include imaging PD-L1-associated cancer cells, for example, by radioimaging, photoluminescence, or fluorescence.

[0101] Therapeutic moieties. In some embodiments, the PD-L1-binding protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, wherein the PD-L1-binding 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 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 further comprises at least one therapeutically active moiety. In other embodiments, the PD-L1-binding protein further comprises two or more therapeutically active moieties. In some embodiments, such therapeutically active moieties may be selected from a monoclonal antibody or fragment thereof, an extracellular domain of a receptor or fragment thereof, a radionuclide, a cytotoxic compound, a cytokine, a chemokine, an enzyme, or derivatives thereof, or any combination of the above. In some embodiments, the PD-L1-binding protein comprising a therapeutically active moiety may be used for targeted delivery of any of the above-listed moieties to PD-L1-expressing tumor cells, where they accumulate, thereby resulting in low levels of toxicity to normal cells.

[0102] Radionuclides. Suitable radionuclides for imaging (e.g., in vitro) applications or radiotherapy include, but are not limited to, gamma-emitting isotopes, positron emitters, beta emitters, and alpha emitters. In some embodiments, suitable binding partners include chelators, 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 imaging agents.

[0103] Pharmacokinetic-modulating moiety. In some embodiments, the protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence 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 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 further comprises at least one pharmacokinetic-modulating moiety. In some embodiments, the pharmacokinetic-modulating moiety is selected from polyethylene glycol, human serum albumin, albumin-binding peptides, immunoglobulin-binding peptides or immunoglobulins or immunoglobulin fragments, sialic acid, or an unstructured amino acid sequence that increases the hydrodynamic radius, such as transferrin, transferrin receptor-binding protein, polysaccharides (e.g., hydroxyethyl starch), or multimers containing the amino acids alanine, glycine, serine, or proline. In various embodiments, the moiety increases the half-life of the PD-L1-binding protein by at least 1.5-fold. Several techniques for generating PD-L1-binding proteins with extended half-lives are known in the art, such as direct fusion of a pharmacokinetic-modifying moiety to the above-mentioned PD-L1-binding proteins, or chemical conjugation methods. The pharmacokinetic-modifying moiety can be attached to one or several sites on the PD-L1-binding protein, for example, via a peptide linker sequence or via one of the above-mentioned attachment sites.

[0104] Conjugation of protein or non-protein moieties to PD-L1 binding proteins can be achieved by applying chemical methods well known in the art. In some embodiments, conjugation chemistries specific to derivatization of cysteine ​​or lysine residues may be applicable. Chemical conjugation can be achieved by chemistries well known to those skilled in the art, including, but not limited to, substitution, addition, cycloaddition, or oxidation chemistry (e.g., disulfide formation).

[0105] Purification / detection molecules. In some embodiments, PD-L1-binding proteins can be extended with additional amino acids at either the N-terminus or C-terminus, or both. Additional sequences may include, for example, sequences introduced for purification or detection. In one embodiment, the additional amino acid sequence comprises one or more peptide sequences that confer affinity to a particular chromatography column material. Typical examples of such sequences include, but are not limited to, a Strep tag, an oligohistidine tag, glutathione S-transferase, maltose-binding protein, an intein, an intein fragment, or the albumin-binding domain of protein G.

[0106] Compositions. Various embodiments relate to compositions comprising an amino acid sequence having at least 90% identity 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 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 protein has a binding affinity for human PD-L1 of less than 100 nM. Various embodiments relate to compositions comprising a PD-L1-binding protein as defined above for use in medicine. Various preferred embodiments relate to compositions comprising a PD-L1-binding protein as defined above for use in the diagnosis or treatment of various PD-L1-associated (PD-L1-positive) tumors or infectious diseases, such as chronic viral infections. Compositions comprising the above PD-L1-binding proteins may be used in clinical applications for both diagnostic and therapeutic purposes. In particular, compositions comprising the above-described PD-L1 binding proteins may be used in clinical applications to image, monitor, and eliminate or inactivate pathogenic cells that express PD-L1.

[0107] Various embodiments relate to diagnostic compositions for diagnosing PD-L1-associated cancers, comprising a PD-L1 binding protein as defined herein and a diagnostically acceptable carrier and / or diluent, including, but not limited to, stabilizers, surfactants, salts, buffers, coloring agents, etc. The composition may be in the form of a liquid formulation, a lyophilizate, a granular formulation, an emulsion, or a liposomal formulation.

[0108] Diagnostic compositions comprising the PD-L1 binding proteins described herein can be used to diagnose PD-L1 associated cancers as described above.

[0109] Various embodiments relate to pharmaceutical (e.g., therapeutic) compositions for the treatment of diseases, 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 comprise further auxiliary substances and excipients known per se. These include, but are not limited to, stabilizers, surfactants, salts, buffers, coloring agents, etc.

[0110] Pharmaceutical compositions comprising PD-L1 binding proteins as defined herein may be used in the treatment of diseases as described above.

[0111] The compositions comprise an effective amount 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 further factors known per se. Depending on the galenic preparation, these compositions can be administered parenterally by injection or infusion, systemically, intraperitoneally, intramuscularly, subcutaneously, transdermally, or by other conventionally used administration methods.

[0112] The composition may be in the form of a liquid formulation, a lyophilisate, a cream, a lotion for topical application, an aerosol form, a powder, a granular form, an emulsion or a liposomal formulation. The type of formulation 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 arts.

[0113] The various components of the composition may be packaged as a kit together with instructions for use.

[0114] Use in Medicine. Various embodiments relate to the PD-L1-binding proteins disclosed herein for use in medicine. In one embodiment, the PD-L1-binding proteins are used in medicine to diagnose or treat cancers associated with PD-L1 expression. Accordingly, disclosed herein are methods for diagnosing or treating PD-L1-associated cancers or infectious diseases. The PD-L1-binding proteins disclosed herein enable the selective diagnosis and treatment of PD-L1-associated cancer cells or cancer tissues, such as melanoma, NSCLC, head and neck cancer, bladder cancer, and breast cancer. PD-L1 is known to be upregulated in tumor cells, possibly resulting in uncontrolled tumor cell proliferation and the formation of metastases. The PD-L1-binding proteins disclosed herein further enable the selective diagnosis and treatment of PD-L1-associated infectious diseases, such as chronic viral infections (HIV, HBV, HCV). In one embodiment, the PD-L1-binding proteins are used to diagnose PD-L1-associated cancers or infectious diseases by applying in vitro methods.

[0115] One embodiment is a method of diagnosing (including monitoring) a subject with a PD-L1-associated cancer or an infectious disease, such as a chronic viral infection, where the diagnostic (monitoring) method comprises administering to the subject a PD-L1-binding protein described herein, optionally conjugated to a radioactive molecule. In various embodiments, the PD-L1-binding proteins disclosed herein may be used to diagnose a PD-L1-associated cancer or an infectious disease, such as a chronic viral infection, where the PD-L1-binding protein is optionally conjugated to a radioactive molecule. In some embodiments, the PD-L1-binding proteins are used in (in vitro) imaging methods using labels, such as radioactive or fluorescent labels, to visualize PD-L1 on specific tissues or cells, which can be used, for example, to assess the presence of PD-L1-associated tumor cells (PD-L1-positive tumor cells), the distribution of PD-L1-associated tumors, the recurrence of PD-L1-associated tumors (PD-L1-positive tumors), and / or to assess a patient's response to therapeutic treatment.

[0116] One embodiment is a method of treating a subject having a PD-L1-associated (positive) cancer (tumor) or an infectious disease, such as a chronic viral infection, comprising administering to the subject a PD-L1-specific binding protein described herein, optionally conjugated to a radioactive molecule and / or a cytotoxic agent, or as an immuno-oncology agent. In various embodiments, the PD-L1-binding proteins disclosed herein may be used to treat PD-L1-associated cancer or an infectious disease, such as a chronic viral infection, optionally conjugated to a cytotoxic agent and / or a radioactive molecule, or expressed on the surface of target-specific CarT cells. Some embodiments relate to the use of PD-L1-binding proteins labeled with a suitable radioisotope or cytotoxic compound, or to treat PD-L1-associated (positive) tumor cells, particularly to control or kill PD-L1-associated (positive) tumor cells, e.g., malignant cells. In one embodiment, a curative dose of radiation is delivered selectively to PD-L1-associated (positive) tumor cells, but not to normal cells.

[0117] As further described herein, in various embodiments, the PD-L1-associated cancer is characterized by cancer cells that express or overexpress PD-L1. In various embodiments, the PD-L1-associated cancer is a solid tumor that expresses or overexpresses PD-L1. Use as a cancer immunotherapy agent refers to the use of the PD-L1 binding proteins disclosed herein as therapeutic compositions for the treatment of disease, i.e., to promote an immune response against cancer. Certain cancer cells act to inhibit immune responses against them, such as by inhibiting T cell responses against them. The PD-L1 binding proteins disclosed herein as part of a pharmaceutical composition for use as a therapeutic agent can interfere with the inhibition of the immune response.

[0118] Production of PD-L1 Binding Proteins. The PD-L1 binding proteins described herein may be prepared by any of a number of conventional, well-known techniques, such as straightforward organic synthesis strategies, solid-phase assisted synthesis techniques, fragment ligation techniques, or by commercially available automated synthesizers. However, they may also be prepared by conventional recombinant techniques, either alone or in combination with conventional synthetic techniques. Additionally, they may also be prepared by cell-free in vitro transcription / translation.

[0119] Various embodiments relate to polynucleotides encoding the PD-L1 binding proteins disclosed herein. One embodiment further provides an expression vector comprising such a polynucleotide, and a host cell comprising such an isolated polynucleotide or expression vector.

[0120] Various embodiments relate to methods of producing a PD-L1 binding protein disclosed herein, comprising the steps of a) culturing a host cell under suitable conditions that allow for expression of the protein, and b) isolating the protein.

[0121] For example, one or more polynucleotides encoding a PD-L1-binding protein may be expressed in a suitable host, and the resulting PD-L1-binding protein may be isolated. Host cells include the nucleic acid molecules or vectors described above. Suitable host cells include prokaryotic and eukaryotic organisms. A vector refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that can be used to transfer protein-coding information into a host cell. Various cell culture systems, including, but not limited to, mammalian, yeast, plant, or insect, can also be used to express recombinant proteins. Suitable 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 can be carried out on any scale, starting from small shaker flasks to large fermenters, using techniques well known to those skilled in the art.

[0122] One embodiment relates to a method of producing a protein as detailed above, comprising the steps of: (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) exposing the host cell to culture conditions such that the PD-L1-binding protein is expressed, thereby producing a PD-L1-binding protein as defined herein; (f) optionally isolating the PD-L1-binding protein produced in (e); and (g) optionally conjugating the PD-L1-binding protein to a further functional moiety as defined herein.

[0123] Generally, isolation of purified PD-L1-binding proteins from the culture mixture can be achieved by applying conventional methods and techniques well known in the art, such as centrifugation, precipitation, aggregation, various forms of chromatography, filtration, dialysis, concentration, and combinations thereof. Chromatographic methods are well known in the art and include, but are not limited to, ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), hydrophobic interaction chromatography, or affinity chromatography.

[0124] To simplify purification, the PD-L1 binding protein can be fused to other peptide sequences that have a high affinity for the separation material. Preferably, fusions are selected that do not adversely affect the function of the PD-L1 binding protein or that can be separated after purification by introducing a specific protease cleavage site. Such methods are also known to those skilled in the art.

[0125] Methods for detecting PD-L1 in a sample. Some embodiments relate to detecting PD-L1 in a sample, the methods comprising contacting the sample with the PD-L1-binding protein and detecting binding of PD-L1 in the sample to a PD-L1-binding protein described above. In some embodiments, the proteins described herein are used in methods for determining the presence of PD-L1. Some embodiments relate to methods for analyzing the presence of PD-L1 in a sample, the methods comprising: (i) providing a sample containing PD-L1; (ii) providing a binding protein for PD-L1; (iii) contacting the sample containing PD-L1 with a binding protein for PD-L1 described herein under conditions allowing at least one protein to bind to PD-L1; (iv) isolating (eluting) the complex between PD-L1 and the binding protein for PD-L1; and (v) determining the amount of the binding protein for PD-L1 indicative of the amount of PD-L1 in the sample of (i). In some embodiments, the sample may be a liquid sample, such as a blood sample, a urine sample, or a liquid tumor sample. [Example]

[0126] The following examples are provided to further illustrate the present invention, but the present invention is not limited thereto, and the following examples merely demonstrate the feasibility of carrying out the present invention based on the above description.

[0127] Example 1. Expression and purification of PD-L1 binding proteins 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 and purified and analyzed as described below. All proteins were expressed and highly purified by affinity chromatography and gel filtration. After affinity chromatography, size-exclusion chromatography (SEC) was performed using an Äkta system and a Superdex™ 200 HiLoad 16 / 600 column (GE Healthcare). The column had a volume of 120 ml and was equilibrated with 2 CV. PBS was used as the running buffer, and the sample was applied at a flow rate of 1 ml / min. Fraction collection began when the signal intensity reached 10 mAU. After SDS-PAGE analysis, positive fractions were pooled and their protein concentrations were determined.

[0128] Purity was confirmed by SDS-PAGE, SE-HPLC, and RP-HPLC. Purity was at least 98% as determined by RP-HPLC. Protein concentration was determined by absorbance measurement at 280 nm using the specific molar extinction coefficient. RP chromatography (RP HPLC) was performed using an Ultimate3000 HPLC system (Thermo Fisher Scientific) and a PLRP-S (5 μm, 300 A) column (Agilent).

[0129] Example 2: Mammalian expression of human and mouse PD-L1 Expi293-F cells were cultured in shake flasks in Expi293-F Expression Medium (Fisher Scientific, 13489756) at 135 rpm, 37°C, 8% CO2, and 95% humidity at 0.5-1 million cells / ml. One day before transfection, cells were seeded at a density of 2 million cells / ml. On the day of transfection, cells were seeded at a density of 2.5 million cells / ml. One microgram of hPD-L1-Fc or mPD-L1-Fc extracellular domain plasmid DNA was diluted in Opti-MEM I Reduced Serum Medium (Life Technologies, 31985-062) per ml of culture volume. ExpiFectamine was diluted in Opti-MEM I Reduced Serum Medium according to the manufacturer's instructions and incubated at room temperature for 5 minutes. The DNA solution was then added to the ExpiFectamine mixture, incubated at room temperature for 20 minutes, and mixed with the cells. After 16 hours, enhancers were added to the cells. After 96–120 hours, supernatants were collected, centrifuged, and filtered through a 0.45 μm membrane.

[0130] Example 3. Stability at high temperatures (above 60°C) Protein thermal stability was measured 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 at an appropriate dilution. A temperature increase from 25°C to 95°C was programmed at a heating rate of 1°C / min. Fluorescence was continuously measured (ViiA7 Real-Time PCR System (Thermo Scientific)) at an excitation wavelength of 520 nm and an emission wavelength of 623 nm. The midpoint of the thermal unfolding transition (T m , melting point) were determined and are shown in Table 1.

[0131] Example 4. Biochemical Binding Analysis (Surface Plasmon Resonance, SPR) Recombinant protein A was immobilized onto a High Capacity Amine sensor chip (Bruker) after NHS / EDC activation, yielding approximately 1000 RU using a Sierra SPR-32 system (Bruker). The chip was equilibrated with SPR running buffer (PBS 0.05%, Tween pH 7.3). After ligand immobilization, unreacted NHS groups were blocked using an ethanolamine injection. Fc-tagged extracellular domains of hPD-L1 and mPD-L1 were injected at 30 nM, followed by PD-L1 binding proteins at a flow rate of 30 μl / min. A flow cell without ligand was used as a reference. Upon ligand binding, the protein analyte accumulated on the surface, increasing the refractive index. This change in refractive index was measured in real time and plotted as response or resonance units (RU) versus time. Serial dilutions of the analyte were applied to the chip. Binding was allowed for 120 seconds, followed by dissociation for 180 seconds. After each run, the chip surface was regenerated with 30 μl of regeneration buffer (10 mM glycine pH 2.0) and equilibrated with running buffer. Binding studies were performed using a Sierra SPR-32 system (Bruker), and data evaluation was performed using the Sierra Analyser software provided by the manufacturer.

[0132] Table 1 shows the binding affinity to PD-L1. 211828 was able to detectably bind to mouse PD-L1, but the effective K D could not be determined by SPR.

[0133] TIFF2025534423000002.tif56170

[0134] Example 5. Functional characterization: specific binding to cell surface-expressed hPD-L1 and mPD-L1 (flow cytometry) 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 the native hPD-L1-expressing H460 cell line were trypsinized, resuspended in medium containing FCS, and washed with pre-chilled FACS blocking buffer. A cell concentration of 1 million cells / ml was prepared for cell staining, and 100μL / well of each cell line was loaded in triplicate into a 96-well plate (Greiner). A dilution series of the protein or 1μg / ml avelumab (Merck) as a positive control was added to the PD-L1-expressing and control cells. After 45 minutes, the supernatant was removed, and 100 μl / well of rabbit anti-Strep-Tag antibody (GenScript; A00626) diluted 1:300 in FACS blocking buffer was added to the wells containing the diluted bound protein. Avelumab was detected with anti-human IgG-Alexa488 (Invitrogen; A-11013) diluted 1:1000. After removing the anti-Strep-Tag antibody, goat anti-rabbit IgG Alexa Fluor 488 antibody (Invitrogen; A11008) was applied at a 1:1000 dilution to the other wells. Flow cytometry measurements were performed using a Guava easyCyte 5HT device (Merck-Millipore) at an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

[0135] 224039, 224058, and 224180 (SEQ ID NOs: 5-7) showed specific binding to HEK293 cells overexpressing human PD-L1 or mouse PD-L1. No binding was detected on HEK293-pEntry cells. 224039, 224058, and 224180 also showed significant binding to cells expressing native human PD-L1, whereas 224121 and 211828 showed only weak binding. In Table 2, Pos6 and Pos8 indicate positions in the second (C-terminal) monomer of the protein.

[0136] TIFF2025534423000003.tif51170

[0137] Example 6. Binding affinity to PD-L1 in human serum after long-term incubation (cell binding assay—flow cytometry) 3.3 × 10 of 224058 (SEQ ID NO: 6) -6 M to 7 x 10 -13 Dilution series up to M and 1 x 10 of 224180 (SEQ ID NO: 7) or 224039 (SEQ ID NO: 5) -5 M to 7 x 10 -13 A dilution series up to 100kJ / mL was incubated in human serum at 37°C for 0 and 24 hours. PD-L1-overexpressing HEK293 cells were trypsinized, washed with FACS blocking buffer, and seeded into a 96-well round-bottom plate at a density of 100,000 cells / 100μl. The protein dilution series was added to the cells. After 45 minutes, the supernatant was removed and the cells were washed. Binding was demonstrated using 100μl / well of rabbit anti-Strep-Tag antibody diluted 1:300 in FACS blocking buffer in the first step, and goat anti-rabbit IgG Alexa Fluor 488 antibody diluted 1:1000 in FACS blocking buffer in the second step. Readout was as described above. The protein is stable in human serum for at least 24 hours (Figure 1), with only a slight decrease in binding affinity for PD-L1 expressed on cells (K for human PD-1 after 24 hours). D 2.4 nM, 224180: K for human PD-1 after 24 hours D 224039 was stable in human serum after 24 hours of incubation and exhibited a K D is 1.3 nM.

[0138] Example 7. Serum stability of PD-L1 binding proteins (ELISA) High-binding plates (Greiner, 781061) were immobilized with 2.5μg / ml recombinant human PD-L1-Fc overnight at 4℃. 224058 (SEQ ID NO: 6), 224180 (SEQ ID NO: 7), and 224039 (SEQ ID NO: 5) were added at concentrations ranging from 1μM to 7x10-8 Dilution series down to 1 μM were incubated in 100% mouse serum or 100% rat serum at 37°C for 24 hours. ELISA plates were washed three times with PBST (PBS, 0.1% Tween) and blocked with 3% BSA / 0.5% Tween / PBS for 2 hours at room temperature. After 0 or 24 hours of serum incubation, the dilution series was incubated on the ELISA plate for 1 hour at room temperature. After washing with PBST, wells were incubated with biotinylated anti-ubiquitin antibody (1:300) for 1 hour at room temperature. Binding was visualized using streptavidin-HRP (1:5,000). PD-L1 binding proteins are stable in rat and mouse serum. They were found to be stable at K after 24 hours of serum incubation. D There was no significant change in the α-amyloid β ...

[0139] TIFF2025534423000004.tif43170

[0140] Appendix - Arrays SEQ ID NO: 1(224039-2) MQIFVFTDTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIWPRRLLHLVLRLRAA Sequence number 2 (224058-2) MQIFVFTDTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIQPRRLLHLVLRLRAA SEQ ID NO: 3 (224180-2) MQIFVFTDTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIAPRRLLHLVLRLRAA Sequence number 4 (224039-1 / 224058-1 / 224180-1 / 224121-1 / 211828-1) MQIFVDTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIRYPAFLHLVLRLRAA Sequence number 5 (224039) MQIFVDTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIRYPAFLHLVLRLRAAMQIFVFTDTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIWPRRLLHLVLRLRAA Sequence number 6 (224058) MQIFVDTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIRYPAFLHLVLRLRAAMQIFVFTDTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIQPRRLLHLVLRLRAA Sequence number 7 (224180) MQIFVDTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIRYPAFLHLVLRLRAAMQIFVFTDTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIAPRRLLHLVLRLRAASAWSHPQFEK Sequence number 8 (224121) MQIFVDTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIRYPAFLHLVLRLRAAMQIFVHTATGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIWPRRLLHLVLRLRAA SEQ ID NO: 9 (211828) MQIFVDTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIRYPAFLHLVLRLRAAMQIFVRTTTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIWPRRLLHLVLRLRAA SEQ ID NO: 10 (ubiquitin) MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRAA SEQ ID NO: 11 (extracellular domain of hPD-L1) FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITV KVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER SEQ ID NO: 12 (224121-2) MQIFVHTATGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIWPRRLLHLVLRLRAA SEQ ID NO: 13 (211828-2) MQIFVRTTTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIWPRRLLHLVLRLRAA

Claims

1. 1. A dimeric PD-L1 binding protein comprising the 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, (i) having 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; (ii) exhibits a binding affinity for human programmed death-ligand 1 (PD-L1) of less than 15 nM, as measured by surface plasmon resonance; Dimeric PD-L1 binding protein.

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

1.

3. 3. The dimeric PD-L1 binding protein of claim 1 or 2, which is stable in serum after incubation at 37°C for 24 hours.

4. 4. The dimeric PD-L1 binding protein of any one of claims 1 to 3, further comprising one or more binding sites for attachment of a chemical moiety, preferably selected from any one of a chelator, a drug, a toxin, a dye, and a small molecule.

5. 5. The dimeric PD-L1 binding protein of any one of claims 1 to 4, further comprising at least one diagnostically active moiety or at least one therapeutically active moiety.

6. 8. The dimeric PD-L1 binding protein of any one of claims 1 to 7, further comprising at least one pharmacokinetic-modulating moiety, preferably selected from any one of serum albumin, albumin-binding proteins, immunoglobulin-binding proteins, immunoglobulins or immunoglobulin fragments, polysaccharides, unstructured amino acid sequences comprising the amino acids alanine, glycine, serine, and proline, polyethylene glycol, sialic acid, transferrin, and transferrin receptor-binding proteins, or any combination thereof.

7. 10. The dimeric PD-L1 binding protein of any one of claims 1 to 6 for use in the diagnosis or treatment of a PD-L1 positive tumor, or for use in the diagnosis or treatment of an infectious disease, such as a chronic viral infection.

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

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

10. 10. The composition for use in medicine according to claim 9, for use in the diagnosis or treatment of a PD-L1 positive tumor, or for use in the diagnosis or treatment of an infectious disease such as a chronic viral infection.

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

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

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