NZ741586B2

NZ741586B2 - New polypeptide having affinity to pd-l1

Info

Publication number: NZ741586B2
Application number: NZ741586A
Authority: NZ
Inventors: Elin Gunneriusson; Elisabet Wahlberg
Original assignee: Affibody Ab
Priority date: 2015-10-30
Filing date: 2016-10-28
Publication date: 2022-03-01

Abstract

The present disclosure relates to a class of engineered polypeptides having a binding affinity for programmed death-ligand 1 (PD-L1), and provides a PD-L1 binding polypeptide comprising the sequence ERNX4AAX7EIL X11LPNLX16X17X18QX20WAFIWX26LX28D. The present disclosure also relates to the use of such a PD-L1 binding polypeptide as a therapeutic, prognostic and/or diagnostic agent. Wherein specific embodiments the PD-L1 binding motif is a binding polypeptide, antibody, fusion protein or conjugate. h a PD-L1 binding polypeptide as a therapeutic, prognostic and/or diagnostic agent. Wherein specific embodiments the PD-L1 binding motif is a binding polypeptide, antibody, fusion protein or conjugate.

Description

NEW POLYPEPTIDE HAVING AN AFFINITY TO PD-L1 Field of the invention The t disclosure relates to a class of engineered polypeptides having a binding affinity for programmed death-ligand 1(in the following referred to as PD-L1). The present disclosure also relates to the use of such a PD-L1 binding polypeptide as a therapeutic, prognostic and/or diagnostic agent. ound Under normal physiologic conditions, the immune checkpoints are crucial for ining self-tolerance (i.e. prevent autoimmunity) and for modulating the immune response to protect against tissue damage when the immune system is responding to pathogenic infections. At times, tumor cells can co-opt certain immune checkpoint pathways to escape from immunesurveillance mechanisms.

Inhibition of immune oints has therefore emerged as a promising ch in cancer immunotherapy. The two immune checkpoint ors that have been most actively studied in this context are the cytotoxic T-lymphocyteassociated antigen (CTLA-4; also known as CD152) and programmed cell death protein 1 (PD-1; also known as CD279), which regulate the immune response at different . CTLA-4 primarily regulates immune responses early in T-cell activation, whereas PD-1 primarily limits the ty of T-cells in the effector phase within tissues and tumors (Pardoll, 2012, Nat. Rev. Cancer, 12:252-64).

PD-1 has two known ligands: programmed death-ligand 1 (PD-L1; also known as human B7 homolog 1, B7-H1, or cluster of differentiation 274, CD274) and programmed ligand 2 ; also known as B7-DC and CD273).

Both ligands belong to the B7 immunoglobulin superfamily and are type I transmembrane glycoproteins composed of IgC- and IgV-type extracellular domains. r, it was recently reported that PD-L1 and PD-L2, as well as PD-1, also exist in soluble forms in addition to being membrane bound. PD-L1 and PD-L2 share approximately 40 % amino acid residue identity. Whereas the expression of PD-L2 is mainly limited to antigen presenting cells, PD-L1 is expressed in both hematopoietic and non- hematopoietic cells. High tumor expression of PD-L1 is associated with increased aggressiveness and worse prognosis (Dai et al, 2014, Cellular Immunology, 290:72-79).

The clinical significance of targeting immune checkpoint pathways has been demonstrated with several monoclonal antibodies inhibiting , PD-1 and PD-L1, which work by restoring protective immune responses to tumor cells. The anti—CTLA—4 antibody ipilimumab (Yervoy®, Bristol Myers Squibb) was approved by FDA in 2011 for the treatment patients with metastatic ma where a durable response was observed in 10-15 % of the patients. However, ipilimumab is ated with -related toxicities, potentially due to its role in the priming phase of the immune se thereby also affecting normal tissues. A safer approach may be to target the D-L1 pathway to restore umor immunity selectively within the tumor microenvironment. Inhibition of the PD-1/PD-L1 pathway has demonstrated durable response in 30-35 % of patients with advanced melanoma, which in 2014 resulted in the FDA approval of the anti-PD-1 antibodies lizumab (formerly lambrolizumab; Keytruda®, Merck) and nivolumab (Bristol Myers Squibb and Ono Pharmaceutical) (Shin and Ribas, 2015, Curr. Opin. |mmunol., 33:23-35; Philips and Atkins, 2015, International Immunology, 27:39-46). The first PD-L1 targeting antibody investigated in clinical trials was MDX-1105 which was evaluated in a Phase I study in patients with advanced solid tumors including melanoma, non-small cell lung cancer (NSCLC), colorectal cancer, renal cell carcinoma, n cancer, pancreatic cancer, gastric cancer and breast cancer (Momtaz and Postow, 2014, Pharmgenomics Pers Med. 7:357-65). The results demonstrated potential benefits of PD-L1 de. Other antibodies against PD—L1 that are currently in Phase III al trials include atezolizumab (MDPL3280A, Genentech), durvalumab (MEDI4736, Medlmmune/Astra Zeneca, Celgene), and avelumab (MSBOO10718C, EMD Serono, Pfizer).

To e the efficacy and increase the number of patients that d to immunotherapy, it may be beneficial to target the antitumor immune response at multiple levels. This may be ed through 2016/076040 synergistic combinations. For instance, preclinical studies combining CTLA-4 and PD-1 blocking antibodies (ipilimumab and nivolumab) has demonstrated or antitumor activity, but with a toxicity similar to TLA-4 monotherapy (Shin and Ribas, 2015, supra). Furthermore, PD-L1 is speculated to be a potential biomarker, due to its abundance in the tumor microenvironment and because tumor expression of PD-L1 has a strong association with se to anti-PD-1/PD-L1 therapy.

The high prevalence of cancer and infectious diseases, together with a high unmet medical need, warrants the development of new modes of treatment. Since tissue penetration rate is negatively associated with the size of the molecule, a relatively large antibody molecule inherently has poor tissue distribution and ation capacity.

Thus, the use of monoclonal antibodies is not always optimal for therapy and there is continued need for ion of agents with a high affinity for PD-L1. Ofgreat st is also the provision of uses of such molecules in the treatment, sis and prognosis of PD—L1 related disorders.

Summary of the invention It is an object of the present disclosure to provide new PD-L1 binding agents, which could for example be used for therapeutic, prognostic and diagnostic applications.

It is an object of the present disclosure to e a new multispecific agent, such as a bispecific agent, which has affinity for PD-L1 and at least one additional antigen.

It is an object of the present disclosure to provide a molecule allowing for efficient therapy of for example various forms of cancer and ious disease, while alleviating the abovementioned and other drawbacks of current therapies.

It is an object of the present disclosure to provide a molecule suitable for prognostic and diagnostic applications, for example stic and diagnostic application in relation to various forms of cancer and infectious disease.

These and other s, which are evident to the skilled person from the present disclosure, are met by the different aspects of the invention as claimed in the appended claims and as generally disclosed herein.

Thus, in the first aspect of the disclosure, there is provided a PD-L1 binding ptide, comprising a PD-L1 binding motif BM, which motif consists of an amino acid sequence selected from: i) ERNX4AAX7E|L X11LPNLX16X17X1sQX20 WAFIWX25LX28D wherein, independently from each other, X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and Y; X7 is selected from A, E, F, H, N, Q, S, T, V, W and Y; X11 is selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W and Y; X16 is selected from N and T; X17 is ed from A, H, K, N, Q, R and S; X18 is selected from A, D, E, G, H, K, L, N, Q, R, S, T, V and Y; X20 is selected from H, I, K, L, N, Q, R, T, V and Y; X25 is selected from K and S; and X28 is selected from A, D and E; ii) an amino acid sequence which has at least 96 % identity to the sequence defined in i).

The above definition of a class of sequence related, PD-L1 binding polypeptides is based on a statistical analysis of a number of random polypeptide ts of a parent scaffold, that were selected for their interaction with PD-L1 in selection experiments. The identified PD-L1 binding motif, or ’, corresponds to the target binding region of the parent scaffold, which region constitutes two alpha helices within a three-helical bundle protein domain. In the parent scaffold, the varied amino acid residues of the two BM helices constitute a binding surface for ction with the nt Fc part of antibodies. In the present sure, the random variation of binding surface residues and subsequent selection of ts have replaced the Fc interaction capacity with a capacity for interaction with PD-L1.

As the skilled person will realize, the function of any ptide, such as the PD-L1 binding capacity of the polypeptide of the present disclosure, is dependent on the tertiary structure of the polypeptide. It is therefore possible to make minor s to the sequence of amino acids in a polypeptide without affecting the function thereof. Thus, the disclosure encompasses modified variants of the PD-L1 binding polypeptide, which have retained PD- L1 binding characteristics.

In this way, encompassed by the present disclosure is a PD-L1 binding polypeptide sing an amino acid sequence with 96 % or greater ty to a polypeptide as defined in i). For e, it is possible that an amino acid residue belonging to a certain functional grouping of amino acid residues (e.g. hydrophobic, hydrophilic, polar etc) could be exchanged for another amino acid residue from the same functional group.

In some embodiments, such changes may be made in any position of the sequence of the PD-L1 g polypeptide as disclosed herein. In other embodiments, such changes may be made only in the non-variable positions, also denoted scaffold amino acid residues. In such cases, changes are not d in the variable positions. In other embodiments, such changes may be only in the variable positions. According to one definition of such “variable positions”, these are positions denoted with an “X” in sequence i) as defined above. According to another definition, “variable positions” are those positions that are randomized in a selection library of Z variants prior to selection, and may thus for example be positions 2, 3, 4, 6, 7, 10, 11, 17, 18, 20, 21, 25 and 28 in sequence i), as illustrated in Example 1.

The term ”% identity”, as used throughout the specification, may for e be calculated as follows. The query sequence is aligned to the target sequence using the L W algorithm (Thompson et al., (1994) Nucleic Acids Research, 22: 4673-4680). A comparison is made over the window corresponding to the shortest of the aligned sequences. The shortest of the aligned sequences may in some instances be the target sequence. In other instances, the query sequence may constitute the shortest of the aligned sequences. The amino acid es at each position are compared and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as % identity.

In another embodiment, there is provided a PD-L1 binding ptide wherein in sequence i) X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and Y; X7 is selected from E, F, H, N, Q, S, T, V, W and Y; X11 is selected from A, D, H, L, Q, R, T, V, W and Y; X15 is selected from N and T; X17 is ed from A, H, K, N, Q, R and S; X18 is selected from A, D, E, G, H, K, L, N, Q, R, S, T, V and Y; X20 is selected from H, l, K, L, Q, R, T, V and Y; X26 is selected from K and S; and X28 is selected from A, D and E.

In r embodiment, there is provided a PD-L1 binding polypeptide, wherein in sequence i) X4 is selected from A, D, E, F, H, |, K, L, N, Q, R, S, T, V and Y; X7 is selected from A, E, F, H, N, Q, S, T, V, W and Y; X11 is selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W and Y; X15 is selected from N and T; X17 is selected from A, H, K, N, Q, R and S; X18 is selected from A, D, E, G, H, K, L, N, Q, R, S, T, V and Y; X20 is selected from H, l, K, L, N, Q, R, T, V and Y; X25 is selected from K and S; and X28 is ed from A, D and E.

In yet another embodiment, there is ed a PD-L1 binding polypeptide, wherein in sequence i) X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T and V; X7 is ed from F, H, Q and Y; X11 is ed from H, Q, W and Y; X16 is selected from N and T; X17 is selected from A, H, K, N, Q and S; X18 is ed from A, E, G, H, K, L, N, Q, R, S, T, V and Y; X20 is selected from H, l, K, Q, R and V; X26 is selected from K and S; and X28 is selected from A and D.

As used herein, “Xn” and “Xm” are used to indicate amino acids in positions n and m in the sequence i) as defined above, wherein n and m are integers which te the position of an amino acid within said sequence as counted from the N-terminal end of said sequence. For example, X3 and X7 indicate the amino acid in position three and seven, respectively, from the N- terminal end of sequence i).

In embodiments according to the first aspect, there are provided polypeptides wherein Xn in sequence i) is independently selected from a group of possible residues according to Table 1. The skilled person will appreciate that Xn may be selected from any one of the listed groups of possible residues and that this selection is independent from the selection of amino acids in Xm, wherein n¢m. Thus, any of the listed le residues in position Xn in Table 1 may be independently combined with any of the listed possible residues any other variable position in Table 1.

The skilled person will appreciate that Table 1 is to be read as follows: In one embodiment according to the first aspect, there is provided a polypeptide wherein amino acid residue “Xn” in sequence i) is selected from “Possible residues”. Thus, Table 1 discloses several ic and individualized embodiments of the first aspect of the t disclosure. For example, in one embodiment according to the first aspect, there is provided a polypeptide wherein X4 in sequence i) is selected from A, D, E, l, K, L, N, Q, S and T and in another embodiment according to the first aspect, there is provided a polypeptide n X4 in sequence i) is selected from A, D, E, l, K, Q, S and T. For avoidance of doubt, the listed embodiments may be freely combined in yet other embodiments. For e, one such ed embodiment is a polypeptide in which X4 is selected from A, D, E, l, K, Q, S and T, while X7 is selected from F, H, Q and Y, and X18 is selected from A, L, K and S.

Table 1 Xn Possible residues Xn Possible residues X4 A, D, E, F, H, l, K, L, N, Q, X4 E R, S, T and V X4 A X4 A, D, E, F, H, l, K, L, N, Q, X4 D R, S and T X4 K X4 A, D, E, F, H, l, K, L, N, Q, X4 S S and T X4 L X4 A, D,E, K,L, N,Q,SandT X4 T X4 A, D, E, l, K, L, N, Q, S and X7 E, F, H, N, Q, S, T, V, W T and Y X4 A, D, E, K, L, N, Q, and S X7 E, F, H, N, Q, S, V, W and X4 A, D, E, l, K,Q,SandT Y X4 A, E, K, L, N, Q, and S X7 E, F, H, N, Q, S, T, V and Y X4 E, l, K, L, N, Q, S and T X7 E, F, H, N, Q, S, T, Wand X4 A, D, K, L, N, and S Y X4 A, K, L, N, and S X7 E, F, H, Q, S, T, V, W and Y X4 A, D, E, Q and S X7 E, F, H, N, Q, S, V and Y X4 A, D, E, K, and S X7 E, F, H, N, Q, S and Y X4 A, K, L, and S X7 E, F, H, Q, S and Y X4 A, D, E and S X7 E, F, H, Q and Y X4 A, E,QandS X7 F,H,QandY X4 A, E, K and S X7 F, Q and Y X4 L, N,SandT X7 H,QandY X4 L, S and T X7 F, Y X4 A, D and K X7 F, Q X4 A, E and S X7 Q, Y X4 A, E and K X7 F X4 A, D X7 Q X4 A, K X7 Y X4 D, K X7 H X4 A, E X11 A, D, E, F, H, K, N, Q, R, S, X4 A, S T, W a d Y X4 E, S X11 D, E, F, H, K, N, Q, R, S, T, X4 E, K Wand X4 L,S X11 D, E, H, K,N,Q, R, S,T,W X4 L, T and Y x4 8, T X11 E, H, K, N, Q, R, S, T, W Xn Possible residues Xn le residues and Y X18 8 X11 E, H, K, N, Q, S, T, W and X18 H Y X20 H, I, K, L, Q, R, T, V and Y X11 E, H, K, N, Q, W and Y X20 H, I, K, L, Q, R, V and Y X11 A, D, H, L, Q, R, T, V, W X20 H, I, K, Q, R and V and Y X20 H, I, K an R X11 H, O; W and Y X20 H, I and K X11 Q, Y X20 H, K and R X11 W, Y X20 K and R X11 H, Y X20 I, K X11 H X20 I, H X11 Y X20 H: K X16 N X20 H X16 T X20 I X17 A, H, K, N, Q and S X20 K X17 A, K, N, Q, R and S X20 R X17 A, K, N, Q and S X26 K X17 A, N, Q and S X26 8 X17 K, N, Q and S X28 A, D X17 N, Q and S X28 A, E X17 N, Q X28 D, E X17 N, S X28 A X17 N X28 D X17 Q X28 E X17 S X18 A, E, G, H, K, L, N, Q, R, S, T, V and Y X18 A, E, G, H, K, L, N, Q, R, S, T and Y X18 A, E, G, H, K, L, N, Q, R, S, and Y X18 A, E, G, H, K, L, N, Q, R and S X18 A, E, G, K, N, Q, R and S X18 A, G, H, L, N, Q, S and Y X18 A, G, N, Q and S X18 A, H, Q and S X18 A, H and Q X18 A, L, K and S X18 A, K, Q and S X18 A, Q and S X18 A, G X18 A, Q X18 A X18 Q X18 G In one particular embodiment ing to the first aspect, there is provided a polypeptide wherein sequence i) fulfills at least four of the seven conditions l-Vll: l. X7 is selected from F, H, Q and Y; H. X11 is selected from H and Y; lll. X16 is T; IV. X17 is selected from N, Q and S; V. X20 is ed from H, I, K and R; VI. X26 is K; and VII. X28 isAor D.

In one embodiment, sequence i) fulfills at least five of the seven conditions l-Vll, such as least six of the seven conditions l-Vll. In one particular embodiment, sequence i) fulfills all of the seven conditions l-Vll.

In some embodiments of a PD-L1 binding polypeptide according to the first aspect, X7X11X20 is selected from FYK and WK. In some embodiments, X11X17X20 is selected from YNK and YQK. In some embodiments, X11X18X20 is YAK.

As described in detail in the experimental section to follow, the selection of PD-L1 binding polypeptide variants has led to the identification of a number of individual PD-L1 binding motif (BM) sequences. These ces constitute individual embodiments of sequence i) according to this aspect. The sequences of individual PD-L1 binding motifs correspond to amino acid positions 8-36 in SEQ ID NO:1-808 presented in Figure 1. Hence, in one ment of the PD-L1 binding polypeptide according to this aspect, sequence i) ponds to the sequence from position 8 to position 36 in a ce selected from the group consisting of SEQ ID NO:1-808. In one embodiment, sequence i) corresponds to the sequence from on 8 to position 36 in a sequence ed from the group consisting of SEQ ID NO:1-93 and 774-796, such as the group consisting of SEQ ID NO:1-93 and 774-787. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781 and 784-786, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784 and 786, such as the group ting of SEQ ID NO:1—93, 776, 781 and 784, such as the group ting of SEQ ID NO:1-93, 776 and 784 or the group consisting of SEQ ID NO:1-93, 776 and 781, for example the group consisting of SEQ ID NO:1- 93 and 776 or the group consisting of SEQ ID NO:1-93 and 781 or the group consisting of SEQ ID NO:1-93 and 784. In one embodiment, sequence i) corresponds to the sequence from position 8 to on 36 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774, 775 and 780- 786, such as the group consisting of SEQ ID NO:1-93, 775, 780, 781, 784 and 786. In one embodiment, sequence i) corresponds to the sequence from position 8 to on 36 in a sequence selected from the group consisting of SEQ ID NO:1, 2, 17,776 and 781, such as the group consisting ofSEQ ID NO: 1, 2 and 776. In one embodiment, sequence i) corresponds to the sequence from on 8 to position 36 in SEQ ID NO:1, SEQ ID N02 or SEQ ID NO:776. In one embodiment, sequence i) corresponds to the sequence from position 8 to on 36 in a sequence selected from the group ting of SEQ ID NO:1-93. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, 3-8, 11, 13, 16, 18, 20, 22, 23, 43 and 73. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a ce selected from the group consisting of SEQ ID NO:1-24. For example, in one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a ce selected from the group consisting of SEQ ID NO:1-16, such as the group consisting of SEQ ID NO:1, 2, 4, 5, 7, 9 and 10. In another embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, 3-6, 9-10, 12-21, 23 and 24, such as the group consisting ofSEQ ID NO:1, 4-6, 9, 14 and 18-21. In one embodiment, sequence i) corresponds to WO 72280 2016/076040 the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-12, 14 and 17-21. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-12 and 17, such as the group consisting of SEQ ID NO:1-5 and 17, such as the group consisting of SEQ ID NO:1, 2 and 17. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, 4, 5, 6, 9, 14 and 18-21, such as the group consisting of SEQ ID NO:4, 5, 18 and 21, such as the group consisting of SEQ ID NO:4, 5 and 21. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group ting of SEQ ID NO:1, 2, 4, 5 and 21, such as the group ting of SEQ ID NO:1 and 2. In one embodiment, sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:21.

In some ments of the present disclosure, the BM as defined above “forms part of” a three-helix bundle protein domain. This is understood to mean that the sequence of the BM is “inserted” into or “grafted” onto the sequence of the al three-helix bundle domain, such that the BM replaces a similar structural motif in the original . For example, without wishing to be bound by , the BM is thought to constitute two of the three helices of a three-helix bundle, and can therefore replace such a two-helix motif within any three-helix bundle. As the skilled person will realize, the replacement of two s of the three-helix bundle domain by the two BM helices has to be performed so as not to affect the basic structure of the polypeptide. That is, the overall folding of the Co backbone of the polypeptide according to this embodiment of the invention is ntially the same as that of the three-helix bundle protein domain of which it forms a part, e.g. having the same elements of secondary structure in the same order etc.

Thus, a BM according to the present disclosure “forms part” of a three—helix bundle domain if the polypeptide ing to this embodiment has the same fold as the original domain, implying that the basic structural properties are shared, those properties e.g. resulting in similar CD spectra. The skilled person is aware of other parameters that are relevant.

In particular embodiments, the PD-L1 binding motif (BM) thus forms part of a three-helix bundle protein domain. For example, the BM may essentially constitute two alpha helices with an interconnecting loop, within said helix bundle n domain. In particular embodiments, said three- helix bundle protein domain is selected from domains of bacterial receptor proteins. Non—limiting examples of such domains are the five ent three- helical domains of Protein A from Staphylococcus aureus, such as domain B, and derivatives thereof. In some embodiments, the three-helical bundle protein domain is a variant of protein Z, which is derived from domain B of staphylococcal Protein A (Wahlberg E eta], 2003, PNAS 100(6):3185-3190).

In some embodiments where the PD-L1 binding ptide as disclosed herein forms part of a three-helix bundle protein domain, the PD-L1 binding polypeptide may comprise a binding module (BMod), the amino acid sequence of which is selected from: iii) K-[BM]—DPSQSXaXbLLXC dXeXfQ; wherein [BM] is a PD-L1 binding motif as defined ; X3 is selected from A and S; Xb is selected from N and E; XC is selected from A, S and C; Xd is ed from E, N and S; X6 is selected from D, E and S; and Xf is selected from A and S; and iv) an amino acid sequence which has at least 93 % identity to a sequence defined in iii).

In some embodiments, said polypeptide may beneficially exhibit a high structural stability, such as resistance to chemical modifications, to s in physical conditions and to proteolysis, during production and storage, as well as in vivo.

As sed above, polypeptides comprising minor changes as compared to the above amino acid sequences, which do not largely affect the tertiary structure and the function of the polypeptide, are also within the scope of the present disclosure. Thus, in some embodiments, sequence iv) has at least 93 %, such as at least 95 %, such as at least 97 % identity to a sequence defined by iii).

In one embodiment, Xa in sequence iii) is A.

In one embodiment, Xa in sequence iii) is S.

In one embodiment, Xb in sequence iii) is N.

In one embodiment, Xb in ce iii) is E.

In one embodiment, XC in ce iii) is A.

In one embodiment, XC in sequence iii) is S.

In one embodiment, XC in sequence iii) is C.

In one embodiment, Xd in sequence iii) is E.

In one embodiment, Xd in sequence iii) is N.

In one embodiment, Xd in sequence iii) is S.

In one embodiment, X6 in sequence iii) is D.

In one embodiment, X6 in sequence iii) is E.

In one ment, X6 in ce iii) is S.

In one embodiment, dee in sequence iii) is selected from EE, ES, SD, SE and SS.

In one embodiment, dee in sequence iii) is ES.

In one embodiment, dee in sequence iii) is SE.

In one embodiment, dee in sequence iii) is SD.

In one embodiment, Xf in sequence iii) is A.

In one embodiment, Xf in sequence iii) is S.

In one embodiment, in sequence iii), X8] is A; Xb is N; XC is A and Xf is A.

In one embodiment, in sequence iii), X8 is S; Xb is E; XC is A and Xf is A.

WO 72280 In one embodiment, in sequence iii), Xa is A; Xb is N; XC is C and Xf is A.

In one embodiment, in sequence iii), Xa is S; Xb is E; XC is S and Xf is S.

In one embodiment, in sequence iii), X3 is S; Xb is E; XC is C and Xf is S.

In one embodiment, in sequence iii), Xa is A; Xb is N; XC is A; dee is ND and Xf is A.

In one embodiment, in sequence iii), Xa is S; Xb is E; XC is A; dee is ND and X is A.

In one embodiment, in sequence iii), Xa is A; Xb is N; XC is C; dee is ND and Xf is A.

In one ment, in sequence iii), Xa is S; Xb is E; XC is S; dee is ND and Xf is S.

In one ment, in sequence iii), Xal is S; Xb is E; Xc is C; XdXe is ND and X is S.

In one embodiment, in sequence iii), Xa is A; Xb is N; XC is A; dee is SE and Xf is A.

In one embodiment, in sequence iii), Xa is S; Xb is E; XC is A; dee is SE and X is A.

In one embodiment, in sequence iii), Xa is A; Xb is N; XC is C; dee is SE and Xf is A.

In one embodiment, in sequence iii), Xa is S; Xb is E; XC is S; dee is SE and X is S.

In one embodiment, in sequence iii), Xa is S; Xb is E; XC is C; dee is SE and Xf is S.

In one embodiment, in sequence iii), Xa is A; Xb is N; XC is A; dee is SD and Xf is A.

In one embodiment, in sequence iii), Xal is S; Xb is E; Xc is A; XdXe is SD and X is A.

In one embodiment, in sequence iii), Xa is A; Xb is N; XC is C; dee is SD and Xf is A.

In one embodiment, in sequence iii), Xa is S; Xb is E; XC is S; dee is SD and X is S.

In one embodiment, in sequence iii), Xa is S; Xb is E; XC is C; dee is SD and Xf is S.

In yet a further embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1—808 presented in Figure 1. Hence, in one embodiment of the PD-L1 binding polypeptide according to this aspect, sequence iii) corresponds to the sequence from on 7 to position 55 in a ce selected from the group ting of SEQ ID NO:1-808. In one embodiment, sequence iii) corresponds to the sequence from on 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-93 and 774-796, such as the group consisting of SEQ ID NO:1-93 and 774-787. In one embodiment, sequence iii) corresponds to the ce from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781 and 784—786, such as the group consisting of SEQ ID NO:1—93, 776, 780, 781, 784 and 786, such as the group consisting of SEQ ID NO:1-93, 776, 781 and 784, such as the group consisting of SEQ ID NO:1—93, 776 and 784 or the group consisting of SEQ ID NO:1-93, 776 and 781, for example the group consisting of SEQ ID NO:1- 93 and 776 or the group consisting of SEQ ID NO:1—93 and 781 or the group consisting of SEQ ID NO:1—93 and 784. In one ment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774, 775 and 780— 786, such as the group consisting of SEQ ID NO:1-93, 775, 780, 781, 784 and 786. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence ed from the group consisting of SEQ ID NO:1, 2, 17,776 and 781, such as the group consisting ofSEQ ID NO: 1, 2 and 776. In one embodiment, sequence iii) corresponds to the ce from position 7 to position 55 in SEQ ID NO:1, SEQ ID N02 or SEQ ID NO:776. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a ce selected from the group consisting of SEQ ID NO:1—93. In one embodiment, sequence iii) corresponds to the sequence from on 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, 3-8, 11, 13, 16, 18, 20, 22, 23, 43 and 73. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-24. For example, in one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a ce selected from the group consisting of SEQ ID NO:1-16, such as the group consisting of SEQ ID NO:1, 2, 4, 5, 7, 9 and 10. In another embodiment, sequence iii) corresponds to the sequence from position 7 to on 55 in a sequence selected from the group consisting of SEQ ID NO:1, 3-6, 9-10, 12-21, 23 and 24, such as the group consisting of SEQ ID NO:1, 4-6, 9, 14 and 18-21. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group ting of SEQ ID NO:1—12, 14 and 17—21. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID 2 and 17, such as the group consisting of SEQ ID NO:1-5 and 17, such as the group ting of SEQ ID NO:1, 2 and 17. In one embodiment, sequence iii) corresponds to the sequence from on 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1, 4, 5, 6, 9, 14 and 18-21, such as the group consisting of SEQ ID NO:4, 5, 18 and 21, such as the group consisting of SEQ ID NO:4, 5 and 21. In one embodiment, sequence iii) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, 2, 4, 5 and 21, such as the group consisting of SEQ ID NO:1 and 2. In one embodiment, sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:21.

Also, in a further embodiment, there is provided a PD—L1 binding polypeptide, which comprises an amino acid sequence selected from: v) YA-[BMod]—AP; n [BMod] is a PD-L1 binding module as defined herein; and vi) an amino acid sequence which has at least 90 % identity to a sequence defined in v).

Alternatively, there is provided a PD-L1 binding polypeptide, which comprises an amino acid sequence selected from: vii) FN-[BMod]—AP; n [BMod] is a PD-L1 g module as defined herein; and viii) an amino acid sequence which has at least 90 % identity to a sequence defined in vii).

For example, in one embodiment there is provided a PD-L1 binding ptide ed from the group consisting of ix) FNK-[BM]-DPSQS ANLLXC EAKKL NDAQA P; wherein [BM] is a PD-L1 binding motif as defined above and XC is selected from A and C; and x) an amino acid sequence which has at least 90 % identity to a sequence defined in ix).

In r embodiment, there is provided a PD-L1 binding polypeptide ed from the group consisting of xi) FAK-[BM]—DPSQS SELLXC EAKKL SESQA P; wherein [BM] is a PD-L1 binding motif as defined above and XC is selected from A, S and C; and xii) an amino acid sequence which has at least 90 % identity to a sequence defined in xi).

In another embodiment, there is provided a PD-L1 binding polypeptide selected from the group consisting of xiii) FAK-[BM]-DPSQS SELLXC EAKKL NDSQA P; wherein [BM] is a PD-L1 binding motif as defined above and XC is selected from A, S and C; xiv) an amino acid sequence which has at least 90 % identity to a sequence defined in xiii).

In yet another embodiment, there is provided a PD-L1 binding polypeptide selected from the group consisting of xv) YAK-[BM]-DPSQS SELLXC EAKKL NDSQA P; wherein [BM] is a PD-L1 binding motif as defined above and XC is selected from A, S and C; xvi) and an amino acid ce which has at least 90 % identity to a ce defined in xv).

As discussed above, polypeptides comprising minor s as compared to the above amino acid sequences, which do not largely affect the tertiary structure and the function of the polypeptide, also fall within the scope of the present disclosure. Thus, in some embodiments, sequence vi), viii), x), xii), xiv) or xvi) may for example be at least 90 %, such as at least 92 %, such as at least 94 %, such as at least 96 %, such as at least 98 % identical to a sequence defined by v, vii), ix), xi), xiii) and xv), tively.

In some embodiments, the PD-L1 binding motif may form part of a polypeptide sing an amino acid sequence ed from ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK; ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK; ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK; WO 72280 AQHDE—[BM]—DPSQSANVLGEAQKLNDSQAPK; VDNKFNK-[BM]—DPSQSANLLAEAKKLNDAQAPK; K-[BM]—DPSESSELLSEAKKLNKSQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLNDAQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLNDSQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLNDSQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLNDSQAP; AEAKFAK-[BMJ-DPSQSSELLSEAKKLNDSQAPK; AEAKFAK-[BM]—DPSQSSELLSEAKKLNDSQAP; AEAKYAK-[BM]—DPSQSSELLAEAKKLNDAQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLSESQAP; AEAKFAK-[BM]—DPSQSSELLSEAKKLSESQAPK; AEAKFAK-[BM]—DPSQSSELLSEAKKLSESQAP; AEAKYAK-[BM]—DPSQSSELLAEAKKLSEAQAPK; AEAKYAK-[BMj-QPEQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLESSQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLESSQAP; AEAKYAK-[BM]—DPSQSSELLAEAKKLESAQAPK; AEAKYAK-[BM]—QPEQSSELLSEAKKLESSQAPK; AEAKYAK-[BMj-DPSQSSELLSEAKKLSDSQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLSDSQAP; AEAKYAK-[BMj-DPSQSSELLAEAKKLSDSQAPK; AEAKYAK-[BMj-DPSQSSELLAEAKKLSDAQAPK; AEAKYAK-[BM]—QPEQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]—DPSQSSELLSEAKKLNDSQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLNDAQAPK; VDAKYAK-[BM]—DPSQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLSEAQAPK; VDAKYAK-[BM]—QPEQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]—DPSQSSELLSEAKKLESSQAPK; K-[BM]—DPSQSSELLAEAKKLESAQAPK; VDAKYAK-[BM]—QPEQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]—DPSQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLSDSQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLSDAQAPK; VDAKYAK-[BM]—QPEQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLNKAQAPK; K-[BMj-DPSQSSELLAEAKKLNKAQAPK; and ADAKYAK-[BM]—DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is a PD—L1 binding motif as defined herein.

In one embodiment, the PD-L1 binding ptide comprises an amino acid sequence selected from: xvii) VDAKYAK-[BM]—DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is a PD—L1 binding motif as defined herein; and xviii) an amino acid sequence which has at least 89 % identity to the ce d in xvii).

In one embodiment, the PD-L1 binding polypeptide comprises an amino acid sequence selected from: xix) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK; wherein [BM] is a PD-L1 binding motif as defined herein; and xx) an amino acid sequence which has at least 89 % identity to the sequence defined in xix).

In one embodiment, the PD-L1 binding polypeptide comprises an amino acid sequence selected from: xxi) AEAKYAK-[BM]—DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is a PD-L1 binding motif as defined herein; and xxii) an amino acid sequence which has at least 89 % identity to the sequence defined in xxi).

In one embodiment, the PD-L1 g polypeptide comprises an amino acid sequence selected from: xxiii) AEAKFAK-[BM]—DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is a PD-L1 binding motif as d herein; and xxiv) an amino acid sequence which has at least 89 % ty to the sequence defined in xxiii).

Again, polypeptides comprising minor changes as compared to the above amino acid sequences, which do not largely affect the tertiary structure and the on of the polypeptide, also fall within the scope of the t disclosure. Thus, in some embodiments, sequence xviii), xx), xxii) or xxiv) may for example be at least 89 %, such as at least 91 %, such as at least 93 %, such as at least 94 %, such as at least 96 %, such as at least 98 % identical to a sequence defined by xvii), xix), xxi) and xxiii), respectively.

Sequence xvii) or xxi) in such a ptide may be selected from the group consisting of SEQ ID NO:1-814 presented in Figure 1. In one embodiment of the PD-L1 binding polypeptide according to this aspect, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a ce selected from the group consisting of SEQ ID NO:1-808. In one embodiment, sequence xvii) or xxi) ponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774-796 and 4, such as the group consisting of SEQ ID NO:1-93, 774-787 and 809-814. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781, 784-786 and 809-814, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784, 786 and 809-814, such as the group consisting of SEQ ID NO:1-93, 776,781,784 and 809-814, such as the group consisting of SEQ ID NO:1-93, 776,784, 809 and 811-814 or the group consisting of SEQ ID NO:1- 93, 776, 781,809 and 811—814. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 776, 809 and 811-814 or the group consisting of SEQ ID NO:1-93, 781, 809 and 811-814 or the group consisting of SEQ ID NO:1-93, 784 and 4. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to on 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774, 775, 780-786 and 810-814, such as the group ting of SEQ ID 3, 775, 780, 781, 784, 786 and 810-814. In one embodiment, ce xvii) or xxi) corresponds to the sequence from position 1 to on 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 776, 781 and 809— 813, such as the group consisting of SEQ ID NO:1-93, 781 and 810-813. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to on 58 in a sequence selected from the group consisting of SEQ ID NO:1, 2, 17,776, 781, 809-812, such as the group consisting ofSEQ ID NO: 1, 2, 776, 809, 811 and 812. In one embodiment, sequence xvii) corresponds to the sequence from on 1 to position 58 in a sequence selected from the group ting of SEQ ID 3, 776 and 781, such as the group consisting of SEQ ID NO:1-93 and 781. In one embodiment, sequence xvii) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1, 2, 17, 776 and 781, such as the group consisting of SEQ ID NO: 1, 2 and 776. In one embodiment, sequence xvii) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:1, SEQ ID N02 or SEQ ID NO:776. In one ment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1—93 and 811—813. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1, 3-8, 11, 13, 16, 18, 22, 23, 43, 73 and 811-813. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-24 and 811-813. For example, in one embodiment, ce xvii) or xxi) ponds to the sequence from position 1 to position 58 in a sequence ed from the group consisting of SEQ ID NO:1-16 and 811-813, such as the group ting of SEQ ID NO:1, 2, 4, 5, 7, 9, 10, 811 and 812. In another embodiment, ce xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1,3-6, 9-10, 12-21, 23,24,811 and 812, such as the group consisting of SEQ ID NO:1, 4-6, 9, 14,18-21,811 and 812. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-12, 14, 17-21 and 811-812. In one ment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a ce selected from the group consisting of SEQ ID NO:1-12, 17, 811 and 812, such as the group consisting of SEQ ID NO:1-5, 17,811 and 812, such as the group consisting of SEQ ID NO:1, 2, 17,811 and 812. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1, 4, 5, 6, 9, 14, 18, 19, 20, 21 and 811, such as the group consisting of SEQ ID NO:4, 5, 18 and 21, such as the group consisting of SEQ ID NO:4, 5 and 21. In one embodiment, sequence xvii)) corresponds to the ce from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, 2, 4, 5 and 21, such as the group consisting of SEQ ID NO:1 and 2. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:1 or 811. In one embodiment, sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID N02 or 812. In one embodiment, sequence xvii) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:4, SEQ ID N05 or SEQ ID NO:21.

The terms “PD-L1 binding” and ng affinity for PD-L1” as used in this specification refer to a property of a polypeptide which may be tested for example by ELISA or by the use of surface plasmon resonance (SPR) technology.

For example as described in the examples below, PD-L1 binding ty may be tested in an experiment in which samples of the polypeptide are captured on antibody-coated ELISA plates and biotinylated PD-L1 is added followed by streptavidin-conjugated HRP. TMB substrate is added and the absorbance at 450 nm is measured using a multi-well plate reader, such as Victor3 (Perkin Elmer). The d person may then interpret the results obtained by such ments to establish at least a qualitative measure of the binding ty of the ptide for PD-L1. If a quantitative measure is desired, for example to ine the EC50 value (the half maximal effective concentration) for the interaction, ELISA may also be used. The response of the polypeptide t a dilution series of biotinylated PD—L1 is measured using ELISA as described above. The skilled person may then interpret the results obtained by such experiments, and EC50 values may be calculated from the s using for example GraphPad Prism 5 and non-linear regression.

PD-L1 binding affinity may also be tested in an experiment in which PD-L1, or a fragment thereof, is immobilized on a sensor chip of a surface plasmon resonance (SPR) instrument, and the sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on a sensor chip of the instrument, and a sample containing PD-L1, or a fragment thereof, is passed over the chip. The skilled person may then interpret the s obtained by such experiments to establish at least a qualitative measure of the g affinity of the polypeptide for PD-L1. If a quantitative measure is desired, for e to determine a KD value for the interaction, surface plasmon resonance methods may also be used. Binding values may for example be defined in a Biacore (GE care) or ProteOn XPR 36 (Bio-Rad) instrument. PD-L1 is suitably immobilized on a sensor chip of the instrument, and samples of the polypeptide whose ty is to be determined are prepared by serial dilution and injected in random order. KD values may then be calculated from the results using for example the 1:1 Langmuir g model of the BlAevaluation 4.1 software, or other suitable software, provided by the instrument manufacturer.

The terms “albumin binding” and “binding affinity for n” as used in this disclosure refer to a property of a polypeptide which may be tested for example by the use of SPR technology in a e ment or ProteOn XPR36 instrument, in an analogous way to the example described above for PD-L1.

In one embodiment, the PD-L1 binding polypeptide is capable of binding to PD-L1 such that the KD value of the interaction with PD-L1 is at most 2 x10'8 M, such as at most 1 x10"8 M, such as at most 1 x10"9 M, such as at most 5 x10'10 M, such as at most 3 x10'10 M. 2016/076040 In one embodiment, the PD-L1 g polypeptide is capable of binding to PD-L1 such that the kd value of the interaction with PD-L1 is at most 1 x10"3 s'1, such as at most 6 x104 s'1.

In one embodiment, there is provided a PD-L1 g polypeptide according to any preceding item which is e of binding to PD-L1 such that the E050 value of the interaction is at most 1 x 10'9 M, such as at most 1 x10'10 M, such as at most 7 X 10'11 M.

Binding of a polypeptide as defined herein to PD-L1 may interfere either with signaling via PD-L1 in vivo or in vitro. When PD—L1 binds to PD-1, the ligand/receptor interaction dampens the T-Iymphocyte response by e.g. inhibiting kinases involved in T-lymphocyte activation. Thus, blocking the binding of PD—L1 to PD-1 restores the hocyte response. ng activity may for example be quantified by the half maximal inhibitory concentration (ICSO), which is a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function. This tative measure indicates how much of a particular substance is needed to inhibit a given biological process by half, and is ly used in the art.

Thus, in one embodiment, there is provided a PD-L1 binding polypeptide as d herein which is capable of blocking PD-L1 dependent signaling. In one embodiment, the half maximal inhibitory concentration (IC50) of the blocking is at most 5 x 10'8 M, such as at most 1 x 10'8 M, such as at most 5 x10'9 M, such as at most 3.5 x10'9 M, such as at most 1 x 10'9 M, such as at most 5 x10'10 M, such as at most 1 x10"10 M. In one embodiment, the PD-L1 binding polypeptide is capable of blocking the interaction of PD-L1 with PD-1.

In one embodiment, said PD-L1 is human PD-L1. In another embodiment, said PD-L1 is rhesus monkey PD-L1.

The skilled person will understand that various modifications and/or additions can be made to a PD—L1 binding polypeptide according to any aspect disclosed herein in order to tailor the ptide to a specific application without departing from the scope of the present disclosure.

WO 72280 For example, in one embodiment, there is provided a PD-L1 binding polypeptide as described herein, which polypeptide has been extended by and/or comprises additional amino acids at the C terminus and/or N terminus.

Such a polypeptide should be understood as a polypeptide having one or more additional amino acid residues at the very first and/or the very last position in the polypeptide chain. Thus, a PD-L1 binding polypeptide may se any suitable number of onal amino acid residues, for example at least one additional amino acid residue. Each additional amino acid e may individually or collectively be added in order to, for example, improve and/or simplify production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide. Such additional amino acid residues may comprise one or more amino acid residues added for the purpose of chemical coupling. One example of this is the addition of a cysteine residue. Additional amino acid residues may also provide a ”tag” for purification or detection of the ptide, such as a Hise tag, a (HisGlu)3 tag (“HEHEHE” tag) or a ”myc” ) tag or a ”FLAG” tag for interaction with antibodies specific to the tag or immobilized metal affinity chromatography (IMAC) in the case of a Hise-tag.

In one embodiment, there is provided a PD-L1 binding polypeptide as described herein which comprises additional amino acids at the C-terminal and/or N-terminal end. For example, in one embodiment of the PD-L1 binding polypeptide as disclosed herein, it consists of any one of the sequences disclosed herein, having from 0 to 15 onal C-terminal and/or N-terminal residues, such as from 0 to 7 additional inal and/or N-terminal residues. In one embodiment, the PD-L1 binding polypeptide consists of any one of the sequences disclosed herein, having from O to 15, such as from 0 to 4, such as 3 additional C-terminal residues. In one particular embodiment, the PD-L1 binding polypeptide as described herein comprises the additional C- terminal residues VDC or VEC.

The further amino acids as discussed above may be coupled to the PD-L1 g polypeptide by means of chemical conjugation (using known organic chemistry methods) or by any other means, such as expression of the PD-L1 binding polypeptide as a fusion protein orjoined in any other fashion, either directly or via a linker, for example an amino acid linker.

A further polypeptide domain may moreover provide another PD-L1 binding moiety. Thus, in a further embodiment, there is provided a PD-L1 g polypeptide in a multimeric form. Said multimer is understood to comprise at least two PD-L1 binding polypeptides as disclosed herein as monomer units, the amino acid sequences of which may be the same or different. Multimeric forms of the polypeptides may se a suitable number of domains, each having a PD—L1 g motif, and each forming a monomer within the multimer. These domains may have the same amino acid sequence, but alternatively, they may have ent amino acid sequences. In other words, the PD—L1 binding polypeptide of the invention may form homo- or heteromultimers, for example homo- or heterodimers. In one ment, there is provided a PD-L1 binding ptide, wherein said monomer units are covalently coupled together. In another ment, said PD-L1 binding polypeptide monomer units are expressed as a fusion protein. In one embodiment, there is provided a PD-L1 g polypeptide in dimeric form.

In one particular embodiment, said c form is a homodimeric form. In another embodiment, said dimeric form is a heterodimeric form. For the sake of clarity, throughout this disclosure, the term “PD-L1 binding ptide” is used to encompass PD-L1 binding polypeptides in all forms, i.e. monomeric and multimeric forms.

The further amino acids as discussed above may for example comprise one or more further polypeptide (s). A further polypeptide domain may provide the PD-L1 binding dimer with another function, such as for example another binding function, or an enzymatic function, or a toxic function or a fluorescent signaling function, or combinations thereof.

Furthermore, it may be beneficial that the PD—L1 binding polypeptide as defined herein is part of a fusion protein or a ate comprising a second or further moieties. Second and further moiety/moieties of the fusion polypeptide or ate in such a protein may suitably have a desired biological activity.

Thus, in a second aspect of the present disclosure, there is provided a fusion protein or a conjugate, comprising a first moiety consisting of a PD-L1 binding polypeptide according to the first aspect, and a second moiety ting of a polypeptide having a desired biological ty. In another embodiment, said fusion n or conjugate may additionally comprise further moieties, comprising desired biological activities that can be either the same as or different from the biological activity of the second moiety.

Non-limiting examples of a d biological activity se a therapeutic activity, a binding activity and an enzymatic activity. In one embodiment, the second moiety having a desired biological activity is a therapeutically active polypeptide. In one embodiment, said second moiety is an immune response modifying agent. In another embodiment, said second moiety is an anti-cancer agent.

In one embodiment of either the first or second aspect of the present disclosure, there is provided a PD-L1 binding polypeptide, fusion protein or ate which comprises an immune response ing agent. Non- limiting examples of additional immune response modifying agents include immunomodulating agents or other anti—inflammatory agents In one embodiment of either the first or second aspect of the present disclosure, there is provided a PD-L1 binding polypeptide, fusion protein or conjugate which comprises an anti-cancer agent. Non-limiting examples of anti-cancer agents include agents ed from the group consisting of auristatin, anthracycline, calicheamycin, combretastatin, doxorubicin, duocarmycin, the CC—1065 anti-tumor-antibiotic, ecteinsascidin, geldanamycin, maytansinoid, methotrexate, xin, taxol, ricin, bouganin, gelonin, pseudomonas exotoxin 38 (PE38), diphtheria toxin (OT), and their analogues, and derivates thereof and combinations thereof. A d person would appreciate that the non-limiting examples of anti-cancer agents include all le variants of said agents, for example the agent auristatin is ed to include for example auristatin E, auristatin F, auristatin PE, and tives thereof.

Non-limiting examples of therapeutically active polypeptides are biomolecules, such as molecules selected from the group consisting of human endogenous enzymes, hormones, growth s, chemokines, cytokines and lymphokines.

Non-limiting examples of binding activities are binding activities which increase the in vivo half-life of the fusion protein or conjugate, and binding activities which act to block a biological activity. One example of such a binding activity is a binding activity, which ses the in vivo half-life of a fusion protein or conjugate. In one embodiment of said fusion protein or conjugate, the in vivo half—life of said fusion protein or conjugate is longer than the in vivo half—life of the polypeptide having the desired biological activity per so. In one embodiment, said in vivo half—life is sed at least 10 times, such as at least 25 times, such as at least 50 times, such as at least 75 times, such as at least 100 times compared the in vivo ife of the fusion protein or conjugate per se.

The fusion n or conjugate may comprise at least one further moiety. In one ular embodiment, said target is albumin, binding to which increases the in vivo half-life of said fusion protein or conjugate. In one embodiment, said albumin binding activity is provided by an albumin binding domain (ABD) of streptococcal protein G or a derivative thereof. Thus, said fusion protein may for example comprise a PD-L1 binding polypeptide in ric or multimeric form (such as a homodimeric or heterodimeric form) as defined herein and an albumin binding domain of streptococcal protein G or a derivative f.

In another embodiment, said there is provided a fusion protein or a ate wherein said second moiety having a desired binding ty is a protein based on protein Z, derived from the B domain of protein A from lococcus aureus, which has a binding affinity for a target other than PD-L1.

For example, said fusion protein or conjugate, comprising at least one further moiety, may comprise [PD-L1 binding polypeptide] — [albumin binding moiety] — [moiety with affinity for selected target]. It is to be understood that the three moieties in this e may be arranged in any order from the N- to the inal of the ptide.

The d person is aware that the construction of a fusion protein often involves the use of linkers between the functional moieties to be fused, and there are different kinds of linkers with ent properties, such as flexible amino acid linkers, rigid amino acid linkers and cleavable amino acid linkers. s have been used to for example increase stability or improve folding of fusion proteins, to increase expression, improve biological activity, enable targeting and alter pharmacokinetics of fusion proteins. Thus, in one embodiment, the polypeptide according to any aspect disclosed herein further comprises at least one linker, such as at least one linker selected from flexible amino acid s, rigid amino acid linkers and cleavable amino acid linkers.

In one embodiment, said linker is arranged between said PD—L1 binding polypeptide and a further polypeptide domain, such as between a PD-L1 binding domain as disclosed herein and an antibody or n binding fragment thereof (as described in further detail below). Flexible linkers are often used in the art when the joined domains require a certain degree of movement or interaction, and may be ularly useful in some embodiments of the complex. Such linkers are generally composed of small, non-polar (for example G) or polar (for example 8 or T) amino acids. Some flexible linkers primarily consist of stretches of G and 8 residues, for example (GGGGS)p. ing the copy number “p” allows for optimization of linker in order to e appropriate separation between the functional moieties or to maintain necessary moiety interaction. Apart from G and S linkers, other flexible linkers are known in the art, such as G and S linkers containing additional amino acid residues, such as T and A, to maintain flexibility, as well as polar amino acid residues to improve solubility. Additional non-limiting examples of linkers include GGGGSLVPRGSGGGGS, (GS)3, (GS)4, (GS)8, GGSGGHMGSGG, GGSGGSGGSGG, GGSGG, GGSGGGGG, GGSEGGGSEGGG, AAGAATAA, GGGGG, GGSSG, GSGGGTGGGSG, GSGGGTGGGSG, GSGSGSGSGGSG, WO 72280 GSGGSGGSGGSGGS and GSGGSGSGGSGGSG, corresponding to SEQ ID NO:820-836, respectively, and GT. The skilled person is aware of other suitable linkers.

In one ment, said linker is a flexible linker comprising glycine (G), serine (S) and/or threonine (T) residues. In one embodiment, said linker has a general formula selected from p and (SnGm)p, wherein, independently, n = 1-7, m = 0-7, n + m S 8 and p = 1-7. In one ment, n = 1-5. In one embodiment, m = 0—5. In one embodiment, p = 1-5. In a more specific embodiment, n = 4, m = 1 and p = 1-4. In one embodiment, said linker is selected from the group ting of S4G, (S4G)3 and (S4G)4_ In one ment, said linker is selected from the group consisting of G4S and (G4S)3. In one particular embodiment, said linker is G48 and in another embodiment said linker is (G4S)3.

With regard to the description above of fusion proteins or conjugates incorporating a PD-L1 binding polypeptide according to the disclosure, it is to be noted that the designation of first, second and further es is made for clarity reasons to distinguish n PD-L1 binding polypeptide or polypeptides according to the invention on the one hand, and moieties exhibiting other functions on the other hand. These designations are not intended to refer to the actual order of the different domains in the polypeptide chain of the fusion protein or conjugate. Similarly, the designations first and second monomer units are made for clarity reasons to distinguish between said units. Thus, for example, said first moiety (or monomer unit) may without restriction appear at the N-terminal end, in the middle, or at the C-terminal end of the fusion protein or conjugate.

Recently, considerable progress has been made in the development of multispecific , such as antibodies with the y to bind to more than one antigen, for e through engineering of the complementarity determining regions (CDRs) to address two antigens in a single antibody combining site (Bostrom etal, 2009, Science 323(5921):1610-1614; Schaefer et al, 2011, Cancer Cell 20(4):472—486), via construction of heterodimeric antibodies using engineered Fc units (Carter, 2001, J Immunol Methods 248(1-2):7-15; Schaefer etal, 2011, Proc Natl Acad Sci USA ):11187- 11192) and via genetic fusion of auxiliary recognition units to N- or C-termini of light or heavy chains of full-length antibodies (Kanakaraj et al, 2012, MAbs 4(5):600-613; LaFleur et al, 2013, MAbs 5(2):208—218). Thus, it may be beneficial for a molecule incorporating an affinity for PD-L1 as disclosed herein to also exhibit affinity for another factor, such as a factor associated with cancer or an immune se associated factor.

Thus, in third aspect of the present disclosure, there is provided a x comprising at least one PD-L1 binding polypeptide and at least one antibody or an antigen binding fragment thereof, wherein the PD-L1 binding polypeptide is as described .

When used herein, the term “complex” is intended to refer to two or more associated polypeptide chains, at least one having an affinity for PD-L1 and at least one being an antibody or an antigen binding fragment thereof.

These polypeptide chains may each contain different n domains, and the resulting multiprotein complex can have multiple functions. ex” intends to refer to two or more polypeptides as defined herein, connected by covalent bonds, for example two or more ptide chains ted by covalent bonds through expression thereof as a inant fusion n, or associated by chemical conjugation.

As is well known, antibodies are immunoglobulin molecules capable of specific binding to a target (an antigen), such as a carbohydrate, polynucleotide, lipid, polypeptide or other, through at least one antigen recognition site d in the variable region of the immunoglobulin molecule.

As used herein, the term “antibody or an antigen binding fragment thereof’ encompasses not only full-length or intact polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof, such as Fab, Fab', F(ab’)2, Fab3, Fv and variants thereof, fusion proteins comprising one or more antibody portions, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear dies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin le that comprises an antigen recognition site of the ed specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies and covalently modified antibodies. Further examples of ed antibodies and antigen binding fragments thereof include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engager), TandAbs (tandem diabodies), DAFs (dual acting Fab), two—in-one antibodies, SMlPs (small r immunopharmaceuticals), bs (fynomers fused to antibodies), DVD-lgs (dual variable domain immunoglobulin), Con—bodies (peptide modified antibodies), duobodies and triomAbs. This listing of variants of antibodies and antigen binding nts thereof is not to be seen as limiting, and the skilled person is aware of other suitable variants.

A full-length antibody comprises two heavy chains and two light chains.

Each heavy chain ns a heavy chain variable region (VH) and first, second and third constant regions (CH1, CH2 and CH3). Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). ing on the amino acid sequence of the nt domain of its heavy , antibodies are assigned to different classes. There are six major classes of antibodies: lgA, lgD, lgE, lgG, lgM and ng, and several of these may be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2. The term “full-length antibody” as used herein refers to an antibody of any class, such as lgD, lgE, lgG, lgA, lgM or ng (or any sub—class thereof). The subunit structures and three-dimensional configurations of different classes of antibodies are well known.

An “antigen binding fragment” is a portion or region of an antibody le, or a derivative thereof, that retains all or a significant part of the n binding of the corresponding full-length antibody. An antigen g fragment may comprise the heavy chain variable region (VH), the light chain variable region (VL), or both. Each of the VH and VL typically contains three complementarity determining regions CDR1, CDR2 and CDR3. The three CDRs in VH or VL are flanked by framework regions (FR1, FR2, FR3 and FR4). As briefly listed above, es of antigen binding fragments include, but are not limited to: (1) a Fab fragment, which is a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a Fab’ fragment, which is a Fab fragment with the heavy chain hinge region, (3) a 2 fragment, which is a dimer of Fab’ fragments joined by the heavy chain hinge , for e linked by a disulfide bridge at the hinge region; (4) an Fc fragment; (5) an Fv fragment, which is the minimum antibody fragment having the VL and VH domains of a single arm of an antibody; (6) a single chain Fv (scFv) fragment, which is a single polypeptide chain in which the VH and VL domains of an scFv are linked by a peptide linker; (7) an (scFv)2, which comprises two VH domains and two VL domains, which are associated through the two VH domains via disulfide bridges and (8) domain antibodies, which can be antibody single variable domain (VH or VL) polypeptides that ically bind antigens.

Antigen g fragments can be prepared via e methods. For example, 2 nts can be produced by pepsin digestion of a full- length antibody molecule, and Fab nts can be generated by reducing the disulfide bridges of F(ab’)2 fragments. Alternatively, fragments can be prepared via recombinant technology by expressing the heavy and light chain fragments in suitable host cells (e.g., E. coli, yeast, mammalian, plant or insect cells) and having them led to form the desired antigen-binding fragments either in vivo or in vitro. A single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. For example, a flexible linker may be incorporated between the two variable regions. The skilled person is aware of methods for the preparation of both ength antibodies and antigen binding fragments thereof.

Thus, in one embodiment, this aspect of the disclosure es a complex as defined herein, wherein said at least one antibody or antigen binding fragment thereof is selected from the group consisting of full-length antibodies, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fc fragments, Fv fragments, single chain Fv fragments, (scFv)2 and domain antibodies. In one ment, said at least one antibody or antigen binding fragment thereof is selected from full—length antibodies, Fab fragments and scFv WO 72280 nts. In one particular embodiment, said at least one antibody or antigen binding fragment thereof is a ength antibody.

In one embodiment of said complex as d herein, the antibody or antigen binding fragment thereof is selected from the group consisting of monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and antigen-binding fragments f.

The term “monoclonal antibodies” as used herein refers to antibodies having monovalent ty, meaning that each antibody molecule in a sample of the monoclonal antibody binds to the same epitope on the antigen, whereas the term “polyclonal antibodies” as used herein refers to a collection of antibodies that react against a specific antigen, but in which collection there may be different dy molecules for example identifying different epitopes on the antigen. Polyclonal antibodies are typically ed by inoculation of a suitable mammal and are purified from the ’s serum. Monoclonal antibodies are made by identical immune cells that are clones of a unique parent cell (for example a hybridoma cell line). The term “human antibody” as used herein refers to antibodies having variable and constant regions corresponding substantially to, or derived from, antibodies obtained from human subjects. The term “chimeric antibodies” as used herein, refers to recombinant or genetically engineered antibodies, such as for example mouse monoclonal antibodies, which contain polypeptides or domains from a different species, for example human, introduced to reduce the antibodies’ immunogenicity. The term “humanized antibodies” refers to antibodies from non—human species whose protein sequences have been modified to se their similarity to antibody variants ed naturally in humans, in order to reduce immunogenicity.

The complex as described herein may for example be present in the form of a fusion n or a conjugate. Thus, said at least one PD-L1 binding ptide and said at least one dy, or antigen binding fragment thereof, may be coupled by means of chemical conjugation (using known organic chemistry methods) or by any other means, such as expression of the complex as a fusion protein orjoined in any other fashion, either directly or via a linker, for example an amino acid linker. The skilled person will appreciate that the above description of linker ces in relation to fusion polypeptides is equally relevant for the complex as disclosed herein.

Thus in one embodiment, there is provided a complex as defined herein, wherein said complex is a fusion protein or a conjugate. In one embodiment, said complex is a fusion protein. In another embodiment, said complex is a conjugate. In one embodiment of said x, said PD-L1 binding polypeptide is attached to the N—terminus or C—terminus of the heavy chain of said antibody or antigen binding fragment thereof. In another embodiment, said PD-L1 g ptide is ed to the N—terminus or C-terminus of the light chain of said antibody or antigen binding fragment thereof. In one ment, said PD-L1 binding polypeptide is attached to the N-terminus and/or C-terminus of the light chain and heavy chain of said antibody or antigen binding fragment thereof. For example, the PD-L1 binding polypeptide may be attached to only the N-terminus of the heavy chain(s), only the N-terminus of the light chain(s), only the C-terminus of the heavy chain(s), only the C-terminus of the light chain(s), both the N-terminus and the C-terminus of the heavy chain(s), both the inus and the C-terminus of the light chain(s), only the C-terminus of the light chain(s) and the N-terminus of the heavy chain(s), only the C-terminus of the heavy chain(s) and the N- terminus of the light chain(s), of said antibody or antigen binding fragment thereof.

In one embodiment there is provided a complex, wherein said PD-L1 binding polypeptide is attached either to the C-terminus or the inus of the heavy chain or the light chain of said antibody or antigen binding fragment thereof.

In one particular embodiment, there is provided a complex according to any preceding item, wherein said antibody or antigen g fragment thereof has affinity for an antigen, for example an antigen associated with an ious e, or an antigen ated with cancer. For example, said n may be PD—1 or CTLA—4.

In one embodiment there is provided a fusion protein, conjugate or complex as bed herein, wherein the said second or further moiety/moieties or dy or antigen binding fragment thereof is an inhibitor selected from the group consisting of inhibitors of: PD-1, CTLA-4, T-cell immunoglobulin and mucin containing protein—3 (TIM—3), galectin—9 (GAL—9), cyte activation gene-3 (LAG-3), PD-L2, B7 homolog 3 (B7-H3), B7 homolog 4 (B7-H4), V-domain lg suppressor of T-cell tion (VISTA), carcinoembryonic antigen-related cell on molecule 1 (CEACAMI), B and T lymphocyte attenuator (BTLA), colony stimulating factor 1 receptor (CSF1 R), herpes virus entry mediator (HVEM), killer immunoglobulin receptor (KIR), adenosine, adenosine A2a receptor (A2aR), CD200-CD200R and T cell lg and ITIM domain.

In one ment, said second moiety or antibody or antigen binding fragment thereof is an inhibitor of PD-1, such as an inhibitor selected from the group consisting of nivolumab, pidilizumab, BMS , MPDL328OA (Roche) and pembrolizumab. In a specific embodiment, the inhibitor is pembrolizumab.

In one embodiment, said second moiety or antibody or antigen g fragment thereof is an inhibitor of CTLA—4, such as an inhibitor ed from the group consisting of belatacept, abatacept, tremelimumab and ipilimumab.

In a specific embodiment, the tor is ipilimumab.

In one embodiment there is provide a fusion protein, conjugate or complex as described herein, wherein said second moiety or antibody or n binding fragment thereof is an agonist selected from the group consisting of agonists of CD134, CD40, 4-1 BB and glucocorticoid-induced TNFR—related protein (GITR).

The above aspects rmore encompass polypeptides in which the PD-L1 binding polypeptide according to the first aspect, the PD-L1 binding polypeptide as comprised in a fusion protein or conjugate according to the second aspect or in a complex according to the third aspect, further comprises a label, such as a label selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds, bioluminescent proteins, enzymes, radionuclides, ctive particles and pretargeting recognition tags. Such labels may for example be used for detection of the polypeptide. For example, in some embodiments, such labeled polypeptide may for example be used for labeling or targeting tumors which have a high expression of PD—L1.

Indirect labeling of a Z variant polypeptide was ly shown using pretargeting ition tags (Westerlund et al (2015), Bioconjugate Chem 4-1736). Similarly, the disclosure provides a PD-L1 binding polypeptide as described herein labeled with a pretargeting moiety, which may then be used for indirect labeling with a moiety complementary to the pretargeting moiety. When comprising a pretargeting moiety, a PD-L1 binding agent of the present disclosure is able to associate with a complementary pretargeting moiety, and such complementary pretargeting moiety may then comprise or be attached to a suitable radionuclide. The skilled person is aware of suitable radionuclides for therapeutic, stic and/or prognostic purposes. Such a radionuclide may be chelated to said complementary geting moiety via a chelating nment as generally described for the PD-L1 binding agent below.

In one embodiment, the complementary pair of pretargeting moieties comprise stept(avidin)/biotin, oligonucleotide/complementary oligonucleotide such as DNA/complementary DNA, RNA/complementary RNA, phosphorothioate nucleic acid/ complementary phosphorothioate nucleic acid and peptide nucleic acid (PNA)/complementary peptide nucleic acid (cPNA) and morpholinos/ complementary morpholinos. In one ular embodiment, said geting moiety is a PNA oligonucleotide, such as a 10mer PNA ce, such as a 15—mer PNA sequence.

In ments in which the polypeptide, fusion protein, conjugate or complex is labeled, directly or indirectly (e.g. via pretargeting as described above), with an imaging agent (e.g. radioactive agent), measuring the amount of labeled polypeptide present in a tumor may be done using imaging ent, such as through acquiring radioactivity counts or images of radiation density, or derivatives thereof such as ion concentration. Non— limiting examples of radionuclides, suitable for either direct labeling of the PD- L1 binding agent according to any aspect disclosed herein, or for indirect labeling by ng of a complementary pretargeting moiety, include 686a, 110mm, 18F, 45-“, 4480, 61CU, 66GB, 64CU, 5500’ 72AS, 86Y, BQZF, 124', 768F, 111m, gngc, 123', 131' and 67Ga- In one embodiment, the imaging equipment used in such measurements is positron emission tomography (PET) equipment, in which case the radionuclide is selected such that it is suitable for PET. The skilled person is aware of radionuclides suitable for use with PET. For example, a PET radionuclide is selected from the group consisting of 686a, 110mIn, 18F, 45Ti, 44Sc, 61Cu, 666a, 64Cu, 550o, 72As, 86v, 892r, 124i and 76Br.

In another embodiment, the imaging equipment used is single-photon emission computed tomography (SPECT) equipment, in which case the uclide is selected such that it is suitable for SPECT. The skilled person is aware of radionuclides suitable for use with SPECT. For example, a SPECT uclide is selected from the group consisting ofmln, 9ngc, 123l, 131| and 676a.

Thus, in one embodiment there is provided a PD-L1 binding ptide, fusion protein or x as bed herein, which comprises a direct or indirect radionuclide label, such as a radionuclide selected from the group consisting of 68Ga, “0min, 18F, 45Ti, 44Sc, 61Cu, 66Ga, 64Cu, 55Co, 72As, 86Y, 89Zr, 124l, 76Br, 111In, QQmTc, 123I, 131l and 676a, such as the group consisting of 686a, “0min, 18F, 45Ti, 44Sc, 61Cu, 66Ga, 64Cu, 55Co, 72As, 86v, 89Zr, 124| and 76Br, such as 18F.

In some embodiments, the d PD—L1 binding polypeptide is present as a moiety in a fusion protein, conjugate or complex also comprising a second moiety having a d biological ty. The label may in some instances be coupled only to the PD-L1 binding polypeptide, and in some instances both to the PD-L1 binding polypeptide and to the second moiety of the fusion protein or conjugate and/or the antibody or antigen binding fragment thereof the complex. Furthermore, it is also possible that the label may be coupled to a second moiety, or antibody or antigen binding fragment thereof only and not to the PD-L1 binding moiety. Hence, in yet another embodiment, there is provided a PD-L1 binding polypeptide comprising a second moiety, n said label is coupled to the second moiety only. In another embodiment, there is provided a complex as defined herein, n said label is coupled to the antibody or antigen binding fragment thereof only.

When reference is made to a labeled polypeptide, this should be understood as a reference to all aspects of polypeptides as described herein, including PD-L1 binding polypeptides, fusion proteins, conjugates and complexes comprising a PD-L1 binding polypeptide. Thus, a labeled polypeptide may contain only the PD-L1 binding polypeptide and e.g. a eutic radionuclide, which may be chelated or covalently coupled to the PD-L1 binding ptide, or contain the PD—L1 binding polypeptide, a therapeutic uclide and a second moiety such as a small molecule having a desired biological activity, for example a therapeutic efficacy. A labeled polypeptide may contain a PD-L1 binding polypeptide in heterodimeric form and e.g. a therapeutic uclide, which may be chelated or covalently coupled to the PD-L1 binding polypeptide, or contain the PD-L1 binding polypeptide in dimeric form, a eutic radionuclide and a second moiety such as a small molecule having a desired biological activity, for example a therapeutic efficacy. Also envisioned is a complex which contains a PD-L1 binding ptide as defined herein, an antibody or antigen binding fragment thereof and a e.g. a therapeutic radionuclide, which may be chelated or covalently coupled to the PD-L1 binding ptide or to the antibody or antigen binding fragment thereof.

The skilled person is aware of other possible variants. ln embodiments where the PD—L1 binding polypeptide, fusion n, ate or complex is radiolabeled, such a radiolabeled polypeptide may se a uclide. A majority of radionuclides have a ic nature and metals are typically incapable of forming stable covalent bonds with elements presented in proteins and peptides. For this reason, labeling of proteins and peptides with radioactive metals is performed with the use of chelators, i.e. multidentate ligands, which form non-covalent compounds, called chelates, with the metal ions. In an embodiment of the PD—L1 binding polypeptide, fusion protein, conjugate or complex, the incorporation of a radionuclide is enabled through the ion of a chelating environment, through which the radionuclide may be coordinated, chelated or xed to the polypeptide. One example of a chelator is the polyaminopolycarboxylate type of chelator. Two classes of such polyaminopolycarboxylate chelators can be distinguished: macrocyclic and acyclic chelators.

In one embodiment, the PD-L1 binding polypeptide, fusion n, conjugate or complex ses a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the PD-L1 binding polypeptide via a thiol group of a cysteine residue or an epsilon amine group of a lysine residue.

The most commonly used macrocyclic chelators for radioisotopes of indium, gallium, yttrium, bismuth, radioactinides and radiolanthanides are different derivatives of DOTA (1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10- tetraacetic acid). In one embodiment, a chelating environment of the PD-L1 g ptide, PD-L1 binding polypeptide in heterodimeric form, fusion protein, ate or complex is provided by DOTA or a derivative thereof.

More specifically, in one embodiment, a chelating polypeptide encompassed by the present disclosure is obtained by reacting the DOTA tive 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid—10- maleimidoethylacetamide (maleimidomonoamide-DOTA) with said polypeptide. In one embodiment, a chelating polypeptide encompassed by the present disclosure is obtained by reacting the DOTA derivative DOTAGA (2,2’,2”—(10-(2,6-dioxotetrahydro-2H-pyranyl)—1 ,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetic acid) with said ptide.

Additionally, triazacyclononane—1,4,7-triacetic acid (NOTA) and derivatives thereof may be used as chelators. Hence, in one embodiment, a chelating environment of the PD—L1 g polypeptide, PD-L1 binding polypeptide in heterodimeric form, fusion protein, conjugate or x is provided by NOTA or a derivative thereof. In one embodiment, a chelating ptide encompassed by the present disclosure is obtained by reacting the NOTA derivative NODAGA (2,2’-(7-(1-carboxy((2,5-dioxopyrrolidin yl)oxy)oxobutyl)—1,4,7—triazonane-1,4-diyl)diacetic acid) with said polypeptide. The most commonly used acyclic polyaminopolycarboxylate chelators are different tives of DTPA (diethylenetriamine-pentaacetic acid). Hence, polypeptides having a chelating nment provided by diethylenetriaminepentaacetic acid or derivatives f are also encompassed by the present disclosure.

In further aspects of the present disclosure, there is provided a polynucleotide encoding a PD-L1 binding polypeptide, fusion protein or complex as described herein; an expression vector comprising said polynucleotide; and a host cell comprising said expression vector.

Also encompassed by this disclosure is a method of producing PD-L1 binding polypeptide, fusion protein or complex as described above, comprising culturing said host cell under conditions permissive of expression of said polypeptide from its expression vector, and isolating the polypeptide.

The PD—L1 g polypeptide, fusion protein or complex of the present sure may atively be produced by non-biological peptide synthesis using amino acids and/or amino acid derivatives having protected ve side-chains, the non-biological peptide synthesis comprising - step—wise coupling of the amino acids and/or the amino acid derivatives to form a polypeptide, fusion protein or complex as bed herein having protected reactive hains, - removal of the protecting groups from the reactive side-chains of the polypeptide fusion protein or x, and - folding of the ptide in aqueous solution.

A complex as disclosed herein may also be produced by the conjugation of at least one PD-L1 binding polypeptide or fusion protein as described herein to at least one antibody or antigen binding fragment thereof.

The skilled person is aware of conjugation methods, such as conventional chemical conjugation methods, for example using charged succinimidyl esters or carbodiimides. it should be understood that the PD-L1 binding polypeptide according to the present disclosure may be useful as a therapeutic, stic and/or prognostic agent in its own right or as a means for targeting other therapeutic or diagnostic agents, with eg. direct or indirect effects on PD—L1 . A direct eutic effect may for example be accomplished by inhibiting PD-L1 signaling. An indirect therapeutic effect may for example be accomplished by pretargeting using PD-L1 binding polypeptides as described above.

Thus, in another aspect, there is provided a composition comprising a PD-L1 g polypeptide, fusion protein, conjugate or complex as described herein and at least one pharmaceutically acceptable excipient or carrier. In one embodiment, said ition further comprises at least one additional active agent, such as at least two additional active agents, such as at least three additional active agents. Non-limiting examples of additional active agents that may prove useful in such combination are immune response modifying agents and anti-cancer agents as described herein.

The small size and robustness of the PD—L1 binding polypeptides of the present disclosure confer l advantages over conventional monoclonal antibody based therapies. Such advantages include advantages in ation, modes of administration, such as alternative routes of administration, stration at higher doses than antibodies and absence of Fc-mediated side effects. The agents of the present disclosure are contemplated for oral, topical, intravenous, intraperitoneal, subcutaneous, ary, ermal, intramuscular, asal, buccal, sublingual or suppository administration, such as for topical administration. Also, many diseases and disorders, such as cancers and infectious disease, are associated with more than one factor. Thus, a complex as defined herein confers the advantage of targeting an additional antigen together with PD-L1.

In another aspect of the present sure, there is provided a PD-L1 binding polypeptide, fusion protein, conjugate, complex or ition as described herein for use as a medicament, a prognostic agent and/or a stic agent. In one embodiment, there is provided a PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use in the treatment, diagnosis or prognosis of a PD-L1 related disorder.

In one embodiment, said PD-L1 binding polypeptide, fusion protein, conjugate, x or composition is provided for use as a medicament. In a 2016/076040 more specific embodiment, there is provided a PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition as described herein, for use as a ment to modulate PD—L1 function in vivo. As used herein, the term “modulate” refers to changing the activity, such as rendering PD-L1 function hypomorph, partially inhibiting or fully inhibiting PD—L1 function.

In one embodiment, there is ed a PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use in the treatment of a PD-L1 related disorder.

In one ment, there is provided a PD-L1 binding polypeptide, fusion n, conjugate, complex or composition for use in the diagnosis of a PD-L1 related disorder.

In one embodiment, there is provided a PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use in the prognosis of a PD-L1 related disorder.

As used herein, the term “PD-L1 d disorder” refers to any disorder, disease or condition in which PD-L1 signalling plays a regulatory role. Examples of such PD-L1 related disorder include infectious diseases and cancers.

It is to be understood that said PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition may be used as the sole diagnostic or prognostic agent or as a companion diagnostic and/or stic agent.

In one embodiment, said PD-L1 related disorder is selected from the group consisting infectious diseases and cancers. Non-limiting es of ious diseases include chronic viral infection, for example selected from the group consisting of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV). The d person will appreciate that a cancer suitable for treatment, diagnosis and/or sis using PD-L1 binding polypeptide, fusion protein, conjugate, x or composition may be a solid tumor cancer or a non-solid tumor cancer characterized by over—expression of PD-L1. Non-limiting examples of such cancers include skin cancer; such as melanoma and nonmelanoma skin cancer (NMSC); lung cancers such as small cell lung cancer, non—small cell lung cancer (NSCLC); head and neck cancer, renal cell carcinoma (RCC), r cancer, breast cancer, colorectal cancer, gastric , ovarian cancer, pancreatic cancer, te cancer, glioma, glioblastoma, liver carcinoma, gallbladder cancer, thyroid cancer, bone cancer, cervical cancer, uterine cancer, vulval , endometrial cancer, testicular , kidney cancer, esophageal carcinoma, brain/CNS cancers, neuronal cancers, mesothelioma, as, small bowel adenocarcinoma and pediatric malignancies; leukaemia, acute myeloid leukaemia, acute lymphoblastic leukaemia and multiple myeloma.

In one particular embodiment, said cancer is selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC, r cancer, breast cancer, colorectal cancer, gastric cancer, n cancer, pancreatic cancer and prostate cancer, such as a cancer selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC and bladder cancer.

In one ment, it may be beneficial to administer a therapeutically effective amount of a PD—L1 binding polypeptide, fusion protein, conjugate, complex or composition as described herein together with at least one second drug substance, such as an anti—cancer agent or an immune response modifying agent.

In one embodiment, there is provided a PD-L1 binding polypeptide, fusion protein, conjugate, complex or ition for use in prognosis and/or diagnosis together with at least one cell proliferation marker. Non-limiting es of contemplated cell proliferation markers are those selected from the group consisting of Ki-67, AgNOR, choline, claspin, cyclin A, CYR61, Cdk1, histone H3, HsMCM2, IL-2, Ki-S‘I, Ki-SZ, LigI, MCM2, MCM6, MCM7, mitosin, p120, PCNA, PDPK, PLK, STK1, TK-1, topoisomerase II alpha and TPS.

In a related aspect, there is provided a method of treatment of a PD-L1 related disorder, sing stering to a subject in need thereof an ive amount of a PD—L1 binding polypeptide, fusion protein, conjugate, complex or composition as described . In a more specific embodiment of said method, the PD-L1 binding polypeptide, fusion protein, conjugate, complex or ition as described herein modulates PD-L1 function in vivo. The skilled person will appreciate that any description in relation to the use of PD-L1 g polypeptide, fusion protein, conjugate, complex or ition as bed herein for treatment of a disease or disorder is equally relevant for the related eutic method. For the sake of brevity, such description will not be repeated here.

In one particular embodiment, said method of treatment, particularly relevant for the treatment of PD-L1 related cancers, comprises the steps of - contacting the subject with a PD-L1 binding polypeptide, fusion protein, conjugate or complex comprising a pretargeting moiety as described herein, or with a composition comprising said PD-L1 binding polypeptide, fusion protein, conjugate or complex comprising a pretargeting moiety, and - contacting the subject with a mentary pretargeting moiety comprising a radionuclide.

In another aspect of the present disclosure, there is provided a method of detecting PD-L1, comprising providing a sample ted to contain PD- L1, contacting said sample with a PD-L1 binding polypeptide, fusion protein, ate, complex or composition as described herein, and detecting the binding of the PD-L1 binding polypeptide, fusion protein, ate, complex or composition to indicate the presence of PD-L1 in the sample.

In one embodiment, said method further comprises an intermediate washing step for removing non-bound polypeptide, fusion protein, conjugate, complex or ition, after ting the sample.

In another embodiment, said method is a diagnostic or prognostic method for determining the presence of PD-L1 in a subject, the method comprising the steps: a) contacting the subject, or a sample isolated from the t, with a PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition as described herein, and b) ing a value corresponding to the amount of the PD-L1 binding ptide, fusion protein, conjugate, complex or composition that has bound in said t or to said sample.

In one embodiment, said method further comprises an intermediate washing step for removing non-bound polypeptide, fusion protein, conjugate or composition, after contacting the subject or sample and before obtaining a value.

In one embodiment of this diagnostic or prognostic , said PD—L1 binding ptide, fusion protein, conjugate or complex comprises a pretargeting moiety as described herein, and the contacting step a) of the method further comprises contacting the subject with a complementary pretargeting moiety labeled with a detectable label, such as a radionuclide label.

In one embodiment, said method further comprises a step of comparing said value to a reference. Said reference may be by a numerical value, a threshold or a visual indicator, for e based on a color on.

The skilled person will appreciate that different ways of comparison to a reference are known in the art and may be suitable for use.

In one embodiment of such a method, said t is a mammalian subject, such as a human subject. In one embodiment, said method is performed in vivo. In another embodiment, said method is performed in vitro.

In one embodiment, the diagnostic or prognostic method is a method for medical imaging in vivo as discussed above. Such a method comprises the systemic stration of a PD-L1 binding entity as disclosed herein (i.e. the polypeptide per se, or the fusion protein, conjugate, complex or ition containing it) to a mammalian subject. The PD-L1 binding entity is directly or ctly labelled, with a label comprising a uclide suitable for medical imaging (see above for a list of contemplated radionuclides).

Furthermore, the method for medical imaging comprises obtaining one or more images of at least a part of the subject’s body using a medical imaging instrument, said image(s) indicating the presence of the radionuclide inside the body.

While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the ion. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to any particular embodiment contemplated, but that the invention will include all embodiments g within the scope of the appended claims.

Brief description of the figures Figure 1 is a listing of the amino acid sequences of es of PD-L1 binding polypeptides of the present disclosure (SEQ ID NO:1-814), as well as the heavy chain (HCLam; SEQ ID NO:815) and the light chain (LCLam; SEQ ID NO:816) of the PD—1 binding monoclonal dy Lam and the heavy chain (HCIpi; SEQ ID NO:817) and the light chain (LClpi; SEQ ID NO:818) of the CTLA—4 binding monoclonal antibody lpi. In the PD-L1 binding polypeptides of the present disclosure, the deduced PD-L1 binding motifs (BM) extend from residue 8 to residue 36 in each ce. The amino acid sequences of the 49 amino acid residues long polypeptides (BMod) ted to constitute the te three-helix bundle within each of these Z variants extend from residue 7 to residue 55.

Figure 2 shows binding of two first tion polypeptides to human PD-L1 analyzed by Biacore as described in Example 3. Z variants (A) 213091 (SEQ ID NO:776) and (B) 213156 (SEQ ID NO:781 were injected at concentrations of 50 nM (black), 5 nM (dark grey) and 0.5 nM (light grey) over a CM5 chip with immobilized hPD-L1.

Figure 3 shows e of SPR response against (A) hPD-L2, (B) hB7—H3 and (C) hB7-H4, here illustrated with the PD—L1 binding polypeptides 213091 (SEQ ID NO:776) and 213156 (SEQ ID ) ed at concentrations of 50 nM, 5 nM and 0.5 nM.

Figure 4 shows circular dichroism (CD) spectra of two first generation PD-L1 binding ptides. The CD a at ngs ranging from 250 to 195 nm collected at 20 °C before (broken line) and after (solid line) variable temperature measurement (VTM) are shown for (A) 215168—Cys (SEQ ID NO:809) and (B) 215169-Cys (SEQ ID NO:810).

Figure 5 shows binding of two second generation polypeptides to human PD-L1 analyzed by e as described in Example 7. Z variants (A) 217964 (SEQ ID N02) and (B) 218064 (SEQ ID NO:1) were injected at trations of 135 nM (black), 45 nM (dark grey) and 15 nM (light grey) over a CM5 chip with lized hPD—L1.

Figure 6 shows circular dichroism (CD) spectra of two second generation PD-L1 binding polypeptides. The CD spectra at wavelengths ranging from 250 to 195 nm ted at 20 °C before (broken line) and after (solid line) the variable temperature measurement (VTM) are shown for (A) 218064 (SEQ ID NO:1) and (B) 218090 (SEQ ID NO:17).

Figure 7 is a schematic representation of the design of complexes according to the disclosure, produced as described in Example 8. “Z” denotes the PD—L1 targeting Z variant 215170 (SEQ ID NO:814), which was genetically fused to the N—termini (7A and 7B) or the C-termini (7C and 7D) of the heavy (7A and 7D) or the light (7B and 7C) chains of the D-1 monoclonal antibody Lam or the anti-CTLA-4 monoclonal antibody lpi via a 15 residue )3-linker.

Figure 8 shows dual binding specificity of complexes analyzed in a Biacore capture assay as described in Example 8. (A) 215170-HCLam and (B) 215170-LCLam were injected for 5 min over chip surfaces immobilized with PD-1, followed by ion of PD-L1 at a concentration of 100 andfor 500 nM, respectively. (C) 215170-HClpi and (D) 215170—LClpi were injected for 5 min over chip surfaces immobilized with CTLA-4, followed by injection of PD-L1 at a concentration of 100 and 500 nM, respectively.

Figure 9 shows the result of inhibition of PD-L1 and CTLA-4 by Ipi— based complexes, ed in a mixed lymphocyte assay as described in Example 8. (A) Reduction in the number of MDA-MB231 cells and (B) increasing number of CD3+ T-cells with increasing concentrations of the Ipi- based complexes HCipi-Z15170, LCipi-Z15170, Z15170-Hcipi and 215170- LCipi.

Figure 10 shows PET maximum intensity projection (MIP) images of aft mice. (A) MIPs of mice with LOX tumor (left) and SUDHL6 tumor (right) afts, 30-90 min after administration of [18F]AlF—NOTA-Z15168.

(B) MIPs of mice with LOX tumor xenografts, 30-90 min after administration of [18F]AlF-NOTA-Z15168 (left) at baseline and (right) following pre-block with 400 pg NOTA—Z15168.

Figure 11 shows ex vivo biodistribution results for LOX and SUDHL6 mouse aft models, as analyzed directly after PET data acquisition. The results are displayed in units of (A) Standard Uptake Value (SUV) and (B) blood ratio. Error bars represent standard deviation.

Figure 12 shows the result of whole body scan of rhesus monkeys.

MIPs d over 90-180 min; colour inverted images) of rhesus monkeys administered with (A) [18F]AlF-NOTA-Z15168 and (B) [18F]AlF—NOTA-Z18609.

(C) Average tracer uptake over z 120-180 min in different organs displayed in the units of SUV. Error bars represent standard deviation.

Examples Summary The following Examples disclose the development of novel 2 variant molecules targeted to human programmed death-ligand 1 (PD-L1), also known as human B? homolog 1 (B7-H1) and cluster of differentiation 274 (CD274), based on phage display technology. The PD-L1 binding polypeptides described herein were sequenced, and their amino acid sequences are listed in Figure 1 with the sequence identifiers SEQ ID NO:1- 808. The Examples further describe the characterization of PD-L1 binding polypeptides and trate in vitro functionality of said polypeptides.

Example 1 Selection and screening of PD-L1 binding Z variants In this e, human PD-L1 (hPD-L1)was used as target in phage display ions using a phage library of Z variants. Selected clones were DNA sequenced, produced in E. coli periplasmic fractions and assayed against PD-L1 in ELISA (enzyme-linked sorbent assay).

Materials and methods ylation of target protein: hPD-L1 (human PD-L1 Fc a, R&D Systems, cat. no. 156-B7-100) was biotinylated using No-Weigh EZ-Link NHS-LC-Biotin (Thermo Scentific, cat. no. 21327) at a 10x molar excess, according to the manufacturer’s recommendations. The reaction was performed at room temperature (RT) for 40 min. Subsequent buffer exchange to PBS (10 mM phosphate, 137 mM NaCl, 2.68 mM KCI, pH 7.4) was performed using a Slide-a-Iyzer dialysis cassette (10000 MWCO, Thermo Scientific, cat. no. 66383) according to the manufacturer’s instructions.

Phage display selection of PD-L1 binding Z variants: A library of random ts of protein Z displayed on bacteriophage, constructed in phagemid pAY02592 essentially as described in Grénwall etal. (2007) J hnol, 128:162-183, was used to select PD-L1 binding Z variants. In this library, an albumin binding domain (ABD, GA3 of protein G from Streptococcus strain G148) is used as fusion partner to the Z variants. The library is denoted Zlib006Naive.ll and has a size of 1.5 x 1010 library members (Z variants). E. coli RRIAM15 cells (RUther et a/., (1982) Nucleic Acids Res :5765-5772) from a glycerol stock ning the phagemid library Zlib006Naive.ll were inoculated in 20 l of a defined proline free medium [3 g/l KH2PO4, 2 g/l K2HPO4, 0.02 g/l uracil, 6.7 g/l YNB (DifcoTM Yeast Nitrogen Base w/o amino acids, Becton Dickinson), 5.5 g/l glucose monohydrate, 0.3 g/l ine, 0.24 g/l L-arginine monohydrochloride, 0.11 g/l L-asparagine drate, 0.1 g/l L-cysteine, 0.3 g/l L-glutamic acid, 0.1 g/l L—glutamine, 0.2 g/l glycine, 0.05 g/l L-histidine, 0.1 g/l L-isoleucine, 0.1 g/l L-leucine, 0.25 g/l L-lysine monohydrochloride, 0.1 g/l L-methionine, 0.2 g/l L-phenylalanine, 0.3 g/l L—serine, 0.2 g/l L-threonine, 0.1 g/l L-tryptophane, 0.05 g/l L—tyrosine, 0.1 g/l ne], supplemented with 100 ug/ml ampicillin. The cultivations were grown at 37 °C in a fermenter (Belach nik, BR20). When the cells d an optical density at 600 nm (ODeoo) of 0.75, approximately 2.6 l of the cultivation was infected using a 10 x molar excess of M13K07 helper phage (New England Biolabs, cat. no. N0315S). The cells were incubated for min, whereupon the fermenter was filled up to 20 l with cultivation medium (2.5 g/l (NH4)2SO4; 5.0 g/l Yeast Extract (Merck 1037530500); 25 g/l Peptone lau 07-119); 2 g/l K2HPO4; 3 g/l ; 1.25 g/l Na3C5H507 - 2 H20; 0.1 ml/l Breox FMT30 aming agent) supplemented with 100 uM isopropyl-B-Dthiogalactopyranoside (IPTG) for induction of expression and with 50 ug/ml llin, 12.5 ug/ml carbenicillin, 25 ug/ml kanamycin, 35 ml/l of 1.217 M MgSO4 and 10 ml of a trace element solution [129 mM FeCI3; 36.7 mM ZnSO4; 10.6 mM CuSO4; 78.1 mM MnSO4; 94.1 mM 08012, dissolved in 1.2 M HCI]. A glucose-limited fed-batch cultivation was started where a 600 g/l glucose solution was fed to the reactor (15 g/h in the start, 40 g/h at the end of the tation after 17 h). pH was controlled at 7 through the automatic addition of 25 % NH4OH, air was supplemented (10 l/min), and the stirrer was set to keep the dissolved oxygen level above 30 %. The cells in the cultivation were removed by tangential flow filtration.

The phage particles were itated from the supernatant twice in PEG/NaCl (polyethylene glycol/sodium chloride), filtered and dissolved in PBS and glycerol as described in Grénwall et al., supra. Phage stocks were stored at -80 °C before use.

Selections against biotinylated hPD-L1 were performed in four cycles initially divided in two different tracks (1 and 2). As selection proceeded, the tracks were further divided according to target concentration and number and/or time of washes to finally end up in nine tracks in cycle 4. More precisely, the first track (1) was divided in the second to the fourth cycles, resulting in a total of two tracks (1-1 to 1-2) in cycle 2, four tracks (11 to 1- 2-2) in cycle 3 and six tracks (1—11 to 1-2—2—1) in cycle 4. The second track (2) was divided in the third to the fourth cycles, resulting in a total of two tracks (21 to 2-1—2) in cycle 3, three tracks —1 to 2—11) in cycle 4.

In track 1 with dants, Dynabeads® M-280 Streptavidin (SA-beads, ogen, cat. no. 11206D) were used to catch the hPD-L1:Z variant complexes. In track 2, Dynabeads® Protein A (SPA—beads, Invitrogen, cat. no. 10002D) were used instead to catch the hPD-L1:Z variant complexes by g to the Fc part of the hPD—L1 Fc chimeric protein.

Phage stock ation, selection procedure and amplification of phage between selection cycles were performed essentially as described for selection against another biotinylated target in W02009/077175 with the following exception: the selection buffer consisted of PBS supplemented with 10 % Fetal Bovine Serum (FBS, Gibco, cat. no. 10108-165) and 0.1% Tween20 (Acros Organics, cat. no. 233362500).

In order to reduce the amount of background binders, a pre—selection was performed in each cycle. In the pre-selection, the same types of beads were used as during the selection, i.e. ds in track 1 and SPA-beads in track 2. In all tracks of cycle 1-4, pre-selections were performed using SA— or SPA-beads coated with biotinylated human lgG-Fc (Jackson ImmunoResearch Lab, cat. no. 009-060—008). Furthermore in cycle 1, track 1, the pre—selection was performed using SA-beads coated with a mix of hPD-L2 (human PD-L2 Fc Chimera; R&D Systems, cat. no. 1224-PL-100), hB7-H3 (human B7-H3 Fc Chimera; R&D s, cat. no. 1027-B3—100), hB7-H4 (human B7-H4; R&D s, cat. no. 6576-B7-50), ylated previously as described for hPD—L1. In cycle 1, track 2, the pre-selection was performed using SPA-beads coated with a mix of biotinylated PD-L2 and biotinylated B7- H3. During pre-selection the phage stock was incubated with coated beads er end for 30-90 min at RT. A|| tubes and beads used in the pre- selections or selection were pre—blocked with PBS supplemented with 3% Bovine Serum Albumin (BSA, Sigma A3059-100G) and 0.1 % 0.

Selection was performed in solution at RT and the time for selection was approximately 120 min followed by wash with PBS + 0.1 % Tween20 and catch of target-phage complexes on ds or SPA-beads using 1 mg beads per 1.6 or 8.5 pg biotinylated hPD-L1, respectively.

For amplification of phage particles between selection cycle 1 and 2, E. coli strain ER2738 cells (Lucigen, Middleton, WI, USA) were used for infection and grown in medium supplemented with 20 ug/ml tetracycline. A 5 x excess of M13K07 helper phage compared to bacteria were allowed to infect log phase bacteria.

Table 2: Selection against biotinyiated hPD-L1 Fc a Cycle Selection Phage stock Proteins used Target Number on track from library in pre-selection conc. of of last or selection (nM) washes wash track (h) lgG—Fc, hPD—L2, 1 1 Zlib006Naive.ll_ . hB7-H3, hB7-H4 100 2 1 2 lgG-Fc, hPD-L2, Zlib006Naive.l|_ . hB7-H3 100 2 2 1-1 1 lgG-Fc 66 4 2 1-2 1 lgG—Fc 10 4 2 2-1 2 lgG-Fc 66 4 3 1—1-1 1-1 IgG-Fc 44 6 3 12 1—1 lgG-Fc 10 6 3 1—2-1 1-2 lgG-Fc 1 6 3 12 1—2 IgG-Fc 0.5 10 3 21 2-1 lgG-Fc 44 6 3 22 2—1 IgG-Fc 10 6 4 11-1 11 lgG-Fc 30 10 4 11—2 1—1-1 IgG—Fc 10 31 1 4 12-1 12 lgG-Fc 10 31 15 4 1—21 1-2—1 lgG-Fc 0.5 10 4 11-2 1—2-1 lgG-Fc 0.2 31 64 [4 °C] 4 1—21 1-2—2 lgG-Fc 0.05 31 15 4 1 2—1-1 IgG-Fc 30 10 4 21-2 21 lgG-Fc 10 31 1 4 22—1 2—1-2 IgG-Fc 10 31 15 The amplification of phage particles between selection cycles 2 and 4 was done by performing ion of bacteria in solution as follows. After infection of log phase E. coli ER2738 with phage particles, T88 supplemented with 2 % glucose, 10 ug/ml tetracycline and 100 ug/ml ampicillin was added, followed by incubation with rotation for 30 min at 37 °C.

Thereafter, the bacteria were infected with M13K07 helper phage in 5 x excess. The ed bacteria were pelleted by centrifugation, re-suspended in TSB—YE medium mented with 100 uM IPTG, 25 ug/ml kanamycin and 100 ug/ml ampicillin, and grown overnight at 30 °C. The overnight cultures were centrifuged, and phage particles in the supernatant were precipitated twice with PEG/NaCl buffer. Finally, the phage particles were re- suspended in selection buffer before entering the next selection cycle.

In the final selection cycle, log phase ia were infected with eluate and diluted before ing onto TBAB plates (30 g/l tryptose blood agar base, Oxoid, cat. no. B) supplemented with 0.2 g/l ampicillin in order to form single colonies to be used in ELISA screening.

An overview of the selection strategy, describing an increased stringency in subsequent cycles, using a lowered target concentration and an increased number of washes, is shown in Table 2. Washes were performed for 1 min, if nothing else is stated in Table 2, using PBST 0.1 % (PBS supplemented with 0.1 % Tween-20) and elution was carried out as described in WO2009/077175.

Production of Z variants for ELISA: Z variants were ed by inoculating single colonies from the selections into 1 ml TSB-YE medium supplemented with 100 ug/ml ampicillin and 1 mM IPTG in deep-well plates (Nunc, cat. no. 278752). The plates were incubated with rotation for 24 h at 37 °C. Cells were pelleted by centrifugation, re-suspended in 200 pl PBST 0.05 % and frozen at -80 °C to release the periplasmic fraction of the cells.

Frozen samples were thawed in a water bath and the freeze-thawing procedure was repeated eight times. 600 pl PBST 0.05 % was added to the thawed samples and cells were pelleted by centrifugation.

The final supernatant of the periplasmic extract contained the Z variants as fusions to ABD, expressed as LE-[Z#####]—VDYV- [ABD]-YVPG (Gronwall et al., supra). Z##### refers to dual, 58 amino acid residue Z variants.

ELISA screening of Z variants: The binding of Z variants to hPD-L1 was analyzed in ELISA assays. Half-area 96-well ELISA plates (Costar, cat. no. 3690) were coated at 4 °C overnight with 2 ug/ml of an BD goat antibody (produced in-house) diluted in g buffer (50 mM sodium ate, pH 9.6; Sigma, cat. no. . The antibody solution was poured off and the wells were washed in water and blocked with 100 pl of PBSC (PBS supplemented with 0.5 % casein; Sigma, cat. no. C8654) for 1 to 3 h at RT. The blocking solution was discarded and 50 pl periplasmic solutions, diluted 1:1 with PBST 0.05%, were added to the wells and incubated for 1.5 to 2.5 h at RT under slow agitation. As a blank control, PBST 0.05 % was added instead of a asmic sample. The supernatants were poured off and the wells were washed 4 times with PBST 0.05 %. Then, 50 pl of biotinylated hPD-L1 at a concentration of 0.32 nM in PBSC was added to each well. The plates were incubated for 1 h at RT followed by washes as described above. Streptavidin conjugated HRP (Thermo ific, cat. no.

N100) diluted 1:30,000 in PBSC, was added to the wells and the plates were incubated for approximately 1 h. After washing as described above, 50 pl lmmunoPure TMB ate (Thermo Scientific, cat. no. 34021 ) was added to the wells and the plates were treated according to the cturer’s recommendations. The absorbance at 450 nm was measured using a multi- well plate reader, 3 (Perkin Elmer). cing: In parallel with the ELISA screening, all clones were sequenced. PCR fragments were amplified from single colonies, sequenced and analyzed essentially as described in WO2009/077175.

EC50 analysis of Z variants: A selection of PD—L1 binding Z variants was subjected to an analysis of the response against a on series of biotinylated hPD-L1 ing the procedure described above. The Z variants were diluted 1:1 in PBST 0.05 %. Biotinylated hPD-L1 was added at a concentration of 40 nM and diluted stepwise 1:4 down to 32 pM. As a background control, all Z variants were also assayed with no target protein added. Periplasm samples containing the PD-L1 binding Z variant Z131 12 (SEQ |D.NO:777) were included on each plate and analyzed as positive control. Periplasm containing the ABD moiety only was used as negative control. In the same assay, the icity of the Z variants was tested by incubating periplasm samples with the four ent biotinylated control proteins hPD-L2, hB7-H3, hB7-H4 and IgGFc, respectively, added at a concentration of 8 nM. Data were analyzed using ad Prism 5 and non— linear regression, and EC50 values (the half maximal effective concentration) were calculated.

Results Phage display selection of PD—L1 binding Z variants: Individual clones were obtained after four cycles of phage display selections against biotinylated hPD-L1.

ELISA screening of Z variants: The clones obtained after four cycles of selection were ed in 96-well plates and screened for hPD-L1 binding activity in ELISA. Several unique Z variants were found to give a response of 0.3 AU or higher (corresponding to at least 3 X the blank control) t hPD- L1 at a concentration of 0.32 nM. The average response of the blank controls was 0.067 AU.

Seguencing: Sequencing was med for clones obtained after four cycles of selection. Each variant was given a unique identification number #####, and individual variants are referred to as Z#####. The amino acid sequences of the 58 amino acid residues long Z variants are listed in Figure 1 and in the sequence listing as SEQ ID NO:774-808. The deduced PD-L1 binding motifs extend from residue 8 to residue 36 in each ce. The amino acid sequences of the 49 amino acid residues long polypeptides predicted to constitute the complete three-helix bundle within each of these Z variants extend from e 7 to residue 55.

EC50 analysis of Z variants: A subset of Z variants having the highest ELISA values in the ELISA screening experiment described above was selected and subjected to a target titration in ELISA format. Periplasm samples were incubated with a serial dilution of biotinylated hPD—L1 . A asm sample containing 213112 (SEQ ID NO:777), confirmed to bind PD-L1 in the ELISA screen, was selected as a positive l and used to normalize different plates to each other. Obtained values were analyzed and their respective EC50 values were calculated (Table 3).

No icant g was detected to any of the included l proteins of the B7-family (hPD-L2, hB7-H3 and hB7-H4), nor to the control protein lgGFc (included here because Fc chimeric proteins were used in the ion and screening). These results indicate that the selected Z variants are specific to PD-L1.

Table 3: Calculated E050 values from ELISA titration analysis Z variant SEQ ID NO: EC50 (M) Z variant SEQ ID NO: EC50 (M) 213080 774 2.8 x 10'10 213184 783 2.2 x 10'10 213088 775 3.8 x 10‘10 213185 784 2.4 x 10'10 213091 778 2.2 x 10'10 213189 785 1.5 x 10‘10 213104 788 4.1 x10'10 213188 792 4.7 x10'10 213112 777 2.2 x10"10 213190 793 2.8 x10'10 213115 789 4.0 x10'10 213198 788 1.8 x10'10 213117 790 2.9 x10‘10 213210 794 3.5 x10'10 213134 791 4.5 x 10'10 213304 787 3.2 x 10-10 213147 779 2.8 x 10‘10 213388 795 4.8 x 10'10 213154 780 1.1 x 10'10 213447 798 2.9 x 10'10 213158 782 2.5 x10‘10 Example 2 Subcloning and tion of a subset of primary PD-L1 binding Z variants Materials and methods Subcloning of Z variants with a Hise-tag: The DNA of 14 PD—L1 binding Z variants, Z13080 (SEQ ID ), 213088 (SEQ ID NO:775), 213091 (SEQ ID NO:776), Z13112 (SEQ ID NO:777), Z13120 (SEQ ID NO:778), Z13147 (SEQ ID NO:779), Z13154 (SEQ ID NO:780), 213156 (SEQ ID NO:781), Z13158 (SEQ ID NO:782), 213164 (SEQ ID ), Z13165 (SEQ ID NO:784), 213169 (SEQ ID NO:785), Z13198 (SEQ ID ) and 213304 (SEQ ID NO:787) were amplified from the library vector pAY02592. A subcloning strategy for uction of monomeric Z variant molecules with N- terminal Hiss-tag was applied using standard molecular biology techniques (essentially as described in detail in WO2009/O77175 for Z variants binding another target). The Z gene fragments were subcloned into the sion vector pAY01448 resulting in the encoded ce MGSSHHHHHHLQ— [Z#####]—VD.

Subcloning of Z variants with a C-terminal Cys: Two Z variants, Z13091 (SEQ ID NO:776) and 213156 (SEQ ID NO:781)were mutated to WO 72280 start with the N-terminal amino acids AE instead of VD and further subcloned with the C-terminal addition of the amino acids VDC (incorporating a unique cysteine in the polypeptide) using rd molecular biology techniques. The resulting encoding sequences are referred to as Z15168—Cys (SEQ ID NO:809) and Z15169-Cys (SEQ ID NO:810), respectively.

Cultivation: E. coli T7E2 cells (GeneBridges) were transformed with plasmids containing the gene fragments of each respective PD-L1 binding Z variant and cultivated at 37 °C in 940 ml of TSB—YE medium supplemented with 50 ug/ml kanamycin. In order to induce n expression, IPTG was added to a final concentration of 0.2 mM at OD600 = 2 and the cultivation was incubated at 37 °C for r 5 h. The cells were harvested by centrifugation.

Purification of PD-L1 binding Z ts with a Hise-tag: Approximately 1-2 g of each cell pellet was resuspended in 30 ml of binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) supplemented with Benzonase® (Merck, cat. no. 1016540001) to a concentration of 15 U/ml.

After cell disruption by tion, cell debris was d by centrifugation and each supernatant was applied on a 1 ml His GraviTrap IMAC column (GE Healthcare, cat. no. 1199). Contaminants were removed by washing with wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mM imidazole, pH 7.4) and the PD—L1 binding Z variants were subsequently eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4). After the IMAC purification, the protein buffer was exchanged to PBS using PD-10 columns (GE Healthcare, cat. no. 1701).

Purification of PD-L1 binding Z variants with a C-terminal Cys: The respective cell pellet was resuspended in 20 mM Tris-HCI, pH 8 (10 ml buffer /g cell pellet) and lysed by heat ent in a water bath at 90 °C for 10 min, ed by cooling on ice to approximately 20 °C. Benzonase® was added (1 ul/g cell ) and each cell lysate was incubated at RT for 30 min, before cell debris was removed by centrifugation. For reduction of disulfides, dithiothreitol (DTT; Acros organics, cat. no. 165680250) was added to a final concentration of 20 mM followed by incubation at RT for 1 h. Purification was performed by anion ge followed by reverse phase chromatography (RPC). Buffer exchange to 20 mM HEPES, 1 mM EDTA, pH 7.2 was carried out using HiPrep 26/10 columns (GE care, cat. no. 1701).

Finally, each Z variant was purified on EndoTrap® red columns (Hyglos, cat. no. 321063) to ensure low xin t.

For each protein purified by any method described above, the concentration was determined by ing the absorbance at 280 nm, using a NanoDrop® ND-1000 spectrophotometer and the extinction coefficient of the n. The purity was analyzed by SDS-PAGE stained with Coomassie Blue and the identity of each purified Z variant was confirmed using HPLC- MS analysis (HPLC—MS 1100; Agilent Technologies).

Results Cultivation and purification: The PD-L1 binding Z variants with a Hiss- tag or a inal Cys were sed as soluble gene products in E. coli.

GE analysis of each final protein preparation showed that these predominantly contained the PD-L1 binding Z variant. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.

Example 3 Characterization of primary PD-L1 binding Z variants In this Example, a subset of Z variants was characterized in terms of stability and in vitro binding properties. The specificity and affinity for human PD-L1 of the Z variants were analyzed by SPR and binding to PD-L1 expressing cells was analyzed using Fluorescence Activated Cell Sorting . Furthermore, the ability of Z variants to block the binding of PD-L1 to its receptor PD1 was investigated using AlphaLlSA.

Materials and methods Biacore kinetic and specificity is: Kinetic constants (ka and kd) and affinities (KD) for hPD-L1 were determined for 14 Hiss-tagged Z variants using a Biacore 2000 instrument (GE Healthcare). Some of the Z variants WO 72280 were also tested for binding against the sequence-related proteins hPD-L2, hB7-H3, hB7-H4 and mPD-L1 (mouse PD-L1 Fc Chimera, R&D Systems, cat. no. 1019-B7). hPD-L1, hPD-L2, hB7-H3, hB7-H4 and mPD-L1 were immobilized in separate flow cells on the carboxylated dextran layer of different CM5 chip es (GE Healthcare, cat. no. BR100012). The lization was performed using amine coupling chemistry according to the manufacturer’s protocol and using HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005 % v/v Surfactant P20, GE Healthcare, cat. no.

BR100188). The ligand immobilization levels on the surfaces were 4 RU for , 537-742 RU for hPD-L2, 383 RU for hB7-H3, 538-659 RU for hB7-H4 and 482 RU for mPD-L1. One flow cell e on each chip was activated and deactivated for use as blank during analyte injections. In the kinetic experiment, HBS-EP was used as running buffer and the flow rate was 50 ul/min. The analytes, i.e. the Z variants, were each diluted in HBS-EP buffer within a concentration range of 1000 to 0.01 nM and injected for 5 min, followed by dissociation in running buffer for 15-25 min. After dissociation, the surfaces were rated with one or two injections of 0.1 % SDS. Kinetic constants were calculated from the sensorgrams using the Langmuir 1 :1 model of BiaEvaluation software 4.1 (GE Healthcare).

AlphaLlSA blocking assay: The potential of Z variants to t g of PD-L1 to PD—1 was analyzed by AlphaLlSA and recordings in an EnSpire multiplate reader 2300 (Perkin Elmer). hPD-1 (human PD-1 Fc-chimera; R&D Systems, cat. no. 1086-PD-050) was immobilized on AlphaLlSA Acceptor beads (Perkin Elmer, cat. no. 6772002) according to the manufacturer’s recommendations. Stepwise serial dilutions 1:3 of agged Z variants to final concentrations of 250 nM to 12 pM were made in a 384 plate (Perkin Elmer, cat. no. 6005350) and incubated for 1 h with 10 nM ylated hPD- L1 in AlphaLlSA buffer (Perkin Elmer, cat. no. AL000F). hPD—1-coated Acceptor beads were added to a final concentration of 10 ug/ml and incubated for 1 h. Finally, streptavidin coated Donor beads (Perkin Elmer, cat. no. 6772002) were added to a final concentration of 40 ug/ml and incubated for 30 min. All incubations were performed at RT in the dark. The plate was analyzed in the EnSpire instrument and the |C50 values were ated using GraphPad Prism 5.

Cell binding analysis by FACS: The potential on variants to bind PD- L1 expressing cells was igated using Fluorescence Activated Cell Sorting (FACS). THP-1 cells, cultivated in RPMI , cat. no. BE12-702F) containing 10 % FBS were stimulated with 10 ng/ml lFNg (R&D Systems, cat. no. 285-lF-100) overnight which results in up-regulation of PD-L1. 150,000 stimulated and unstimulated cells were pipetted per well of a v-bottomed 96 well plate (Nunc, cat. no. 277143) and the cells in the plate were subsequently pelleted at 400 g for 3 min at RT. The supernatants were removed and the cells were resuspended in 100 pl PBS plus 2.5 % FBS (staining buffer) containing 10 ug/ml of the different His-tagged Z variants. A mouse anti-PD-L1 antibody (R&D Systems, cat. no. MAB1561) at 1 ug/ml was used as a positive control. Cells incubated with buffer alone were used as negative controls. The cells were incubated for 1 h at 8 °C in the dark, washed twice with 100 pl staining buffer and resuspended in 100 pl of ng buffer containing a goat anti-Z antibody (produced in house) at a concentration of 5 ug/ml. Cells stained with the positive control were treated with buffer only. The cells were incubated for 1 h at 8 °C in the dark, washed twice with 100 pl staining buffer and resuspended in 100 pl of staining buffer containing an Alexa Fluor 647 chicken anti-goat lgG antibody (Life technologies, cat. no. A21469) or an Alexa Fluor 647 goat anti-mouse lgG antibody (Life technologies cat. no. A21236). The cells were once again incubated for 1 h at 8 °C in the dark, washed twice with 100 pl staining buffer and ended in 200 pl of staining buffer. Data from 10,000 cells were obtained using a FACS Calibur (Beckman Coulter) and the data was analyzed using Flowing software 2.5.0 (Turku University). Mean scence intensity (MFI) was used as a read out of g capacity.

Circular ism (CD) spectroscopy analysis: Two purified Z ts with a C-terminal cysteine, Z15168—Cys (SEQ ID NO:809) and Z15169-Cys (SEQ ID NO:810), were diluted to 0.5 mg/ml in 20 mM HEPES, 1 mM EDTA, pH 7.2. For each diluted Z variant, a CD spectrum at 250-195 nm was obtained at 20 °C. In addition, a le temperature measurement (VTM) was performed to determine the melting temperature (Tm). In the VTM, the absorbance was measured at 221 nm while the temperature was raised from to 90 °C, with a ature slope of 5 °C/min. A new CD spectrum was obtained at 20 °C after the heating procedure in order to study the refolding ability of the Z variants. The CD measurements were performed on a Jasco J- 810 opolarimeter (Jasco Scandinavia AB) using a cell with an optical path length of1 mm.

Results Biacore kinetic and specificity analysis: The interactions of 14 Hise— tagged PD-L1-binding Z variants with hPD-L1 were analyzed in a e instrument by injecting various concentrations of the Z variants over a surface containing immobilized hPD-L1. All tested Z variants showed binding to hPD- L1. A summary of the kinetic parameters (KD, ka and kd) for binding of the Z variants to hPD—L1 obtained using a 1:1 interaction model is given in Table 4.

Typical resulting curves, where responses from a blank surface were acted, are displayed in Figure 2 for two selected Z variants, 213091 (SEQ ID ) and 213156 (SEQ ID NO:781).

Table 4: Kinetic parameters for binding of Z ts to hPD-L1 z variant SEQ ID NO: hPD-L1 ka (1lMs) k... (1ls) KD (M) 213080 774 1.4 x106 9.2 x10"3 6.6 x10“9 213088 775 1.4 x106 3.7 x10'3 2.6 x10'9 213091 776 3.1 x106 1.4 x10‘3 4.6 x10'10 213112 777 6.1 x105 1.5 x10'3 2.5 x10'9 213120 778 2.0 x106 1.2 x10‘2 6.0 x10'9 213147 779 6 1.7x10'3 1.6x10'9 213154 780 2.3 x106 3.1x10'3 1.4 x10'9 213156 781 1.7x 108 2.5x 10-3 1.5x10'9 213158 782 6.7 x105 5.6 x10'3 8.3 x10'9 213164 783 7.9 x105 8.7 x10‘3 1.1x10'8 213165 784 2.0 x106 1.3 x10'3 6.4 x10'10 213169 785 2.4 x106 3.5 x10‘3 1.5 x10'9 213198 786 5.8 x106 5.6 x10'3 9.7 x10'10 213304 787 1.6 x106 6.4 x10‘3 4.1 x10'9 A subset of the Z variants was also tested for binding against four lized, sequence-related proteins: hPD-L2, hB7-H3, hB7-H4 and mPD- L1. No binding against hPD-L2, hB7-H3, hB7-H4 or mPD—L1 was detected at Z variant concentrations up to 50 nM. When injecting 1000 nM of a few selected Z ts (213088, 213091, 213112, , 213154, 213156, 213165, 213169, 213198) some response against B7—H4 was observed for 213156 and 213165. The result of the g specificity analysis is summarized in Table 5. Typical non-interacting traces from the SPR analysis against hPD-L2, hB7-H3 and hB7-H4 are shown in Figure 3.

Table 5: Binding speciﬁcity against , hPD-L2, hB7—H3 and hB7—H4 Z variant SEQ ID NO: mPD-L1 hPD-L2 hB7-H3 hB7-H4 213080 774 n.a. n.d. n.d. n.a. 213088 775 n.d. n.d. n.d. n.d. 213091 776 n.d. n.d. n.d. n.d. 213112 777 n.a. n.d. n.a. n.d. 213120 778 n.a. n.d. n.d. n.a. 213147 779 n.d. n.d. n.a. n.d. 213154 780 n.d. n.d. n.d. n.d. 213156 781 n.d. n.d. n.d. KD > 5 pM 213158 782 n.a. n.d. n.d. n.a. 213164 783 n.a. n.d. n.d. n.a. 213165 784 n.d. n.d. n.d. KD > 5 pM 213169 785 n.d. n.d. n.d. n.d. 213198 786 n.d. n.d. n.d. n.d. 213304 787 n.a. n.d. n.a. n.d. n.a. not d; n.d. no binding detected AlphaLlSA blocking assay: The y of 14 Hise-tagged 2 variants to inhibit hPD—L1 binding to hPD-1 was tested in an AlphaLlSA blocking assay.

Serial dilutions of the 2 variants were incubated with biotinylated hPD-L1 and the blocking ability of each respective variant was measured after addition of hPD-1 coated Acceptor beads and subsequently streptavidin coated Donor beads. Inhibition could be measured as a decrease in AlphaLlSA counts for positive Z variants. The calculated ICSO values for the 14 variants that were all shown to block PD-L1 binding to PD-1 in this assay are shown in Table 6.

Table 6: IC50 values forZ variants inhibiting binding of PD-L1 to PD-1 Z variant SEQ ID NO: |C50 213080 774 2.7 x10'9 213088 775 8.8 x10'10 213091 776 6.9 x10'9 213112 777 3.9 x10“9 213120 778 4.2 x10'9 213147 779 5.6 x10‘9 213154 780 '9 213156 781 1.4 x10'9 213158 782 2.4 x10'9 213164 783 2.3 x10'9 213165 784 1.3 x10'9 213169 785 2.2 x10'9 213198 786 1.3 x10'9 213304 787 4.2 x10'8 Table 7: Normalized MFI for binding of Z variants to THP-1 cells Z variant SEQ ID NO: MFI (normalized) Z13080 774 1.06 213088 775 1.00 Z13091 776 1.00 Z13112 777 0.76 Z13120 778 0.71 Z13147 779 0.56 Z13154 780 1.04 Z13156 781 1.09 Z13158 782 0.91 Z13164 783 0.81 213165 784 1.24 Z13169 785 0.91 213198 786 0.89 Z13304 787 0.75 anti-PD—L1 antibody - 0.40 2016/076040 Cell binding analysis by FACS: This experiment confirmed binding of the PD-L1 specific Z variants to PD-L1 expressing cells. THP-1 cells stimulated with lFNy overnight, which increases the PD-L1 sion, were stained with 10 pg/ml of each of the agged Z variants. The analyses were performed at two different occasions and the MFI values, normalized against 213091 included in both assays, are presented in Table 7.

CD analysis: The CD spectra determined for two selected PD-L1 binding Z variants with a C—terminal cysteine, Z15168—Cys (SEQ ID NO:809) and Z15169-Cys (SEQ ID NO:810) showed that both variants had an o-helical structure at 20 °C based on the typical minima at 208 and 222 nm. Reversible folding was seen for both Z variants when ying spectra measured before and after heating to 90 °C (Figure 4). The noisy signal observed in the far UV region is expected to result from buffer effects (HEPES, which was used as analysis buffer, absorbs strongly at 200 nM and below).The melting atures (Tm) were determined to 50 °C and 58 °C for Z15168—Cys and Z15169-Cys, respectively (Table 8).

Table 8: g temperatures (Tm) z variant SEQ ID NO: Tm (°C) Z15168—Cys 809 50 Z15169-Cys 810 58 Example 4 Design and construction of a maturated library of PD-L1 binding Z variants In this Example, a maturated library was ucted. The library was used for selections of PD-L1 binding Z variants. Selections from ted libraries may result in binders with increased affinity (Orlova et a/., (2006) Cancer Res 4339-48). In this study, randomized single stranded oligonucleotides are generated, using split-pool DNA synthesis enabling incorporation of defined codons in desired positions in the synthesis.

Materials and methods Library design: The library was based on the sequences of the PD-L1 binding Z variants identified and characterized as described in Example 1 and Example 3. In the new library, 13 variable positions in the Z molecule scaffold were biased towards certain amino acid residues, according to a strategy based on the Z variant sequences defined in SEQ ID NO:774-808. Two ucleotides, one forward and one reverse complementary, with complementary 3’-ends were generated using split-pool synthesis. The two ucleotides were ed and extended by PCR, using outer primers, to yield one gene fragment covering 147 bp corresponding to partially ized helix 1 and 2 of the amino acid sequence: 5’- AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA CGT AAC NNN GCG GCT NNN GAG ATC CTG NNN CTG CCT AAC CTC ACC NNN NNN CAA NNN TGG GCC TTC ATC TGG AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A -3’ (SEQ ID NO:819; randomized codons are illustrated as NNN) flanked by restriction sites Xhol and Sacl. The oligonucleotides were ordered from Ella Biotech GmbH (Martinsried Germany).

Table 9: Design of maturated librar Amino acid Randomization (amino acid No of tion position in the Z abbreviations) amino t molecule acids 50% D, 30% A, 20% E 3 1/2 (D), 3/10 (A), 1/5 (E) The theoretical distributions of amino acid residues in the new library including 7 variable positions (11, 14, 18, 24, 25, 27 and 35) in the Z molecule scaffold are given in Table 9. The resulting theoretical library size is 5.3 x 107 variants.

Library construction: The library was amplified using AmpliTaq Gold polymerase (Life Technologies, cat. no. 4311816) during 12 cycles of PCR and pooled products were ed with QlAquick PCR Purification Kit (QIAGEN, cat. no. 28106) ing to the supplier’s recommendations. The purified pool of randomized library fragments was digested with restriction s Xhol and Sacl-HF (New England Biolabs, cat. no. R0146L, and cat. no. R3156M, respectively) and concentrated using a PCR Purification Kit.

Subsequently, the product was run on a preparative 2.5 % agarose (NuSieve GTG ® Agarose, Lonza, cat. no. 50080) gel electrophoresis and purified using QIAGEN Gel Extraction Kit (QIAGEN, cat. no. 28706) according to the er’s recommendations.

The phagemid vector pAY02592 (essentially as pAffi1 described in Gronwall et a/., supra) was restricted with the same enzymes and ed using phenol/chloroform extraction and ethanol precipitation. The restricted fragments and the restricted vector were ligated in a molar ratio of 5:1 with T4 DNA ligase (Thermo Scientific, cat. no. EL0011) for 2 h at RT, ed by overnight incubation at 4 °C. The ligated DNA was recovered by phenol/chloroform tion and ethanol itation, followed by dissolution in 10 mM Tris-HCI, pH 8.5. Thus, the resulting library in vector pAY02592 d Z variants each fused to an albumin binding domain (ABD) derived from streptococcal protein G.

The ligation ons (approximately 160 ng DNA/transformation) were electroporated into electrocompetent E. coli ER2738 cells (Lucigen, Middleton, WI, USA, 50 pl). Immediately after electroporation, approximately 1 ml of recovery medium (supplied with E. coli ER2738 cells) was added. The ormed cells were incubated at 37 °C for 60 min. Samples were taken for titration and for determination of the number of transformants. The cells were thereafter pooled and cultivated overnight at 37 °C in 1 l of TSB-YE medium, supplemented with 2 % e, 10 ug/ml tetracycline and 100 ug/ml ampicillin. The cells were pelleted for 15 min at 4,000 g and resuspended in a PBS/glycerol solution (approximately 40 % glycerol). The cells were aliquoted and stored at —80 °C. Clones from the library of Z variants were sequenced in order to verify the content and to evaluate the outcome of the constructed library vis-a-vis the library design. Sequencing was performed as described in Example 1 and the amino acid bution was verified.

Preparation of phage stock: Cells from a glycerol stock containing the phagemid y were inoculated in 3.5 l of TSB-YE supplemented with 1 g/l glucose, 100 mg/l ampicillin and 10 mg/l tetracycline. The cells were cultivated at 37 °C with orbital shaking (100 RPM). When the cells d an optical density at 600 nm (OD600) of 0.59, approximately 620 ml of the cultivation was infected using a 5 x molar excess of M13K07 helper phage.

The cells were incubated for 30 min, whereupon the cells were pelleted by centrifugation at 3,000 g and resuspended in 3 | fresh TSB-YE supplemented with 100 mg/l ampicillin, 25 mg/l kanamycin, and 0.1 mM IPTG. The cultivation was split into 6 x 5 l shaker flasks at and incubated at 30 °C with orbital g and after ~18 h the cells were pelleted by centrifugation at 4,700 g. The phage particles were precipitated from the supernatant twice in PEG/NaCl, filtered and ved in PBS and ol as described in Example 1. Phage stocks were stored at -80 °C until use in selection.

Results Library construction: The new library was designed based on a set of PD-L1 binding Z variants with verified binding properties (Example 1 and 3).

The theoretical size of the designed library was 5.3 x 107 Z variants. The actual size of the library, determined by titration after transformation to E. coli.

ER2738 cells, was 2.8 x 109 transformants.

The library quality was tested by sequencing of 116 transformants and by comparing their actual sequences with the theoretical design. The contents of the actual library ed to the designed library were shown to be actory. A maturated library of potential s to PD-L1 was thus successfully constructed.

Selection, screening and characterization of Z variants from a maturated library als and methods Phage display selection of PD-L1 binding Z variants: The target n PD-L1 was biotinylated as described in Example 1. Phage display selections, using the new library of Z variant molecules constructed as described in Example 4, were performed in four cycles against hPD-L1 ially as described in e 1, with the following exceptions: Exception 1: SA-beads were used to catch the PD-L1:Z variant complexes in all selection tracks.

Exception 2: pre-selection was performed against SA—beads coated with biotinylated human lgG-Fc only before cycle 1 and 2. Furthermore in cycle 1, another pre-selection was performed against SA—beads coated with a mix of PD-L2, B7-H3, and B7-H4 as previously described in Example 1. Exception 3: selections t biotinylated human PD-L1 was performed in four cycles initially divided in two different tracks (1 and 2). As selection proceeded, the tracks were further divided according to target concentration and number and/or time of washes to finally end up in 11 tracks in cycle four. More precisely, the first track (1) was divided in the second to the fourth cycles, resulting in a total of2 tracks (1-1 to 1-2) in cycle 2, fourtracks (1-1—1 to 1—2- 2) in cycle 3 and seven tracks (11-1 to 12-2) in cycle 4. The second track (2) was d in the second to the fourth cycles, resulting in a total of 2 tracks (2-1 to 2-2) in cycle 2, four tracks (21 to 22) in cycle 3 and four tracks (21—1 to 2—21) in cycle four. ion 4: during the 19 h washing step in selection cycle 12-3 a 20-fold molecular excess of non-biotinylated hPD-L1 was added to the wash buffer. An overview of the selection strategy, describing an increased stringency in subsequent cycles ed by using a lowered target concentration and an increased number of washes, is shown in Table 10.

Table 10. Selection against biotiny/ated hPD-L1 Fc using a ted library Cycle ion Phage Proteins Target Number Duration Addition track stock from used in conc. of of last to last library or pre- (nM) washes wash (h) wash selection selection buffer track ZIib006PD- | G-Fc, PD-L2, 1 1 50 2 L1 .l Bg7-H3, B7-H4 Zlib006PD- | -F 1 2 gﬁHsc, B7-H4, PD-L2, 25 2 L1 .l 2 1-1 1 lgG-Fc 25 8 2 1-2 1 lgG-Fc 10 12 2 2-1 2 lgG-Fc 2.5 12 2 2-2 2 lgG-Fc 0.5 12 3 11 1-1 no pre-selection 5 20 3 12 1-1 no pre-selection 5 20 3 1-2—1 1-2 no pre-selection 2.5 20 3 12 1-2 no pre-selection 1 20 3 21 2-1 no lection 0.5 20 3 22 2-1 no pre-selection 0.1 20 3 21 2-2 no pre-selection 0.05 20 3 22 2-2 no pre-selection 0.005 20 4 11—1 1—1-1 no pre-selection 2.5 20 4 1 12 no pre-selection 2.5 20 4 12—2 12 no pre-selection 2.5 20 4 12-3 12 no pre-selectlon 2.5 20 22351—1 4 11-1 11 no pre-selection 0.1 20 4 12-1 22 no pre-selection 0.5 20 4 12—2 1—2-2 no pre-selection 0.5 20 4 2-1—1-1 21 no pre-selection 0.05 20 4 1 22 no pre-selection 0.1 30 4 21-1 21 no pre-selection 0.01 30 4 22-1 22 no pre-selection 0.05 20 Production of Z variants for ELISA: The Z variants were produced by inoculating single colonies from the selections into 1.2 ml TSB-YE medium supplemented with 100 pgiml ampicillin and 1 mM IPTG in deep-well plates (Nunc, cat. no. 278752). The plates were incubated with rotation for 24 h at 37 °C. Cells were pelleted by centrifugation at 3300 g and re-suspended in 150 pl PBST 0.05 % and frozen at -80 °C to release the periplasmic fraction of the cells. Frozen samples were thawed in a water bath and the freeze- thawing procedure was repeated eight times before the periplasmic fraction was isolated in deep-well plates (Axygen, cat. no. 391101) by filtration using filter plates (EMD Millipore, cat. no. MSNANLY50). The final supernatant of the periplasmic extract contained the Z variants as fusions to ABD, expressed as AQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (Grc'jnwall et al., supra). Z##### refers to individual, 58 amino acid residue Z variants.

ELISA screening of Z variants: The binding of Z variants to human PD- L1 was analyzed in ELISA assays as described previously in e 1 with the following exceptions. Exception 1: The periplasmic fraction was d 1:8 with PBST 0.05% before added to the wells and incubated for 1.7 h.

Exception 2: d of a blank control a negative l of a periplasmic fraction containing the fusion protein ABD with no Z—fusion partner was used.

Exception 3: periplasm s containing the primary PD-L1 binding Z variant 213091 (SEQ ID.NO:776) was included in ates on each plate and analyzed as ve controls. Exception 4: 50 ul of biotinylated hPD-L1 at a concentration of 40 pM in PBSC was added to each well and the plates were incubated for 1.8 h at RT.

Seguencing: In parallel with the ELISA screening, all clones were sequenced as described in Example 1.

ELISA EC50 analysis: A selection of PD—L1 binding Z variants was subjected to an is of the response against a dilution series of biotinylated human PD-L1 as bed in Example 1 with the following exceptions. Exception 1: the Z variants were diluted 1:8 in PBST 0.05 % before added to the wells. Exception 2: ylated human PD-L1 was added at a concentration of 15 nM and diluted stepwise 1:3 down to 0.25 pM.

Exception 3: a asm sample containing the primary PD—L1 binding Z variant 213091 (SEQ ID.NO:776) was included for comparison and analyzed together with the maturated Z variants.

Results Phage display selection of PD-L1 binding Z ts: Individual clones were obtained after four cycles of phage display selection t biotinylated hPD-L1.

ELISA screening of Z variants: The clones obtained after four cycles of selection were produced in 96-well plates and screened for hPD-L1 g activity in ELISA. A majority of the unique Z variants were found to give a higher response than average response of the positive control Z13091 ge 0.264 AU) against hPD-L1 at a concentration of 40 pM. The average response of the negative controls was 0.051 AU.

Seguencing: Sequencing was performed for clones obtained after four cycles of selection. Each variant was given a unique identification number, Z#####, as described in e 1. The amino acid sequences of the 58 amino acid residues long Z ts are listed in Figure 1 and in the sequence listing as SEQ ID NO:1-773. The deduced PD-L1 binding motifs extend from position 8 to on 36 in each sequence. The amino acid sequences of the 49 amino acid residues long polypeptides predicted to constitute the complete three-helix bundle within each of these Z variants extend from residue 7 to residue 55.

EC50 analysis of Z ts: A subset of Z variants having the highest ELISA values in the ELISA screening experiment described above was selected and subjected to a target titration in ELISA format. Periplasm samples were ted with a serial dilution of biotinylated hPD-L1. A periplasm sample containing Z13091 (SEQ ID NO:776), the isolated primary Z variant that showed the highest binding affinity to hPD-L1, was included as a positive control. Obtained values were analyzed and their respective EC50 values were calculated using GraphPad Prism 5 (Table 11). All maturated Z variants showed lower EC50 values than the top primary Z variant Z13091.

Table 11: ated EC50 values of Z-ABD variants from maturation Z variant SEQ EC50 Z variant SEQ EC50 Z variant SEQ EC50 ID (M) ID (M) ID (M) 217746 218054 218135 217748 218060 218137 217756 218064 218138 217758 218065 218140 217772 218066 218143 217825 218069 218144 217843 218070 218148 217911 218074 218149 217928 218078 218150 217950 218090 218152 217964 218092 218153 217968 218095 218156 217972 218096 218158 217975 218099 218164 217978 218101 218167 217990 218104 218172 217995 218106 218174 217997 218108 218176 217999 218110 218179 218000 218111 218185 218005 218115 218220 218008 218116 218228 218021 218117 218233 218022 218118 218240 218027 218119 218243 218038 41 7.8 x10' 218124 82 8.4 x10' 218252 90 8.5 x10' 218037 42 7.7 x10'11 218128 83 7.7 x10'11 218288 91 8.9 x1011 218038 43 8.9 x10"11 218129 18 8.2 x10"11 218353 15 7.7 x10'ﬁ 218039 20 8.9 x10'11 218130 84 7.4 x10'11 218374 92 1.0 x1010 218048 4 8.5 x10‘11 218131 85 8.7 x10'11 218377 93 7.8 x10'11 218052 14 7.1 x1011 218133 88 7.7x10'11 218418 24 8.0x 10'11 213091 778 1.2 x1010 Example 6 ning and production of a subset of maturated PD-L1 binding Z ts Materials and methods Subcloning of 2 variants with a Hise-tag: The DNA of 24 maturated PD- L1 binding Z variants, (217746 (SEQ ID NO:8), 217748 (SEQ ID , 217756 (SEQ ID NO:7), 217825 (SEQ ID NO:5), 217911 (SEQ ID NO:3), 217964 (SEQ ID NO:2), 217972 (SEQ ID NO:19), 217978 (SEQ ID NO:13), 218022 (SEQ ID NO:9), 218039 (SEQ ID NO:20), 218048 (SEQ ID NO:4), 218052 (SEQ ID NO:14), 218054 (SEQ ID NO:22), 218064 (SEQ ID N011), 218066 (SEQ ID NO:12), 218070 (SEQ ID NO:10), 218074 (SEQ ID NO:6), 218090 (SEQ ID NO:17), 218101 (SEQ ID NO:23), 218129 (SEQ ID NO:16), 218149 (SEQ ID NO:18), 218233 (SEQ ID , 218353 (SEQ ID NO:15) and 218418 (SEQ ID NO:24)) were amplified from the library vector pAY02592 and subcloned with a Hise-tag as described in e 2 above. ning of 2 variants with a C-terminal Cys: Three 2 variants, 218064 (SEQ ID N021), 217964 (SEQ ID N02) and 218090 (SEQ ID NO:17) were mutated to start with the N-terminal amino acids AE instead of VD and further subcloned with the C-terminal addition of the amino acids VDC (incorporating a unique cysteine in the polypeptide) using standard molecular biology techniques. The resulting sequences are referred to as 218608-Cys (SEQ ID NO:811), 218609-Cys (SEQ ID NO:812) and -Cys (SEQ ID ), respectively.

Cultivation: Generally, E. coliT7E2 cells (GeneBridges) were transformed with plasmids containing the gene fragments of each respective PD-L1 binding 2 variant and cultivated at 37 °C in approximately 940 ml of TSB-YE medium supplemented with 50 pg/ml kanamycin. In order to induce protein expression, IPTG was added to a final concentration of 0.2 mM at ODGOO = 2 and the cultivation was incubated at 37 °C for another 5 h. The cells were harvested by centrifugation. Specifically, 218608—Cys and 218609- Cys were fed-batch cultivated at 37 °C in approximately 700 ml of defined l medium supplemented with 50 ug/ml kanamycin. In order to induce protein sion, IPTG was added to a final concentration of 0.5 mM at ODeoo =75 and the cultivation was incubated for another 7 h. The cells were harvested by centrifugation.

Purification of PD—L1 binding Z variants with a Hise—tag: IMAC purifications, buffer exchange to PBS and concentration inations were performed essentially as described in Example 2.

Purification of PD-L1 binding Z variants with a C-terminal Cys: The respective cell pellet was re-suspended in 20 mM Tris-HCI, 0.5 mM EDTA, 0.1 % Tween 80, pH 7.5 (10 ml buffer / g cell pellet) and lysed by heat treatment in a water bath at 80 °C for 10 min, followed by cooling on ice to approximately 20 °C. Benzonase® was added (1 pl/g cell pellet) and each cell lysate was incubated at RT for 30 min, before cell debris was removed by centrifugation. For reduction of disulfides, dithiothreitol (DTT; Acros Organics, cat. no. 250) was added to a final concentration of 10 mM followed by incubation at RT for 20 min. Thereafter, the lysate was filtered through a 0.45 pm syringe filter pore). Purification was performed by anion exchange followed by reverse phase chromatography (RPC). Buffer ge to 20 mM HEPES, 1 mM EDTA, pH 7.2 was carried out using Sephadex G—25 medium (GE Healthcare) packed in an XK-50 column.

For any protein purified by either method described above, the concentration was ined by measuring the absorbance at 280 nm, using a NanoDrop® ND-1000 spectrophotometer and the extinction coefficient of the protein. The purity was analyzed by SDS-PAGE d with Coomassie Blue, and the identity of each purified Z t was confirmed using HPLC- MS analysis (HPLC—MS 1100; Agilent logies).

Results Cultivation and purification: The PD-L1 binding Z variants were expressed as soluble gene ts in E. coli. The amount of purified protein from imately 2.0-2.4 g bacterial pellet was determined spectrophotometrically by measuring the absorbance at 280 nm and ranged from imately 18 mg to 29 mg for the different Hise-tagged PD-L1 binding Z variants. SDS—PAGE analysis of each final protein preparation showed that these predominantly contained the PD-L1 binding Z variant. The correct identity and molecular weight of each Z variant were confirmed by HPLC-MS analysis.

Example 7 Additional characterization of a subset of primary PD-L1 binding Z ts In this e, a subset of Z variants was characterized in terms of stability and various binding properties. The specificity and affinity for PD-L1 of the Z variants were analyzed by Biacore and the ability of Z variants to block the binding of PD-L1 to its receptor PD-1 was investigated using AlphaLlSA.

Materials and methods Biacore kinetic and specificity analysis: Kinetic constants (ka and kd) and affinities (KD) for human PD—L1 and rhesus monkey PD-L1 (RhPD—L1; rhesus PD-L1/Fc Chimera, Sino Biological |nc., cat. no. 90251-C02H) were determined for 24 matured Hiss-tagged Z variants (specified in Example 6).

The Z variants were also tested for binding against the sequence-related proteins , hB7-H3 and hB7-H4. The Biacore analyses were performed essentially as described in Example 3, however a flow rate of 30 ul/min was used. The ligand immobilization levels on the surfaces were 1030 RU for hPD-L1, 1060 RU for RhPD-L1, 1070 RU for hPD-L2, 1090 RU for hBT-H3, and 770 RU for hB7-H4. In a first binding kinetic analysis, the 24 Z variants were ed at concentrations of 5 and 50 nM over chips immobilized with hPD-L1 and 1, respectively. The 12 maturated PD-L1 binding Z variants that showed the t affinity to hPD-L1 in the first ment were analyzed in more detail and injected at concentrations of 135, 45, 15, 5 and 1.67 nM over lized hPD-L1 and RhPD-L1. In the icity test, i.e. the binding analysis against hPD-L2, hBY—H3 and hBT—H4, the 24 Z ts were injected at a concentration of 500 nM.

AlphaLlSA blocking assay: The potential of the Z variants to inhibit binding of PD-L1 to its natural ligand PD-1 was analyzed in an AlphaLlSA assay as described in Example 3 with the following exceptions: Exception 1: stepwise serial ons 1:3 of Hise-tagged Z variants to final concentrations of 250 nM to 4 pM were made in a 384SW plate (Perkin Elmer, cat. no. 6008350) and incubated for 45 min with 8 nM biotinylated hPD-L1 (R&D Systems) in lSA buffer (Perkin Elmer, cat. no. AL000F). Exception 2: hPDcoated Acceptor beads were added to a final concentration of 10 ug/ml and incubated for 50 min.

Circular dichroism (CD) spectroscopy analysis: A subset of the purified Hiss-tagged Z variants were ed by CD spectroscopy as described in Example 3, but with the exceptions that the analysis buffer was PBS and that the temperature was raised to 80 °C in the VTM. s Biacore c and icity analysis: The interactions of 24 maturated Hise-tagged Z variants with human and rhesus monkey PD-L1, were analyzed in a e instrument by injecting various concentrations of the Z variants over surfaces containing immobilized hPD-L1 and RhPD-L1, tively. A first kinetic is was performed in order to rank the Z variants in terms of their affinity for hPD—L1 and RhPD-L1, as well as to e their binding kinetics with the primary PD-L1 binding Z variant 213091. A summary of the approximate affinity constants from the ranking experiment, which were obtained by using a 1:1 interaction model, is given in Table 12.

The 12 maturated Z variants that showed the highest binding affinity to hPD-L1 were further analyzed and the more precisely determined kinetic parameters for these 12 Z variants are given in Table 13. l resulting curves, where responses from a blank surface were subtracted, are displayed for two selected variants in Figure 5.

Table 12: Approximate afﬁnity constants for binding of Z variants to hPD-L1 and RhPD-L1 z variant SEQ ID NO hPD-L1 1 of Z variant KD (M) KD (M) 213091 776 7.4 x10'10 5.2 x10‘9 217746 8 3.8 x10'10 5.2 x10'10 217748 11 4.5 x10'10 7.4 x1010 217756 7 2.4 x10'10 2.1 x10'9 217825 5 2.5 x10'10 1.2 x10‘9 217911 3 3.6 x10'10 2.2 x10'9 217964 2 3.0 x10'10 2.0 x10'9 217972 19 5.4 x10'10 2.7 x10'9 217978 13 4.5 x10'10 1.0 x10'9 218022 9 3.4 x10'10 1.4 x10‘9 218039 20 6.3 x10'10 1.9 x10'9 218048 4 3.5 x10'10 1.8 x10‘9 218052 14 4.5 x10'10 1.2 x10'9 218054 22 7.6 x10'10 1.2 x10‘9 218064 1 1.3 x10'10 1.5 x10'9 218066 12 4.3 x10'10 2.1 x10'9 218070 10 3.1 x10'10 2.5 x10"9 218074 6 3.6 x10'10 2.9 x10'9 218090 17 5.1 x10'10 2.3 x10‘9 218101 23 9.3 x10'10 2.4 x10'9 218129 16 4.8 x10'10 1.2 x10‘9 218149 18 5.3 x10'10 9.6 x10'10 218233 21 7.2 x10'10 4.4 x10'9 218353 15 4.6 x10'10 1.6 x10'9 218418 24 1.9x10'9 4.1 x10'9 Furthermore, all 24 maturated Hise-tagged Z ts were also tested for binding against the three sequence-related proteins, hPD-L2, hB7-H3, and hB7-H4. In line with the results in Example 3, no binding to either of the control proteins were ed at a Z variant concentration of 500 nM.

Table 13: Kinetic ters for binding of Z variants to hPD-L1 and RhPD- ID hPD-L1 RhPD-L1 Zvariant NO: ka (1/Ms) kd (1ls) KD (M) ka (1/Ms) kd (1ls) KD (M) 213091 776 1.8x106 '3 6.3x10'10 1.9x106 1.0x10'3 5.4x10'10 217746 8 1.6 x106 4.6 x10'4 2.8 x10'10 1.4 x106 4.0 x104 2.8 x10'10 217748 11 1.7x106 5.7x104 3.4x10'10 1.8x106 5.1x104 2.9x10'10 217756 7 2.0 x106 5.4 x10'4 2.8 x10'10 2.7 x106 4.5 x104 1.7 x10'10 217825 5 2.0 x106 4.7 x 10*1 2.4 x10'10 1.9 x106 3.9 x10'4 2.1 x10'10 217911 3 6 4.9x10'4 2.1 x1010 2.1 x106 4.2x104 '10 217964 2 2.1 x106 4.3x 10'4 2.1 x10'10 3.4x106 3.8x 10'4 1.1x10'10 218022 9 1.7 x106 4.9 x10'4 2.8 x10'10 1.6 x106 4.6 x104 2.8 x10'10 218048 4 1.9 x106 4.4 x10'4 2.3 x10'10 1.6 x106 3.8 x10“1 2.4 x10'10 218064 1 3.5x106 4.3x104 1.3x10'10 3.5x106 3.4x104 9.6x10'11 218066 12 1.5x106 5.4x10'4 3.7x10'10 1.4x106 4.7x104 3.5x10'10 218070 10 2.0 x106 5.7 x 10*1 2.9 x10'10 1.7 x106 4.7 x10'4 2.8 x10'10 218074 6 2.0 x106 5.4 x10'4 2.7 x10'10 1.7 x106 4.6 x104 2.7 x10'10 AlphaLlSA blocking assay: The ability of 24 maturated Hiss—tagged monomeric Z ts to inhibit hPD-L1 binding to hPD-1 was tested in an AlphaLlSA blocking assay. The primary Z variant 213091 was included as a reference. Serial ons of the Z ts were incubated with biotinylated hPD-L1 and the blocking ability of each respective variant was measured after addition of hPD-1 coated Acceptor beads and subsequently streptavidin coated Donor beads. Inhibition could be measured as a decrease in AlphaLlSA counts for positive Z variants. The calculated IC5O values for the variants that were shown to block PD-L1 binding to PD-1 in this assay are shown in Table 14. 2016/076040 Table 14: IC50 values for Z variants blocking the PD-1/PD-L1 interaction Z variant SEQ ID NO: IC50 AlphaLlSA 213091 776 1.1 x10'9 217746 8 1.3 x10'9 217748 11 1.1x10'9 217756 7 1.5 x10'9 217825 5 1.6 x10'10 217911 3 5.7 x 1010 217964 2 8.9 x10'9 217972 19 2.9 x10‘10 217978 13 6.2 x10‘10 218022 9 3.1 x10'10 218039 20 2.9 x10'10 218048 4 1.5 x10'10 218052 14 3.4 x10'10 218054 22 1.1 x10'9 218064 1 3.9 x 1010 218066 12 8.3 x10'10 218070 10 8.6 x10‘10 218074 6 3.1 x10‘10 218090 17 6.7 x10'10 218101 23 6.2 x10'10 218129 16 6.0 x10'10 218149 18 1.9x10'10 218233 21 1.5 x10'10 218353 15 7.2 x10'10 218418 24 5.7 x10'10 CD analysis: The CD spectra determined for 24 maturated PD-L1 binding 2 variants with a Hise tag showed that each had an d—helical structure at 20 °C. The melting temperatures (Tm) were determined using variable temperature measurements (Table 15). Reversible folding was observed for all PD-L1 g Z variants when overlaying spectra measured at 20 °C before and after heating to 80 °C, as shown for two ed Z variants in Figure 6.

Table 15: Melting temperatures of maturated PD-L1 binding Z variants Z variant SEQ ID NO: Tm (°C) 217746 8 59 217748 11 63 217756 7 63 217825 5 60 217911 3 59 217964 2 62 217978 13 59 218022 9 59 218039 20 55 218048 4 60 218064 1 62 218066 12 58 218070 10 60 218074 6 62 218090 17 68 218129 16 63 218233 21 57 Characterization of anti-PD-L1/PD-1 and anti-PD-L1/CTLA-4 complexes als and methods Production of complexes and control dies: Four different complexes targeting PD-L1 and PD-1, and four ent complexes targeting PD-L1 and CTLA-4 were constructed, as well as a control antibody targeting PD-1. An antibody denoted “Lam”, having the same CDR sequences and icity as the commercially available, PD-1 ing monoclonal antibody pembrolizumab (formerly lambrolizumab), was constructed using the heavy chain (HC) and light chain (LC) sequences HCLam (SEQ ID NO:815) and LCLam (SEQ ID NO:816). An antibody denoted “Ipi”, having the same CDR sequences and specificity as the commercially available, CTLA-4 targeting monoclonal antibody ipilimumab, was constructed using the heavy chain (HC) and light chain (LC) sequences HClpi (SEQ ID NO:817) and LCIpi (SEQ ID NO:818). The PD-L1 targeting Z variant 215170 (SEQ ID NO:814; identical to 213165 (SEQ ID ) but starting with the amino acid residues AE d of VD) with a C-terminal VD sequence was genetically fused, via a flexible 15 residue (GGGGS)3 linker, to the ini of HCLam‘ LCLam, HCIpiand LCIpi, respectively, resulting in the complexes 215170—HCLam, 215170-LCLam, 215170-HCIpi and -LCIpi, respectively; or to the C-termini of the same chains, resulting in the complexes HCLam—215170, LCLam-215170, HCIpi- 215170 and LCM-215170, respectively. Gene synthesis, cloning, production by transient gene expression in CHO cells as well as purification by Protein A chromatography and verification of constructs by gel electrophoresis were med by Evitria AG (Switzerland).

Biacore kinetic analyses: Kinetic constants (ka and kd) and affinities (KD) for hPD-L1, human PD-1 (hPD-1; R&D Systems cat. no. 1086-PD-050) and human CTLA-4 (hCTLA—4; R&D Systems cat. no. 325-CT-200) were determined for all eight complexes ed and using a Biacore 2000 instrument (GE Healthcare). The control antibody Lam was also analyzed for binding against PD-1. 5 ug/ml solutions of each of the proteins hPD-L1, hPD-1 and hCTLA—4 were prepared in 10 mM NaAc buffer (pH 5.0 for PD-L1, and pH 4.5 for PD-1 and CTLA-4) and used for immobilization in separate flow cells on the carboxylated dextran layer of different CM5 chip surfaces (GE Healthcare, cat. no. BR100012). The immobilization was performed using amine coupling chemistry according to the manufacturer’s protocol and using HBS—EP with 500 mM NaCI as g buffer. Immobilization levels obtained were ~110-140 RU. A series of 3.33, 10, 30, 90, 270 nM concentrations of the respective complex and Lam were injected and the responses recorded, except for analysis of binding to PD-L1 for constructs with 215170 positioned on the C-terminus of the respective antibody, for which a concentration series of 30, 90, 270 and 900 nM was used.

In a separate ment, the dual binding specificity was evaluated by a e assay using the Biacore 2000 instrument. The complexes 215170- HCLam, 215170-LCLam, 215170-HCIpi and 215170-LCIpi, Lam and ipilimumab y®, l-Myers Squibb/Astra Zeneca via Apoteket AB, cat. no. 065544, lot no. 4A85968), at a concentration of 300 nM, were injected over chip surfaces lized with PD-1 or CTLA-4 as described above. In all cases, the duration of the injection was 5 min at a flow rate of 30 ul/min with a wait/dissociation step of 5 min before a second injection (5 min) of 100 or 500 nM PD-L1 was made. HBS—EP with 500 mM NaCl was used as a running buffer and for protein dilutions.

Cell binding analysis by FACS: The potential of the complexes to bind PD-L1 expressing cells was investigated using FACS. 150 000 cells of the breast cancer cell line MDA-MB-231, cultivated in DMEM (ATCC cat. no. 30- 2002) containing 10 % FBS, were pipetted per well of a v—bottomed l plate (Nunc, cat. no. 277143) and the cells in the plate were subsequently pelleted at 400 g for 3 min at RT. The supernatants were removed and the cells were resuspended in 100 pl PBS plus 2.5 % FBS (staining buffer) containing 0.625 ug/ml of the complexes Z15170-HCLam, Z15170-LCLam, HCLam-Z15170, LCLam-Z15170, Z15170-HCIpi, Z15170—LCIpi, HCIpi-Z15170 and LCIpi-Z15170, respectively, or 0.625 ug/ml of the antibodies Lam or ipilimumab. A mouse anti-PD-L1 antibody (RnD Systems, cat. no. MAB1561) at a concentration of 1 ug/ml was used as a ve control. Cells incubated with buffer alone were used as negative controls. The cells were incubated for 1 h at 8 °C in the dark, washed twice with 100 pl staining buffer, and resuspended in 100 pl of staining buffer containing 2.5 ug/ml of a goat-anti- human lgG-Alexa488 ular Probes, cat. no. A11013) or, for cells stained with the ve control antibody, goat anti-mouse lgG-Alexa647 antibody (Life Technologies, cat. No. A21236). The cells were incubated for 1 h at 8 °C in the dark, washed twice with 100 pl staining buffer and resuspended in 300 pl of staining . Data from 10,000 cells were ed using a FACS Calibur (Beckman r) and the data was analyzed using g software 2.5.0 (Turku University). Mean fluorescence intensity (MFI) was used as a read out of g capacity.

Co-culture of MDA-MB-231 and PBMC: A mixed lymphocyte assay was used to analyze if the sed complexes could affect proliferation or the cytotoxic effect of T-cells and thereby increase the elimination of cancer cells. Herein, peripheral blood mononuclear cells (PBMC) and MDA-MB—231 cells were co-cultivated for six days and the number of T-cells and cancer cells were assessed. 20000 MDA-MB-231 cells, cultivated in DMEM containing 10 % FBS, were pipetted per well of a ottomed 96—well plate and were left to adhere to the bottom of the well by incubation at 37 °C in a humidified 5 % C02 atmosphere. Day 2 of the experiment, serial ons (200-0064 nM) of the lpi-based complexes were prepared in a separate plate using RPM|1640 with L-glut (Lonza) supplemented with 10 % FCS, and 1 % Pen-Strep (Lonza, cat. no. DE17-603E). The DMEM medium was discarded from the MDA—MB-231 cells and 100 pl of the diluted complexes were added.

PBMC were prepared form a buffy coat using Ficoll Paque PLUS (GE Healthcare, cat no.1702). In brief, the buffy coat was d 2X in PBS. ml of the diluted buffy coat were layered on the top of 5 ml Ficoll in 15 ml falcon tubes and centrifuged at RT for 30 min at 400 g. The lymphocyte layer was collected and the cells were washed twice in the supplemented RPM|1640 medium described above. The cells were counted and adjusted to 1 million cells per ml in supplemented RPMI medium. 100 pl of the cell suspension were added to the plate with the MDA-MB-231 cells. The plates were incubated for 6 days at 37 °C in a humidified 5 % C02 atmosphere. At day 7 of the experiment, the number of MDA-MD-231 cells and CD3+ T-cells were counted by FACS. The PBMC were transferred to a v-bottom plate, washed two times with PBS containing 2 % FBS (also used as staining buffer) and d with a mouse anti-CD3 antibody (EXBIO Praha, cat no. 12 M001) at a concentration of 2 ug/ml for 1 h at 4 °C. The MDA-MB231 cells were nated (20 ul/well) and transferred to another v—bottom shaped plate washed two times with PBS containing 2 % FBS and stained with a rabbit anti-EGFR antibody (Abcam, cat no. -1) at a concentration of 2 ug/ml for 1 h at 4 °C. The cells were washed two times with PBS ning 2 % FBS and an Alexa-fluor 488-goat-anti-rabbit antibody (lnvitrogen, cat no.

A11008) and Alexa-fluor 647-goat—anti-mouse antibody (Life technologies, cat no. A21236) were used as detection antibodies at a concentration of 1 ug/ml and incubated for 1 h at 4 °C.

Results Production of complex constructs: A schematic representation of the design of each of the four types of produced complexes is shown in Figure 7. e kinetic analyses: The affinity to the target proteins PD-L1, PD- 1 and CTLA—4, respectively, were determined for each relevant complex. The control antibody Lam was also analyzed against its target PD-1. The kinetic ters for the ctions with PD—L1 are summarized in Table 16. The capability of the Z moiety of the complex to interact with PD—L1 was maintained, although the affinity was reduced as well as affected by the positioning of the Z moiety on the dy. For comparison, the KB of the Hiss-Z13165 interaction with PD-L1 was 0.64 nM (as presented in Example 3) whereas the KD for xes with inally oned Z moieties was 1.5-2.6 nM and the KO for C-terminally positioned Z es was 12-41 nM.

Thus, N-terminal positioning of the Z moiety was superior to the C-terminal positioning, with approximately 10 times higher affinity. This effect was evident with both Lam- and |pi-based constructs. Whether the fusions were made to the heavy or light chains of the antibodies were of less importance for the N-terminally positioned Z moiety, but had major impact on the C- terminally positioned Z moiety, where the light chain fusions had a KB of 12- 18 nM compared to a KB of 29-41 nM for the heavy chain fusions.

Table 16: Kinetic parameters for binding of indicated complexes to hPD-L1 Analyte ka (1lMs) k,| (1ls) KD (M) HCLam-Z15170 2.44 x104 7.17 x10‘4 2.9 x10‘8 LCLam-Z15170 3.06 x 104 3.58 x 10'4 1.2 x 10'8 z1517o-HcLam 1.16 x105 2.84 x10'4 2.4 x10'9 Z15170—LCLam 2.33 x 105 6.01 x10'4 2.6 x10'9 Hopi-215170 1.78x104 7.27x10'4 4.1 x10'8 Low-215170 3.07 x 104 5.63 x 10'4 1.8 x10‘8 -Hclpi 2.05 x 105 4.83 x10'4 2.4 x10'9 z1517o-LcIpi 2.30 x 105 3.49 x 10'4 1.5 x10'9 The interactions of the complexes with PD-1 and , respectively, followed a bivalent model. The K91, K92, ka1, kaz, km and kdz are summarized in Table 17 and Table 18 for PD—1 and CTLA—4, tively. The affinity nt Km for the interaction of PD-1 with the produced Lam control antibody was determined to 18.6 nM. The affinity was stronger for all the Lam based xes, with a K01 range of 0.8-2.7 nM. A somewhat slower association and rate, ka1, was seen for Z15170-HCLam, but generally the differences between the complexes were small, i.e. the positioning of the Z moiety on the antibody seems to have minor impact on the interaction between the antibody and PD-1.

Table 17: Parameters for binding of indicated complexes and Lam to hPD-1 Analyte ka1 kd1 Km kaz kdz K02 (1lMs) (1ls) (M) (1lRUs) (1ls) (RU) Lam 4.35x104 8.10x10'4 1.9x 10‘8 1.88x10'3 1.83x10'3 0.97 Z15170 1.17 x105 1.64 x10'4 1.4 x10'9 3.35 x 100 1.65 x101 4.9 LCLam-Z1517O 1.78 x105 1.34 x10'4 7.5 x 10-10 2.92 x10'1 7.89 x10'1 2.7 z15170—HcLam 4.27 x104 1.17 x10'4 2.7 x10'9 1.93 x10"2 7.87 x10'2 4.1 215170-LcLam 05 1.56x10'4 1.4x 10'9 2.22x10‘2 1.72x 10-1 7.7 Table 18: Kinetic parameters for binding of indicated complexes to hCTLA-4 Analyte ka1 kd1 Km kaZ kdz K132 (1lMs) (1ls) (M) (1/RUs) (1ls) (RU) Hopi-215170 6.79 x104 5.40 x10'4 8.0 x10'9 6.76 x10‘2 4.10 x 10-1 6.1 Low-215170 5.48 x104 4.41 x10'4 8.0 x10‘9 4.04 x10"2 2.88 x10'1 7.1 215170—HcIpi 3.33 x104 2.79 x10'4 8.4 x10'9 1.38 x10‘2 8.20 x10'2 5.9 z15170-LcIpi 4.55 x104 4.49 x10'4 9.9 x10‘9 9.74 x10'3 4.22 x10'2 4.3 The affinity constant Km for the interaction of complexes with CTLA-4 was in the range of 8-10 nM and this is in line with the reported KD for ipilimumab (5.25 1r 3.62 nM; an Medicines Agency’s assessment report 2011: EMA/CHMP/557664/2011). The kinetic profiles were similar for all lpi-based constructs, but with somewhat slower association and dissociation rates for Z15170-HCIpi.

The Biacore capture assay confirmed the dual binding specificity of all complexes included in the assay, i.e. Z15170-HCLam, Z15170-LCLam, Z15170- HCIpi and -LCIpi. Figure 8 shows that the complexes first bind to immobilized PD-1 or CTLA—4 and that PD-L1 subsequently binds to the respective captured complex. In separate control experiments, it was shown that PD—L1 does not bind to CTLA—4 or lpi and that no additional binding by PD-L1 was seen ing injection of PD-L1 to Lam captured on PD-1.

Cell binding analysis by FACS: This experiment was performed to analyze whether the complexes could bind to PD-L1 expressing cells. MDA- MB-231 cells that naturally express PD—L1 were stained with 0.625 pg/ml of the respective complex. The MFI values are presented in Table 19 and shows that the xes had the y to bind PD-L1 expressing cells. For both the lpi- and Lam-based complexes, the highest MFI values were obtained for N-terminal positioning of the Z moiety on the light chain of the antibody.

Table 19: MFI for binding of complexes to PD-L1 expressing cells e MFI Lam 62 Z15170-HCLam 161 HCLam‘Z15170 274 Z15170-LCLam 327 LCLam-Z1517O 145 umab 64 -HCIpi 488 HCIpi-Z1517O 264 Z15170-LCIpi 582 LCIPi-Z15170 192 Negative control 69 Anti-PD-L1 antibody 610 WO 72280 Co-culture of MDA-MB-231 and PBMC: To assess whether the lpi— based xes could affect the inhibitory mechanisms caused by CTLA-4 and PD-L1, a mixed lymphocyte assay was used. Breast cancer cells MDA- MB-231 were co-cultivated with PBMC for six days and the number of cancer cells and T-cells were ted. The analysis revealed a concentration dependent effect of the complexes, with an increased amount of T—cells and a lowered number of cancer cells. Figure 9A shows the reduction in number of MDA-MB231 cells with increasing concentration of the xes. This reduction was evident for all complexes with the best effect achieved with the uct in which the Z moiety is situated at the N—terminus of the light chain of the antibody. In contrast, the ipilimumab control antibody did not induce a concentration dependent decrease of the cancer cells. Thus, blocking of the interaction PD-1/PD-L1 appears essential to reduce the amount of cancer cells. Figure QB shows the increase in the number of T-cells with an increasing tration of the complexes. Again, the effect is most prominent with the construct in which the Z moiety is situated at the N- terminus of the light chain of the antibody.

Example 9 ation and radiolabeling of PD-L1 binding Z variants This Example describes the conjugation and radiolabeling of 215168- Cys (SEQ ID NO:809), Z18608—Cys (SEQ ID NO:811), Z18609-Cys (SEQ ID NO:812) and Z18610-Cys (SEQ ID NO:813), cloned and produced as described in Example 2 and Example 6, and further used for the in vivo imaging studies described in Example 10 and 11.

Materials and methods Reduction and NOTA coniugation: To 5 mg on t in [20 mM HEPES, 1 mM EDTA, pH 7.2] was added three molar equivalents of tris(2— carboxyethyl)phosphine (TCEP) in 0.5 ml of degassed 0.2 M ammonium e buffer (pH 7.0). The reaction was kept at RT for 60 min before being transferred to an Ultracel 3K Centrifugal Filter and centrifuged at 4000 rpm for 90 min. The flow-through was discarded and an additional 1 ml of0.2 M um acetate buffer added, and the process repeated. The reduced Z variant was then transferred to a second reaction vessel in 2 ml of oxygen free 0.2 M ammonium acetate buffer (pH 7.0). 4 mg of NOTA-maleimide (Macrocyclics) in 0.5 ml of0.2 M ammonium acetate buffer (pH 7.0) was then added, and the reaction vessel purged with argon before heating to 40 °C for 3 h, at which point the reaction mixture was transferred to an Ultracel 3K Centrifugal Filter and centrifuged for 90 min at 4000 rpm. The flow-through was discarded and 2 ml milIiQ water added. fugation was performed for an additional 90 min and the flow-through ded. Purified NOTA- conjugated Z variant was collected in 1 ml miIIiQ water, lyophilized and stored at -70 °C prior to use. Purity of the final t was determined by LC/MS. abeling: A cartridge containing [18F]-f|uoride was first washed with 1.5 ml of ultrapure water, then f|uoride was eluted with 1.0 ml of 0.4 M KHCOg. 100 pl of the eluted [18F]-f|uoride solution was added to a stem vial charged with 10 ul acetic acid, 50 pl AlCl3 (2 mM in 0.1 M NaOAc buffer, pH 4) and 125 pl 0.1 M NaOAc pH 4. The solution was incubated for 2 min at RT before 1 mg of NOTA-conjugated Z variant in 400 pl of a 1:1 solution of acetrontrile and 0.1 M NaOAc pH 4 was added, then heated to 100 °C for 15 min. After g was complete, the sample was transferred to a vial containing 0.7 ml of 0.1 % formic acid, mixed and purified by HPLC [Waters Xselect CSH C18 column (250><10 mm, 130 um)] using a gradient of 10-30 % MeCN over 15 min at a flow rate of 5 ml/min, the balance being 0.1 % formic acid. The peak corresponding to [18F]AIF-NOTA-Z##### was collected, the MeCN was removed in vacuo, and transferred to a sterile vial using physiologic saline as a rinse to give [18F]AIF-NOTA-Z#####. ic ty and radiochemical purity was determined via a Waters Acquity LC/MS system (Milford, MA, USA) and a B—RAM Model 4 Radio—HPLC detector (IN/US Systems, Brandon, FL, USA).

Results The PD-L1 g 2 variants, —Cys (SEQ ID ), 218608- Cys (SEQ ID NO:811), Z18609—Cys (SEQ ID NO:812) and Z18610-Cys (SEQ ID NO:813), were site specifically conjugated with NOTA at their respective unique C-terminal cysteine residue. Subsequent radiolabeling with [18F]AIF typically resulted in radiochemical purities of 97—100 % and specific activities of 14.6 1r 6.5 GBq/mmol. The radiolabeled Z ts will be referred to as [18F]AlF—NOTA—Z[#####].

Example 10 In vivo imaging and biodistribution in tumor bearing mice Materials and methods Animal models: Female SCID Beige mice (6-8 week old, Charles River Laboratories) were housed in a temperature and humidity controlled room and kept on a r diet. LOXIMVI (human melanoma cell line; PD-L1 positive) or SUDHL-6 (PD-L1 negative) cells were cultured in complete growth medium containing RPMI 1640 medium with 10 % fetal bovine serum at 37°C with 5 % 002. The growth medium was changed 2 or 3 times per week and the cells subcultured at a ratio of 1 :10 when . Tumors were implanted at the right shoulder by subcutaneous injection of 1 X 106 LOXIMVI cells in 100 pl PBS or 10 x106 SUDHL-6 cells in 100 pl PBS + Growth Factor Reduced Matrigel (1 :1). The mice were used for micro—PET and ex vivo studies about 5-7 days and 3 weeks after the injection of LOXIMVI and SUDHL-6 cells, respectively, when tumors reached a mass of 100-400 mg.

PET data acguisition: Mice were anesthetized with isoflurane (4-5 % ion, 1-3 % maintenance), prepared with tail vein catheters, and placed in a dedicated small animal PET scanner (microPET Focus220, Siemens Preclinical Solutions). A 20 min transmission scan with 57Co was obtained to correct for photon attenuation and r. Then, 0.2-0.6 MBq of the respective labe||ed Z variant was administered via the tail vein catheters, and PET data were ted for 90 min. In a separate pre-blocking WO 72280 experiment, 400 pg non-labelled NOTA-conjugated Z15168—Cys was administered prior to administration of [18F]AlF-NOTA-Z15168.

Ex vivo biodistribution measurements: Immediately after PET acquisition, mice were ized. Tumor, heart, lung, spleen, liver, kidneys, blood, plasma and muscle were collected and measured using a gamma counter (PerkinElmer). For each mouse, biodistribution measurements were ted into units of Standard Uptake Value (SUV). Regions of Interest (ROI) were drawn on all tumors that could be identified in PET , and time activity curves (TACs) were calculated.

Representative PET images following injection of [18F]-labelled Z variants into tumor-bearing mice showed the highest uptake in kidney and bladder. PD-L1 positive LOX tumors could be clearly seen in images, while PD-L1 negative SUDHL6 tumors were not e. Representative PET images are shown in Figure 10A for [18F]AlF-NOTA-Z15168. Ex vivo biodistribution measurements at 90 min post—injection were in agreement with PET images. The uptake of [18F]-labelled z variants Z15168—Cys (SEQ ID NO:809), Z‘l8608—Cys (SEQ ID NO:811), Z18609-Cys (SEQ ID NO:812) and —Cys (SEQ ID NO:813) was icantly higher in LOX tumors than in SUDHL6 tumors and the tumor uptake increased with the PD-L1 binding affinity of the Z variants (Figure 11A-B). Target specificity was confirmed in a pre-blocking experiment in which pre-administration of 400 ug NOTA-Z15168 caused a reduction in [18F]AlF-NOTA—215168 uptake in LOX tumors (Figure 108). Changes in distibution, including faster clearance as ted by reduced blood uptake at 90 min, was also seen. The kidney tracer retention (average SUV ranged between 57 to 84 in LOX tumor xenographs) is likely due to tubular reuptake of proteins, where [18F]AIF label is trapped after cleavage of the Z variants. To summarize, the results show that Z variant ligands are effective in targeting PD-L1 positive tumors in vivo, exhibiting specific binding and a rapid clearance.

Example 11 In vivo imaging in rhesus monkey Materials and methods Fasted rhesus monkeys were sedated with Ketamine (10 mg/kg, intramuscular). An intrevenous catheter was inserted into the right and left saphenous veins and the animals were maintained on propofol esia (5 mg/kg for ion and 0.45 mg/kg/min throughout the scanning procedure). Following the initial induction with propofol, the animal was intubated and maintained on ventilated oxygen/air gas mixture at approximately 10 cm3/kg/breath, and 23 respirations per minute. s were instrumented with a temperature probe, a pulse er and an end tidal 002 monitor. Body temperature was maintained using K-module heating pads. General fluid y was maintained with Lactated ’s solution (10 ml/kg/h i.v.) throughout the scanning procedure. 84-138 MBq of [18F]AIF- NOTA-Z15168 and 147-227 MBq of [18F]AlF-NOTA-Z18609, respectively, were stered as a 2 min infusion. Whole body dynamic scan was initiated at the start of the tracer injection and aquired for 180 min using a Siemens Biograph 64 TPTV PET/CT scanner. Whole body reconstruction was performed using the PET/CT scanner vendor supplied re. PET image analysis was performed using customized Matlab based software.

Results Representative maximum ity projection images of rhesus monkeys administered with[18F]AIF-NOTA-Z15168 and [18F]AIF-NOTA- 218609, respectively, are shown in Figure 12A—B and graphs of the average tracer uptake (~120—180 min) are shown in Figure 120. As in mice, the highest uptake was seen in kidney (SUV z 100—112) and bladder, but also lymph node and spleen targeting was observed, which is consistent with PD- L1 expression. 2016/076040 ITEMIZED LIST OF EMBODIMENTS 1. PD—L1 binding polypeptide, comprising a PD-L1 g motif BM, which motif consists of an amino acid sequence selected from: i) ERNX4AAX7E|L X11LPNLX15X17X1aQX20 WAFIWX26LX28D wherein, independently from each other, X4 is selected from A, D, E, F, H, l, K, L, N, Q, R, S, T, V and Y; X7 is selected from A, E, F, H, N, Q, S, T, V, W and Y; X11 is selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W and Y; X16 is selected from N and T; X17 is selected from A, H, K, N, Q, R and S; X18 is selected from A, D, E, G, H, K, L, N, Q, R, S, T, V and Y; X20 is selected from H, l, K, L, N, Q, R, T, V and Y; X26 is selected from K and S; and X28 is selected from A, D and E; and ii) an amino acid sequence which has at least 96 % identity to the sequence defined in i). 2. PD-L1 g polypeptide according to item 1, wherein in sequence i) X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and Y; X7 is selected from E, F, H, N, Q, S, T, V, W and Y; X11 is ed from A, D, H, L, Q, R, T, V, W and Y; X15 is selected from N and T; X17 is selected from A, H, K, N, Q, R and S; X18 is selected from A, D, E, G, H, K, L, N, Q, R, S, T, V and Y; X20 is selected from H, l, K, L, Q, R, T, V and Y; X25 is selected from K and S; and X28 is selected from A, D and E. 3. PD-L1 binding polypeptide according to item 1, n in ce i) X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and Y; X7 is selected from A, E, F, H, N, Q, S, T, V, W and Y; X11 is selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W and Y; X15 is selected from N and T; X17 is selected from A, H, K, N, Q, R and S; X18 is selected from A, D, E, G, H, K, L, N, Q, R, S, T, V and Y; X20 is selected from H, l, K, L, N, Q, R, T, V and Y; X26 is selected from K and S; and X28 is selected from A, D and E. 4. PD-L1 binding polypeptide according to item 2 or 3, wherein in sequence i) X4 is ed from A, D, E, F, H, I, K, L, N, Q, R, S, T and V; X7 is selected from F, H, Q and Y; X11 is selected from H, Q, W and Y; X15 is selected from N and T; X17 is selected from A, H, K, N, Q and S; X18 is selected from A, E, G, H, K, L, N, Q, R, S, T, V and Y; X20 is selected from H, l, K, Q, R and V; X25 is selected from K and S; and X28 is selected from A and D.

. PD-L1 binding polypeptide according to any one of item 1-4, wherein sequence i) fulfills at least four of the seven conditions l-VII: l. X7 is selected from F, H, Q and Y; H. X11 is selected from H and Y; lll. X16 is T; IV. X17 is selected from N, Q and S; V. X20 is selected from H, I, K and R; VI. X26 is K; and VII. X28 isAor D. 6. PD-L1 binding ptide according to item 5, wherein sequence i) fulfills at least five of the seven ions I-VII. 7. PD-L1 binding polypeptide according to item 6, wherein sequence i) fulfills at least six of the seven conditions I-VII. 8. PD—L1 binding polypeptide according to item 7, wherein sequence i) fulfills all of the seven conditions I—VII. 9. PD-L1 g polypeptide according to any one of items 1-8, wherein X7X11X20 is selected from FYK and YYK.

. PD-L1 binding polypeptide according to any one of items 1-9, wherein X11X17X20 is selected from YNK and YQK. 11. PD-L1 binding polypeptide according to any one of items 1-10, n X11X18X20 is YAK. 12. PD-L1 binding ptide according to any preceding item, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-808. 13. PD-L1 binding polypeptide according to item 12, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-93 and 774-796. 14. PD-L1 binding polypeptide according to item 13, wherein ce i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group ting of SEQ ID NO:1-93 and 774-787.

. PD-L1 binding polypeptide according to item 14, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group ting of SEQ ID NO:1-93, 775, 776, 779-781 and 784-786, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784 and 786, such as the group consisting of SEQ ID NO:1—93, 776,781 and 784, such as the group consisting of SEQ ID NO:1-93, 776 and 784 or the group consisting of SEQ ID NO:1-93, 776 and 781, for example the group consisting of SEQ ID NO:1—93 and 776 or the group consisting of SEQ ID NO:1-93 and 781 or the group consisting of SEQ ID NO:1-93 and 784. 16. PD-L1 binding polypeptide according to item 15, wherein sequence i) corresponds to the sequence from on 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774, 775, 6, such as the group consisting of SEQ ID NO:1-93, 775, 780, 781, 784 and 786. 17. PD—L1 binding polypeptide according to any one of items 14-16, n sequence i) corresponds to the sequence from on 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-93. 18. PD-L1 g polypeptide according to item 17, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-24. 19. PD-L1 binding polypeptide according to item 18, wherein ce i) corresponds to the sequence from position 8 to on 36 in a sequence selected from the group consisting of SEQ ID NO:1-12, 14 and 17-21.

. PD-L1 binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence from on 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-12 and 17, such as the group consisting of SEQ ID NO:1—5 and 17, such as the group consisting of SEQ ID NO:1, 2 and 17. 21. PD-L1 binding polypeptide according to item 19, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1, 4, 5, 6, 9, 14 and 18-21, such as the group consisting of SEQ ID NO:4, 5, 18 and 21, such as the group consisting of SEQ ID NO:4, 5 and 21. 22. PD—L1 binding polypeptide according to item 20 or 21, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:1. 23. PD-L1 binding polypeptide according to item 20 or 21, wherein ce i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:4. 24. PD—L1 binding polypeptide according to item 20 or 21, wherein sequence i) corresponds to the sequence from position 8 to position 36 in SEQ ID NO:5.

. PD-L1 binding polypeptide according to item 21, wherein sequence i) corresponds to the sequence from position 8 to on 36 in SEQ ID NO:21. 26. PD-L1 binding polypeptide according to any preceding item, wherein said PD-L1 binding motif forms part of a three—helix bundle protein domain. 27. PD—L1 binding polypeptide according to item 26, wherein said PD- L1 binding motif essentially forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain. 28. PD—L1 g polypeptide according to item 27, wherein said three-helix bundle n domain is selected from ial receptor domains. 29. PD—L1 binding polypeptide according to item 28, wherein said three-helix bundle protein domain is selected from domains of n A from Staphylococcus aureus or derivatives f.

. PD—L1 binding polypeptide according to any preceding item, which comprises a binding module BMod, the amino acid ce of which is selected from: iii) K—[BM]—DPSQSXaXbLLXC EAKKLXdXeXfQ; wherein [BM] is a PD-L1 binding motif as defined in any one of items 1-25; X2! is selected from A and S; Xb is selected from N and E; XC is selected from A, S and C; Xd is ed from E, N and S; X9 is selected from D, E and S; and Xf is selected from A and S; and WO 72280 iv) an amino acid sequence which has at least 93 % identity to a sequence defined in iii). 31. PD-L1 binding polypeptide according to any preceding item, n sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-808. 32. PD-L1 binding polypeptide according to item 31, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-93 and 774-796. 33. PD-L1 binding polypeptide according to item 32, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID 3 and 774-787. 34. PD-L1 binding polypeptide according to item 33, wherein sequence iii) ponds to the ce from on 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781 and 784-786, such as the group consisting of SEQ ID NO:1-93, 776, 780, 781, 784 and 786, such as the group consisting of SEQ ID NO:1—93, 776,781 and 784, such as the group consisting of SEQ ID NO:1-93, 776 and 784 or the group consisting of SEQ ID NO:1-93, 776 and 781, for example the group consisting of SEQ ID NO:1—93 and 776 or the group consisting of SEQ ID NO:1-93 and 781 or the group consisting of SEQ ID NO:1-93 and 784.

. PD-L1 binding polypeptide according to item 33, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID 3, 774, 775 and 780- 786, such as the group consisting of SEQ ID NO:1-93, 775, 780, 781, 784 and 786. 36. PD-L1 g polypeptide according to any one of items 33-35, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-93. 37. PD-L1 binding polypeptide according to item 36, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-24.

WO 72280 38. PD-L1 binding polypeptide ing to item 37, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1—12, 14 and 17—21. 39. PD-L1 binding polypeptide according to item 38, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in a sequence selected from the group consisting of SEQ ID NO:1-12 and 17, such as the group consisting of SEQ ID NO:1—5 and 17, such as the group consisting of SEQ ID NO:1, 2 and 17. 40. PD-L1 binding polypeptide according to item 38, wherein sequence iii) corresponds to the ce from position 7 to position 55 in a sequence selected from the group ting of SEQ ID NO:1, 4, 5, 6, 9, 14 and 18-21, such as the group consisting of SEQ ID NO:4, 5, 18 and 21, such as the group consisting of SEQ ID NO:4, 5 and 21. 41. PD-L1 binding polypeptide according to item 40 or 41, n sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:1. 42. PD—L1 g polypeptide according to item 40 or 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:4. 43. PD—L1 binding polypeptide according to item 40 or 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:5. 44. PD-L1 binding polypeptide according to item 41, wherein sequence iii) corresponds to the sequence from position 7 to position 55 in SEQ ID NO:21. 45. PD-L1 binding polypeptide according to any preceding item, which comprises an amino acid sequence ed from: v) YA-[BMod]—AP; wherein [BMod] is a PD-L1 binding module as defined in any one of items 30- 44;and vi) an amino acid sequence which has at least 90 % identity to a sequence defined in v).

WO 72280 46. PD—L1 binding ptide according to any one of items 1-44, which comprises an amino acid sequence selected from: vii) FN-[BMod]—AP; wherein [BMod] is a PD-L1 binding module as defined in any one of items 30- 44;and viii) an amino acid sequence which has at least 90 % identity to a sequence defined in vii). 47. PD—L1 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from: ADNNFNK-[BM]—DPSQSANLLSEAKKLNESQAPK; ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK; ADNKFNK-[BM]—DPSVSKEILAEAKKLNDAQAPK; NFNK-[BM]—DPSQSTNVLGEAKKLNESQAPK; AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK; VDNKFNK-[BMJ-DPSQSANLLAEAKKLNDAQAPK; AEAKYAK-[BM]—DPSESSELLSEAKKLNKSQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLNDAQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLNDSQAPK; AEAKYAK-[BMj-DPSQSSELLSEAKKLNDSQAPK; AEAKYAK-[BMj-DPSQSSELLSEAKKLNDSQAP; AEAKFAK-[BM]—DPSQSSELLSEAKKLNDSQAPK; AEAKFAK-[BMJ-DPSQSSELLSEAKKLNDSQAP; AEAKYAK-[BM]—DPSQSSELLAEAKKLNDAQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLSESQAP; AEAKFAK-[BMj-DPSQSSELLSEAKKLSESQAPK; K-[BMj-DPSQSSELLSEAKKLSESQAP; AEAKYAK-[BM]—DPSQSSELLAEAKKLSEAQAPK; AEAKYAK-[BMJ-QPEQSSELLSEAKKLSESQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLESSQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLESSQAP; AEAKYAK-[BM]—DPSQSSELLAEAKKLESAQAPK; AEAKYAK-[BMj-QPEQSSELLSEAKKLESSQAPK; WO 72280 AEAKYAK-[BM]—DPSQSSELLSEAKKLSDSQAPK; AEAKYAK-[BM]—DPSQSSELLSEAKKLSDSQAP; AEAKYAK-[BM]—DPSQSSELLAEAKKLSDSQAPK; AEAKYAK-[BM]—DPSQSSELLAEAKKLSDAQAPK; AEAKYAK-[BM]—QPEQSSELLSEAKKLSDSQAPK; VDAKYAK-[BMj-DPSQSSELLSEAKKLNDSQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLNDAQAPK; VDAKYAK-[BM]—DPSQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLSEAQAPK; VDAKYAK-[BM]—QPEQSSELLSEAKKLSESQAPK; VDAKYAK-[BM]—DPSQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLESAQAPK; VDAKYAK-[BMj-QPEQSSELLSEAKKLESSQAPK; VDAKYAK-[BM]—DPSQSSELLSEAKKLSDSQAPK; VDAKYAK-[BMJ-DPSQSSELLAEAKKLSDSQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLSDAQAPK; VDAKYAK-[BM]—QPEQSSELLSEAKKLSDSQAPK; VDAKYAK-[BM]—DPSQSSELLAEAKKLNKAQAPK; AEAKYAK-[BM]—DPSQSSELLAEAKKLNKAQAPK; and ADAKYAK-[BMj-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is a PD-L1 binding motif as defined in any one of items 1-25. 48. PD—L1 binding polypeptide according to any one of items 1-47, which comprises an amino acid sequence selected from: xvii) VDAKYAK-[BM]—DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is a PD-L1 binding motif as defined in any one of items 1-25; xviii) an amino acid sequence which has at least 89 % identity to the sequence defined in xvii). 49. PD—L1 binding ptide according to any one of items 1-47, which comprises an amino acid sequence selected from: xix) AEAKFAK-[BM]—DPSQSSELLSEAKKLSESQAPK; wherein [BM] is a PD-L1 g motif as defined in any one of items 1-25; xx) an amino acid sequence which has at least 89 % identity to the sequence defined in xix). 50. PD—L1 binding ptide according to any one of items 1-47, which comprises an amino acid sequence selected from: xxi) AEAKYAK-[BMj-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is a PD—L1 binding motif as defined in any one of items 1-25; xxii) an amino acid sequence which has at least 89 % identity to the sequence defined in xxi). 51. PD—L1 binding polypeptide according to any one of items 1-47, which comprises an amino acid sequence selected from: xxiii) AEAKFAK-[BMj-DPSQSSELLSEAKKLNDSQAPK; wherein [BM] is a PD—L1 binding motif as defined in any one of items 1-25; xxiv) an amino acid ce which has at least 89 % identity to the sequence defined in xxiii). 52. PD-L1 binding polypeptide according to any preceding item, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-814. 53. PD-L1 binding ptide according to item 52, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence ed from the group ting of SEQ ID NO:1-93, 774-796 and 809-814. 54. PD-L1 binding polypeptide according to item 53, wherein sequence xvii) or xxi) corresponds to the ce from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774-787 and 809-814. 55. PD-L1 binding polypeptide according to item 54, wherein sequence xvii) or xxi) ponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 775, 776, 779-781, 784-786 and 4, such as the group consisting of SEQ ID 3, 776, 780, 781, 784, 786 and 809-814, such as the group consisting of SEQ ID NO:1-93, 776, 781, 784 and 809-814, such as the group consisting of SEQ ID NO:1-93, 776, 784, 809 and 811-814 or the group ting of SEQ ID NO:1—93, 776, 781, 809 and 811-814, for e the group consisting of SEQ ID 3, 776, 809 and 811-814 or the group consisting of SEQ ID NO:1-93, 781, 809 and 811—814 or the group consisting of SEQ ID NO:1-93, 784 and 811-814. 56. PD-L1 binding polypeptide according to item 55, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93, 774, 775, 780—786 and 810—814, such as the group consisting of SEQ ID NO:1-93, SEQ ID , 780, 781, 784,786 and 810-814. 57. PD-L1 binding polypeptide according to any one of items 54-56, wherein ce xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-93 and 811-813. 58. PD-L1 binding polypeptide according to item 57, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-24 and 811- 813. 59. PD-L1 binding polypeptide according to item 58, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1-12, 14, 17-21 and SEQ ID NO:811-812. 60. PD-L1 binding polypeptide according to item 59, wherein ce xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a ce selected from the group ting of SEQ ID NO:1-12, 17, 811 and 812, such as the group consisting of SEQ ID NO:1-5, 17,811 and 812, such as the group consisting of SEQ ID NO:1, 2, 17,811 and 812. 61. PD-L1 binding polypeptide according to item 58, wherein sequence xvii) or xxi) corresponds to the ce from position 1 to position 58 in a sequence selected from the group consisting of SEQ ID NO:1, 4, 5, 6, 9, 14, 18, 19, 20, 21 and 811, such as the group consisting of SEQ ID NO:4, 5, 18 and 21, such as the group consisting of SEQ ID NO:4, 5 and 21. 62. PD—L1 binding polypeptide according to item 60 or 61, wherein sequence xvii) or xxi) corresponds to the ce from position 1 to position 58 in SEQ ID NO:1 or 811. 63. PD-L1 g polypeptide according to item 60, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in SEQ ID N02 or 812. 64. PD-L1 binding polypeptide according to item 60 or 61, wherein sequence xvii) corresponds to the sequence from position 1 to on 58 in SEQ ID NO:4. 65. PD—L1 binding polypeptide according to item 60 or 61, wherein sequence xvii) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:5. 66. PD-L1 binding ptide according to item 61, wherein sequence xvii) corresponds to the sequence from position 1 to position 58 in SEQ ID NO:21. 67. PD-L1 binding ptide according to any preceding item, which is capable of blocking PD—L1 dependent signaling. 68. PD—L1 binding polypeptide according to item 67, wherein the half maximal inhibitory concentration (ICSO) of the ng is at most 5 x 10'8 M, such as at most 1 x10'8 M, such as at most 5 x10'9 M, such as at most 3.5 x '9 M, such as at most 1 x10'9 M, such as at most 5 x10'10 M, such as at most 1 x 10-10 M. 69. PD-L1 binding polypeptide ing to any preceding item, which is capable of blocking the interaction of PD-L1 with PD-1. 70. PD—L1 binding polypeptide according to any preceding item, which is capable of binding to PD-L1 such that the KB value of the interaction is at most 2 x10'8 M, such as at most 1 x10'8 M, such as at most 1 x10'9 M, such as at most 5 x10'10 M, such as at most 3 x10'10 M. 71. PD—L1 binding polypeptide according to any preceding item, which is capable of binding to PD-L1 such that the kd value of the interaction is at most 1 x10"3 s'1, such as at most 6 x104 s'1. 72. PD—L1 binding polypeptide according to any preceding item, which is e of binding to PD-L1 such that the E050 value of the interaction is at most 1 X 10'9 M, such as at most 1 x10"10 M, such as at most 7 x10'11 M. 73. PD-L1 binding polypeptide according to any preceding item, wherein said PD-L1 is human PD-L1. 74. PD—L1 binding polypeptide according to any preceding item which comprises additional amino acids at the C-terminal and/or N-terminal end. 75. PD—L1 binding polypeptide according to item 74, wherein said additional amino ) improve(s) production, purification, stabilization in vivo or in vitro, coupling or detection of the polypeptide. 76. PD—L1 binding polypeptide according to any preceding item in multimeric form, comprising at least two PD-L1 binding ptide monomer units, whose amino acid sequences may be the same or different. 77. PD-L1 binding polypeptide according to item 76, wherein said PD- L1 binding polypeptide monomer units are covalently coupled together. 78. PD-L1 binding polypeptide according to item 77, wherein the PD-L1 g polypeptide monomer units are expressed as a fusion protein. 79. PD—L1 binding polypeptide according to any one of items 76-78, in dimeric form. 80. Fusion protein or conjugate comprising - a first moiety consisting of a PD-L1 binding ptide according to any preceding item; and - a second moiety consisting of a polypeptide having a desired biological activity. 81. Fusion protein or conjugate according to item 80, n said d biological activity is a therapeutic activity. 82. Fusion protein or conjugate ing to item 80, wherein said desired biological activity is a binding activity. 83. Fusion protein or conjugate according to item 80, wherein said desired biological activity is an tic activity. 84. Fusion n or ate according to item 82, wherein said binding ty is albumin binding activity which increases in vivo half-life of the fusion protein or conjugate. 85. Fusion protein or conjugate according to item 84, wherein said second moiety comprises the albumin g domain of streptococcal protein G or a derivative thereof. 86. Fusion protein or ate according to item 82, wherein said g activity acts to block a biological activity. 87. Fusion protein or conjugate ing to item 81, wherein the second moiety is a therapeutically active polypeptide. 88. Fusion protein or conjugate according to item 87, wherein the second moiety is an immune response modifying agent. 89. Fusion protein or conjugate according to item 87, wherein the second moiety is an anti-cancer agent. 90. Fusion protein or conjugate according to any one of items 80—83 and 86—89, wherein the second moiety is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines. 91. Fusion protein according to any one of items 80-91, wherein the second moiety further comprises a linker. 92. Complex, comprising at least one PD-L1 binding ptide according to any one of the ing items and at least one antibody or an antigen g fragment thereof. 93. x according to item 92, wherein said at least one antibody or antigen binding fragment f is selected from the group consisting of full-length antibodies, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fc fragments, Fv fragments, single chain Fv (scFv) fragments, (scFv)2 and domain antibodies. 94. Complex according to item 93, wherein said at least one antibody or antigen binding fragment thereof is selected from the group ting of full-length antibodies, Fab fragments and scFv fragments. 95. x according to item 94, wherein said at least one antibody or antigen binding fragment thereof is a ength antibody. 96. Complex according to any one of items 92—95, wherein said antibody or antigen binding fragment thereof is a monoclonal antibody or an antigen binding fragment thereof.

WO 72280 97. Complex according to any one of items 92-96, wherein said antibody or antigen binding fragment thereof is selected from the group consisting of human antibodies, humanized antibodies and chimeric antibodies, and antigen binding fragments thereof. 98. Complex according to item 97, wherein said antibody or antigen binding fragment f is a human or humanized antibody, or an antigen binding fragment thereof. 99. Complex ing to any one of items 92-98, wherein said PD-L1 binding polypeptide is attached at either the inus or the N-terminus of the heavy chain or the light chain of said antibody or antigen binding fragment thereof. 100. Complex ing to any one of items 92-99, further comprising 101. Complex according to any one of items , wherein said antibody or antigen binding fragment thereof has affinity for an antigen, for example an n associated with an infectious disease, or an antigen associated with cancer. 102. Fusion protein or conjugate according to any one of items 79-90 or complex ing to any one of items 92-101, wherein said second moiety or said antibody or antigen binding fragment thereof is an inhibitor selected from the group consisting of inhibitors of: PD-1, CTLA-4, T-cell immunoglobulin and mucin containing protein-3 (TIM-3), galectin-9 (GAL-9), lymphocyte activation gene-3 (LAG-3), PD-L2, B7 homolog 3 (B7-H3), B? homolog 4 ), V-domain lg suppressor of T-cell activation (VISTA), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), B and T lymphocyte attenuator (BTLA), colony stimulating factor 1 receptor (CSF1 R), herpes virus entry or (HVEM), killer immunoglobulin receptor (KIR), adenosine, adenosine A2a receptor (A2aR), CD200R and T cell lg and ITIM domain. 103. Fusion protein, ate or complex according to item 102, wherein said second moiety, antibody or antigen binding fragment thereof is an inhibitor of PD-1, such as an inhibitor selected from the group consisting of nivolumab, zumab, BMS 936559, MPDL328OA and pembrolizumab, such as pembrolizumab. 104. Fusion protein, conjugate or complex according to item 102, wherein said second moiety, antibody or antigen binding fragment thereof is an inhibitor of CTLA—4, such as an inhibitor ed from the group consisting of belatacept, abatacept and ipilimumab, such as ipilimumab. 105. Fusion protein or conjugate according to any one of items 80-91 or complex according to any one of items 92-101, wherein said second moiety or antibody or antigen g fragment thereof is an agonist selected from the group consisting of ts of CD134, CD40, 4—1 BB and glucocorticoid—induced TNFR-related protein (GITR). 106. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-105, further comprising a label. 107. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to item 106, n said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent nds and inescent proteins, enzymes, uclides, radioactive particles and pretargeting recognition tags. 108. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to item 107, comprising a chelating nment provided by a polyaminopolycarboxylate chelator ated to the PD-L1 binding polypeptide via a thiol group of a cysteine residue or an amine group of a lysine residue. 109. PD-L1 binding polypeptide, fusion n, conjugate or complex according to item 106, which comprises a pretargeting recognition tag forming part of a complementary pair of pretargeting moieties, for example selected from stept(avidin)/biotin, oligonucleotide/complementary oligonucleotide such as DNA/complementary DNA, RNA/complementary RNA, phosphorothioate nucleic acid/ complementary phosphorothioate nucleic acid and peptide nucleic acid/complementary peptide nucleic acid and morpholinos/complementary morpholinos. 110. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to item 109, wherein said pretargeting recognition tag is a peptide nucleic acid tag. 111. PD-L1 binding polypeptide, fusion protein, conjugate or x according to any one of item 110, wherein said pretargeting recognition tag is a 10-20—mer peptide c acid sequence, such as a 15-mer peptide nucleic acid sequence. 112. A polynucleotide encoding a polypeptide according to any one of items 1-105. 113. Expression vector comprising a polynucleotide according to item 112. 114. Host cell comprising an sion vector according to item 113. 115. Method of producing a polypeptide according to any one of items 1-105, comprising — ing a host cell according to item 114 under conditions permissive of expression of said polypeptide from said expression vector, and - isolating said polypeptide. 116. Composition comprising a PD-L1 binding polypeptide, fusion protein, conjugate or x according to any one of items 1-111 and at least one pharmaceutically acceptable excipient or carrier. 117. Composition according to item 116, further comprising at least one additional active agent, such as an agent selected from an immune response modifying agent and an anti-cancer agent. 118. PD-L1 binding polypeptide, fusion n, conjugate or complex according to any one of items 1-111 or a composition according to any one of items 116-1 17 for oral, topical, enous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration, such as for topical stration. 119. PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1-111 or a composition according to any one of items 116-1 17 for use as a medicament, a diagnostic agent and/or a prognostic agent.

WO 72280 120. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 119 as a ment. 121. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 119 as a diagnostic agent and/or a prognostic agent. 122. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use as a medicament according to item 120, wherein said polypeptide, fusion protein, conjugate or composition modulates PD-L1 function in vivo. 123. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use according to any one of items 119-121 in the treatment, prognosis or diagnosis of a PD-L1 related disorder. 124. PD-L1 binding polypeptide, fusion protein, conjugate, x or composition for use ing to item 122, wherein said PD-L1 related disorder is selected from the group consisting of infectious diseases and cancers. 125. PD-L1 binding polypeptide, fusion protein, conjugate, complex or composition for use according to item 124, n said PD-L1 related disorder is an ious disease, such as a chronic viral infection, for example selected from the group consisting of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV). 126. PD-L1 binding polypeptide, fusion protein, ate, complex or ition for use according to item 124, wherein said PD-L1 related disorder is cancer, such as a cancer selected from the group consisting of: - s manifesting solid tumors, for example selected from the group consisting of skin cancer, such as melanoma and nonmelanoma skin cancer (NMSC); lung cancers, such as small cell lung cancer, non-small cell lung cancer (NSCLC); head and neck cancer; renal cell carcinoma (RCC); bladder cancer; breast cancer; colorectal cancer; gastric cancer; ovarian cancer; pancreatic cancer; prostate cancer; glioma; astoma; liver carcinoma; gallbladder cancer; thyroid ; bone cancer; al ; uterine cancer; vulval cancer; endometrial cancer; testicular ; kidney cancer; esophageal carcinoma; brain/CNS cancers; neuronal cancers; mesothelioma; sarcomas; small bowel adenocarcinoma; and pediatric ancies; and - cancers manifesting non—solid tumors, for example mia, acute myeloid leukaemia, acute lymphoblastic leukaemia and le myeloma. 127. PD-L1 g polypeptide, fusion protein, conjugate, complex or composition for use according to item 126, wherein said cancer is selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC, bladder cancer, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, pancreatic cancer and prostate cancer, such as selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC and bladder . 128. Method of treatment of a PD-L1 related er, sing administering to a subject in need thereof an effective amount of a PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of items 1—1 11 or a composition ing to any one of items 116-117. 129. Method according to item 128, wherein said PD—L1 d disorder is selected from the group consisting of infectious disease and cancen 130. Method according to item 129, wherein said PD—L1 related disorder is an infectious disease, such as a chronic viral infection, for example selected from the group consisting of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV). 131. Method according to item 129, wherein said PD—L1 related disorder is cancer, such as a cancer ed from the group consisting of: - cancers manifesting solid tumors, for example selected from the group ting of skin cancer, such as melanoma and nonmelanoma skin cancer (NMSC); lung cancers, such as small cell lung cancer, non-small cell lung cancer (NSCLC); head and neck cancer; renal cell carcinoma (RCC); bladder cancer; breast cancer; colorectal cancer; gastric cancer; ovarian cancer; pancreatic cancer; prostate cancer; glioma; glioblastoma; liver carcinoma; gallbladder cancer; d cancer; bone cancer; al cancer; uterine ; vulval cancer; endometrial cancer; testicular cancer; kidney cancer; esophageal carcinoma; brain/CNS cancers; neuronal cancers; mesothelioma; sarcomas; small bowel arcinoma; and pediatric malignancies; and - cancers manifesting non—solid tumors, for e leukaemia, acute myeloid leukaemia, acute lymphoblastic leukaemia and multiple myeloma. 132. Method according to item 131, in which said cancer is selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC, bladder cancer, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, pancreatic cancer and prostate cancer, such as selected from the group consisting of melanoma, NSCLC, head and neck cancer, RCC and bladder cancer. 133. Method according to any one of items 131-132, comprising the steps of: - contacting the subject with a PD-L1 binding polypeptide, fusion protein, conjugate or complex ing to any one of items 109- 111 comprising a pretargeting ition tag, or with a composition comprising such a PD-L1 binding polypeptide, fusion protein, conjugate or complex, and - contacting the subject with a complementary pretargeting moiety, comprising a radionuclide. 134. Method of ing PD-L1, sing providing a sample suspected to contain PD-L1, contacting said sample with a PD-L1 binding polypeptide, fusion n, conjugate or complex according to any one of items 1-111 or a composition according to any one of items 116-117, and detecting the binding of the PD—L1 binding polypeptide, fusion protein, conjugate, complex or ition to indicate the presence of PD-L1 in the sample. 135. Method for determining the presence PD-L1 in a subject, comprising the steps of: a) contacting the subject, or a sample isolated from the subject, with a PD-L1 binding polypeptide, fusion n, ate or complex according to any one of items 1—1 11 or a composition according to any one of items 116-117, and b) ing a value corresponding to the amount of the PD-L1 binding polypeptide, fusion protein, conjugate or ition that has bound in said t or to said sample. 136. Method according to item 135, in which said PD-L1 binding polypeptide, fusion protein, conjugate or complex is according to any one of items 109-111, or said composition comprises such a PD-L1 binding polypeptide, fusion protein, conjugate or complex, and step a) further comprises contacting the subject with a complementary pretargeting moiety labeled with a detectable label, such as a radionuclide label. 137. Method according to item 135 or 136, further comprising a step of comparing said value to a reference. 138. Method according to any one of items 134-137, wherein said subject is a mammalian subject, such as a human subject. 139. Method according to any one of items 134-138, wherein the method is performed in vivo. 140. Method ing to item 139, which is a method for medical imaging in which - step a) comprises the systemic administration of said PD-L1 binding polypeptide, fusion n, conjugate, x or composition to a ian subject; - said PD-L1 binding polypeptide, fusion protein, conjugate, complex, composition or pretargeting moiety comprises a radionuclide label suitable for medical imaging; and - step b) comprises obtaining one or more images of at least a part of the subject’s body using a medical imaging instrument, said image(s) indicating the presence of the radionuclide inside the body.

Claims

1. PD-L1 binding polypeptide, comprising a PD-L1 binding motif BM, which motif consists of an amino acid sequence selected from: i) ERNX4AAX7EIL X11LPNLX16X17X18QX20 26LX28D wherein, independently from each other, X4 is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V and Y; X7 is selected from A, E, F, H, N, Q, S, T, V, W and Y; X11 is selected from A, D, E, F, H, K, L, N, Q, R, S, T, V, W and Y; X16 is selected from N and T; X17 is selected from A, H, K, N, Q, R and S; X18 is selected from A, D, E, G, H, K, L, N, Q, R, S, T, V and Y; X20 is selected from H, I, K, L, N, Q, R, T, V and Y; X26 is selected from K and S; and X28 is selected from A, D and E; ii) an amino acid sequence which has at least 96 % identity to any one of the ces d in i).

2. PD-L1 binding ptide according to claim 1, wherein sequence i) corresponds to the sequence from position 8 to position 36 in a sequence selected from the group consisting of SEQ ID NO:1-808, such as the group consisting of SEQ ID NO:1-93, such as the group consisting of SEQ ID NO:1-24, such as the group consisting of SEQ ID NO:1, 2, 4, 5 and 21, such as the group consisting of SEQ ID NO:1 and 2.

3. PD-L1 binding polypeptide according to any preceding claim, wherein said PD-L1 binding motif forms part of a three-helix bundle n domain.

4. PD-L1 binding polypeptide according to any preceding claim, which comprises a binding module BMod, the amino acid sequence of which is ed from: iii) K-[BM]-DPSQSXaXbLLXc EAKKLXdXeXfQ; wherein Xa is selected from A and S; Xb is selected from N and E; Xc is selected from A, S and C; Xd is selected from E, N and S; Xe is selected from D, E and S; and Xf is ed from A and S; iv) an amino acid sequence which has at least 93 % ty to any one of the sequences d in iii); wherein [BM] is a PD-L1 binding motif as defined in any one of claims 1-2.

5. PD-L1 binding polypeptide according to any preceding claim, which comprises an amino acid sequence selected from: xvii) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; xviii) an amino acid sequence which has at least 89 % identity to any one of the sequences defined in xvii); wherein [BM] is a PD-L1 binding motif as defined in any one of claims 1-2.

6. PD-L1 binding polypeptide according to any one of claims 1-4, which comprises an amino acid sequence selected from: xxi) AEAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK; xxii) an amino acid sequence which has at least 89 % ty to any one of the sequences defined in xxi); wherein [BM] is a PD-L1 binding motif as defined in any one of claims 1-2.

7. PD-L1 binding polypeptide according to claim 5 or 6, wherein sequence xvii) or xxi) corresponds to the sequence from position 1 to position 58 in a sequence selected from the group ting of SEQ ID NO:1-814, such as the group consisting of SEQ ID NO:1-93 and 811-813, such as the group consisting of SEQ ID NO:1-24 and 3, such as the group consisting of SEQ ID NO:1, 2, 4, 5, 21, 811 and 812, such as the group consisting of SEQ ID NO:1 and 2 or SEQ ID NO:811 and 812.

8. PD-L1 binding ptide according to any preceding claim, which is capable of blocking PD-L1 dependent signaling.

9. PD-L1 binding polypeptide according to claim 8, wherein the half maximal tory concentration (IC50) of the blocking is at most 5 x 10-8 M, such as at most 1 x 10-8 M, such as at most 5 x 10-9 M, such as at most 3.5 x 10-9 M, such as at most 1 x 10-9 M, such as at most 5 x 10-10 M, such as at most 1 x 10-10

10. PD-L1 binding polypeptide ing to any preceding claim, which is capable of blocking the interaction of PD-L1 with PD-1.

11. PD-L1 binding polypeptide according to any preceding claim, which is capable of binding to PD-L1 such that the KD value of the interaction is at most 2 x 10-8 M, such as at most 1 x 10-8 M, such as at most 1 x 10-9 M, such as at most 5 x 10-10 M, such as at most 3 x 10-10 M.

12. Fusion protein or conjugate comprising - a first moiety ting of a PD-L1 binding polypeptide according to any preceding claim; and - a second moiety consisting of a polypeptide having a desired biological activity.

13. Complex, comprising at least one PD-L1 binding polypeptide, fusion n or conjugate according to any one of the preceding claims and at least one dy or an antigen binding fragment thereof.

14. A polynucleotide encoding a polypeptide, fusion protein, conjugate or complex ing to any one of claims 1-13.

15. Composition comprising a PD-L1 binding ptide, fusion protein, conjugate or x according to any one of claims 1-13 and at least one pharmaceutically acceptable excipient or carrier.

16. Use of a PD-L1 binding polypeptide, fusion protein, conjugate or complex according to any one of claims 1-13 in the manufacture of a medicament for the treatment, prognosis and/or diagnosis of a PD-L1 related disorder.

17. Use according to claim 16, wherein said medicament modulates PDL1 function in vivo.

18. Use according to any one of claims 16-17, wherein the PD-L1 related disorder is selected from the group consisting of infectious diseases and cancers. H N m \ I \ : I m.“ ! hm !I!I!! 959; ! -mmmwqqmmmommannnmgl_u¢zmomnen4mqqumwgmmmmmwmdmm mumHEmmommn H H H Lmzmom {mzimzmom ngwﬁEo¢ ngmom hﬂEMOQZB. O; .5 .5 Emgmzn Cmﬁmmm Cmﬁnm H hmgmoomB_ngmgkmo¢<ommeg mmgkoooeg oeg EImmzmomoeg 8:82me Q) OW Q) OO OW OW Q) ‘_ LI) k0 OW C) (*7 k0 OW C) \ I OW OW C) C) C) C) \—I H \—I [\ [\ CO CO Q) CO Q) 00 (I) I I I I I | I \ | \ N N EmmaIChLO IE!!!!!!I[\LO !!!III!!!I!!IE!!!!!!I00 QM QM QM QM QM QM QM mmmmqqmmmammm Mmmmggmmmommm Mmmmggmwmommn‘ Mﬁmmggzmmommmw M cozmcgmwo [\ O LO 8:82me 0 l\ LO O (V) m W <1“ l\ (\1 ('7 (V) ('7 <14 (\1 (’7 l\ l\ l\ l\ l\ CO CO [\ [\ [\ [\ [\ I I I | \ | | \ I 5/42 I\ C) (\I <14 [\ CO m LO C) \—I mm W LO \ I (\I (\I (\I (\I (\I m (Y) <14 8:82me <1” (\I O W (Y) [\ Lr) LO [\ [\ 0:) w I.\ l\ I.\ I.\ I.\ I.\ [\ [\ [\ [\ [\ [\ I I I \ I I \ I N N I\ CO N cozmcgmwo MN LO [\ (I) HN (‘0 cozmcgmwo OW m Q) LO (\l [\ 0 C\] m (‘0 <1” Lr) L0 k0 03 w w 03 w 03 OD [\ [\ [\ [\ [\ [\ [\ I I I I \ I I \ | 8/42 wow mom mom Pom mom mom O HNMWDLO 03 CD \ : \:\|x:\:\|\: i \ E (\1 I\ NN N QMMﬂmeQmmemmﬂﬂﬂQMEI M cozmcgmwo w (Y) LO N LO O (Y) KO [\ [\ 0:) w OW m 0) w w w w OD w [\ [\ [\ [\ [\ [\ [\ I | I | \ I I \ I N N