Novel fusion proteins specific for CD137 and PD-L1

文档序号：639071 发布日期：2021-05-11 浏览：17次中文

阅读说明：本技术 对cd137和pd-l1特异性的新型融合蛋白 (Novel fusion proteins specific for CD137 and PD-L1 ) 是由 M·帕夫利杜 L·帕塔里尼 A·斯科勒-达希雷尔 C·罗特 S·奥威尔 R·贝莱巴 M· 于 2019-07-31 设计创作，主要内容包括：本发明提供对CD137和PD-L1特异性的融合蛋白,其中融合蛋白能够用于以PD-L1靶标依赖性方式共刺激淋巴细胞活化。这种融合蛋白可用于许多制药应用中,例如作为用于治疗或预防诸如各种肿瘤的人疾病的抗癌剂和/或免疫调节剂。本发明还涉及制备本文所述的融合蛋白以及包含这种融合蛋白的组合物的方法。本发明进一步涉及编码这种融合蛋白的核酸分子和用于产生这种融合蛋白和核酸分子的方法。此外,本申请公开了这些融合蛋白以及包含一种或多种这种融合蛋白的组合物的治疗性和/或诊断性用途。(The present invention provides fusion proteins specific for CD137 and PD-L1, wherein the fusion proteins are capable of being used to co-stimulate lymphocyte activation in a PD-L1 target-dependent manner. Such fusion proteins are useful in a number of pharmaceutical applications, for example as anti-cancer and/or immunomodulatory agents for the treatment or prevention of human diseases such as various tumors. The invention also relates to methods of making the fusion proteins described herein and compositions comprising such fusion proteins. The invention further relates to nucleic acid molecules encoding such fusion proteins and methods for producing such fusion proteins and nucleic acid molecules. Furthermore, the present application discloses therapeutic and/or diagnostic uses of these fusion proteins and compositions comprising one or more of such fusion proteins.)

1. A fusion protein capable of binding to CD137 and PD-L1, wherein the fusion protein comprises at least two subunits in any order, wherein a first subunit comprises a full-length immunoglobulin or antigen-binding domain thereof and is specific for PD-L1, and wherein a second subunit comprises a lipocalin mutein and is specific for CD 137.

2. The fusion protein of claim 1, further comprising a third subunit comprising a lipocalin mutein specific for CD 137.

3. The fusion protein of claim 1 or 2, wherein the fusion protein is capable of a K of up to about 2nM_DA K that binds PD-L1 or is capable of binding to an immunoglobulin or antigen binding domain thereof comprised in said first subunit alone _DK of comparable or lower value_DValues were combined with PD-L1.

4. The fusion protein of claim 1 or 2, wherein the fusion protein is capable of a K of up to about 7nM_DValue binds to CD137 or(ii) a K alone capable of cleaving the lipocalin mutein specific for CD137 comprised in said second subunit_DK of comparable or lower value_DValues bind to CD 137.

5. The fusion protein of claim 3 or 4, wherein the K is_DValues were determined by Surface Plasmon Resonance (SPR) measurements.

6. The fusion protein of claim 1 or 2, wherein the fusion protein is capable of an EC of up to about 0.5nM₅₀Value binding to PD-L1 or to EC alone capable of binding to an immunoglobulin or antigen binding domain thereof comprised in said first subunit₅₀EC with equivalent or lower value₅₀Values were combined with PD-L1.

7. The fusion protein of claim 1 or 2, wherein the fusion protein is capable of an EC of up to about 0.6nM₅₀Value binding to CD137 or capable of binding to EC alone of a lipocalin mutein specific for CD137 comprised in said second subunit₅₀EC with equivalent or lower value₅₀Values bind to CD 137.

8. The fusion protein of any one of claims 6-7, wherein the EC is₅₀Values were determined by enzyme-linked immunosorbent assay (ELISA) assay.

9. The fusion protein of claim 1 or 2, wherein the fusion protein is cross-reactive with cynomolgus PD-L1.

10. The fusion protein of claim 1 or 2, wherein the fusion protein cross-reacts with cynomolgus CD 137.

11. The fusion protein of claim 1 or 2, wherein the fusion protein is capable of an EC of up to about 10nM when the fusion protein is measured in an ELISA assay₅₀Values bind both CD137 and PD-L1.

12. The fusion protein of claim 1 or 2, wherein the fusion protein is capable of an EC of up to about 60nM₅₀Values bind to CD137 expressed on the cells.

13. The fusion protein of claim 1 or 2, wherein the fusion protein is capable of an EC of up to about 10nM when the fusion protein is measured in a flow cell assay₅₀Values bind to PD-L1 expressed on the cells.

14. The fusion protein of claim 1 or 2, wherein the fusion protein is capable of binding to a tumor cell expressing PD-L1.

15. The fusion protein of claim 1 or 2, wherein the fusion protein is capable of binding to CD137 in the presence of a CD137 ligand.

16. The fusion protein of claim 1 or 2, wherein the fusion protein is capable of competing with PD-1 for binding to PD-L1.

17. The fusion protein of claim 1 or 2, wherein the fusion protein is capable of competing with the antibodies set forth in SEQ ID nos. 28 and 29 for binding to CD 137.

18. The fusion protein of claim 1 or 2, wherein the fusion protein has an overlapping CD 137-binding epitope with the antibodies set forth in SEQ ID nos. 28 and 29.

19. The fusion protein of any one of claims 1-18, wherein the fusion protein is capable of stimulating T cell proliferation and/or response.

20. The fusion protein of any one of claims 1-19, wherein the fusion protein is capable of stimulating CD4+ and/or CD8+ T cell proliferation.

21. The fusion protein of any one of claims 1-20, wherein the fusion protein is capable of inducing increased secretion of IL-2 and/or IFN- γ.

22. The fusion protein of any one of claims 1-21, wherein the fusion protein is capable of inducing increased secretion of a cytotoxic factor.

23. The fusion protein of any one of claims 1-22, wherein the fusion protein is capable of co-stimulating a T cell response in a PD-L1-dependent manner.

24. The fusion protein of any one of claims 1-23, wherein the fusion protein is capable of co-stimulating a T cell response in a tumor microenvironment.

25. The fusion protein of any one of claims 1-24, wherein the fusion protein does not co-stimulate a T cell response in the absence of PD-L1.

26. The fusion protein of any one of claims 1-25, wherein the fusion protein is capable of blocking an inhibitory signal of PD-1.

27. The fusion protein of any one of claims 1-26, wherein the fusion protein has an antibody-like pharmacokinetic profile.

28. The fusion protein of any one of claims 1-27, wherein the fusion protein has an amino acid sequence that is greater than SEQ ID NO: 147 or SEQ ID NO: 148 more favorable pharmacokinetic profile.

29. The fusion protein of any one of claims 1-28, wherein the lipocalin mutein comprises one or more mutated amino acid residues at positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1).

30. The fusion protein according to claim 29, wherein the amino acid sequence of the lipocalin mutein comprises one or more of the following mutated amino acid residues at one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequence of mature hTalc (SEQ ID NO: 1): ala 5 → Val or Thr; arg 26 → Glu; glu 27 → Gly; phe 28 → Cys; pro 29 → Arg; glu 30 → Pro; met 31 → Trp; leu 33 → Ile; glu 34 → Phe; thr 42 → Ser; gly 46 → Asp; lys 52 → Glu; leu 56 → Ala; ser 58 → Asp; arg 60 → Pro; cys 61 → Ala; lys 65 → Arg or Asn; thr 71 → Ala; val 85 → Asp; lys 94 → Arg or Glu; cys 101 → Ser; glu 104 → Val; leu 105 → Cys; his 106 → Asp; lys 108 → Ser; arg 111 → Pro; lys 114 → Trp; lys 121 → Glu; ala 133 → Thr; arg 148 → Ser; ser 150 → Ile and Cys 153 → Ser.

31. The fusion protein according to claim 29 or 30, wherein the amino acid sequence of the lipocalin mutein comprises one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1):

(a) arg 26 → Glu; glu 27 → Gly; phe 28 → Cys; pro 29 → Arg; glu 30 → Pro; met 31 → Trp; leu 33 → Ile; glu 34 → Phe; leu 56 → Ala; ser 58 → Asp; arg 60 → Pro; cys 61 → Ala; cys 101 → Ser; glu 104 → Val; leu 105 → Cys; his 106 → Asp; lys 108 → Ser; arg 111 → Pro; lys 114 → Trp; and Cys 153 → Ser;

(b) ala 5 → Thr; arg 26 → Glu; glu 27 → Gly; phe 28 → Cys; pro 29 → Arg; glu 30 → Pro; met 31 → Trp; leu 33 → Ile; glu 34 → Phe; leu 56 → Ala; ser 58 → Asp; arg 60 → Pro; cys 61 → Ala; lys 65 → Arg; val 85 → Asp; cys 101 → Ser; glu 104 → Val; leu 105 → Cys; his 106 → Asp; lys 108 → Ser; arg 111 → Pro; lys 114 → Trp; lys 121 → Glu; ala 133 → Thr; and Cys 153 → Ser;

(c) arg 26 → Glu; glu 27 → Gly; phe 28 → Cys; pro 29 → Arg; glu 30 → Pro; met 31 → Trp; leu 33 → Ile; glu 34 → Phe; leu 56 → Ala; ser 58 → Asp; arg 60 → Pro; cys 61 → Ala; lys 65 → Asn; lys 94 → Arg; cys 101 → Ser; glu 104 → Val; leu 105 → Cys; his 106 → Asp; lys 108 → Ser; arg 111 → Pro; lys 114 → Trp; lys 121 → Glu; ala 133 → Thr; and Cys 153 → Ser;

(d) Ala 5 → Val; arg 26 → Glu; glu 27 → Gly; phe 28 → Cys; pro 29 → Arg; glu 30 → Pro; met 31 → Trp; leu 33 → Ile; glu 34 → Phe; leu 56 → Ala; ser 58 → Asp; arg 60 → Pro; cys 61 → Ala; lys 65 → Arg; lys 94 → Glu; cys 101 → Ser; glu 104 → Val; leu 105 → Cys; his 106 → Asp; lys 108 → Ser; arg 111 → Pro; lys 114 → Trp; lys 121 → Glu; ala 133 → Thr; and Cys 153 → Ser;

(e) arg 26 → Glu; glu 27 → Gly; phe 28 → Cys; pro 29 → Arg; glu 30 → Pro; met 31 → Trp; leu 33 → Ile; glu 34 → Phe; thr 42 → Ser; leu 56 → Ala; ser 58 → Asp; arg 60 → Pro; cys 61 → Ala; cys 101 → Ser; glu 104 → Val; leu 105 → Cys; his 106 → Asp; lys 108 → Ser; arg 111 → Pro; lys 114 → Trp; ser 150 → Ile; and Cys 153 → Ser;

(f) arg 26 → Glu; glu 27 → Gly; phe 28 → Cys; pro 29 → Arg; glu 30 → Pro; met 31 → Trp; leu 33 → Ile; glu 34 → Phe; lys 52 → Glu; leu 56 → Ala; ser 58 → Asp; arg 60 → Pro; cys 61 → Ala; thr 71 → Ala; cys 101 → Ser; glu 104 → Val; leu 105 → Cys; his 106 → Asp; lys 108 → Ser; arg 111 → Pro; lys 114 → Trp; ala 133 → Thr; arg 148 → Ser; ser 150 → Ile; and Cys 153 → Ser; and

(g) Ala 5 → Thr; arg 26 → Glu; glu 27 → Gly; phe 28 → Cys; pro 29 → Arg; glu 30 → Pro; met 31 → Trp; leu 33 → Ile; glu 34 → Phe; gly 46 → Asp; leu 56 → Ala; ser 58 → Asp; arg 60 → Pro; cys 61 → Ala; thr 71 → Ala; cys 101 → Ser; glu 104 → Val; leu 105 → Cys; his 106 → Asp; lys 108 → Ser; arg 111 → Pro; lys 114 → Trp; ser 150 → Ile; and Cys 153 → Ser.

32. The fusion protein of any one of claims 29-32, wherein the amino acid sequence of the lipocalin mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-40 or a fragment or variant thereof.

33. The fusion protein of any one of claims 29-32, wherein the amino acid sequence of the lipocalin mutein hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 34-40 have at least 85% sequence identity.

34. The fusion protein of any one of claims 1-28, wherein the lipocalin mutein comprises one or more mutated amino acid residues at positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence (SEQ ID NO: 2) of mature human neutrophil gelatinase-associated lipocalin (hNGAL).

35. The fusion protein of claim 34, wherein the amino acid sequence of the lipocalin mutein comprises one or more of the following mutated amino acid residues at positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence (SEQ ID NO: 2) of mature human neutrophil gelatinase-associated lipocalin (hNGAL): gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Arg or Lys; gln 49 → Val, Ile, His, Ser or Asn; tyr 52 → Met; asn 65 → Asp; ser 68 → Met, Ala or Gly; leu 70 → Ala, Lys, Ser or Thr; arg 72 → Asp; lys 73 → Asp; asp 77 → Met, Arg, Thr or Asn; trp 79 → Ala or Asp; arg 81 → Met, Trp or Ser; phe 83 → Leu; cys 87 → Ser; leu 94 → Phe; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu and Lys 134 → Tyr.

36. The fusion protein of any one of claims 1-28, wherein the lipocalin mutein comprises one or more mutated amino acid residues at positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 of the linear polypeptide sequence (SEQ ID NO: 2) of mature human neutrophil gelatinase-associated lipocalin (hNGAL).

37. The fusion protein of claim 36, wherein the amino acid sequence of the lipocalin mutein comprises one or more of the following mutated amino acid residues at positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): gln 20 → Arg; asn 25 → Tyr or Asp; gln 28 → His; val 33 → Ile; leu 36 → Met; ala 40 → Asn; ile 41 → Leu; glu 44 → Val or Asp; gln 49 → His; tyr 52 → Ser or Gly; lys 59 → Asn; ser 68 → Asp; leu 70 → Met; phe 71 → Leu; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln or His; tyr 78 → His; trp 79 → Ile; ile 80 → Asn; arg 81 → Trp or Gln; thr 82 → Pro; cys 87 → Ser; phe 92 → Leu or Ser; asn 96 → Phe; lys 98 → Arg; tyr 100 → Asp; pro 101 → Leu; leu 103 → His or Pro; phe 122 → Tyr; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly.

38. The fusion protein of any one of claims 34-37, wherein the amino acid sequence of the lipocalin mutein comprises one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):

(a) Gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Lys; gln 49 → Asn; tyr 52 → Met; ser 68 → Gly; leu 70 → Thr; arg 72 → Asp; lys 73 → Asp; asp 77 → Thr; trp 79 → Ala; arg 81 → Ser; cys 87 → Ser; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu; and Lys 134 → Tyr;

(b) gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Arg; gln 49 → Ile; tyr 52 → Met; asn 65 → Asp; ser 68 → Met; leu 70 → Lys; arg 72 → Asp; lys 73 → Asp; asp 77 → Met; trp 79 → Asp; arg 81 → Trp; cys 87 → Ser; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu; and Lys 134 → Tyr;

(c) gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Arg; gln 49 → Asn; tyr 52 → Met; asn 65 → Asp; ser 68 → Ala; leu 70 → Ala; arg 72 → Asp; lys 73 → Asp; asp 77 → Thr; trp 79 → Asp; arg 81 → Trp; cys 87 → Ser; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu; and Lys 134 → Tyr;

(d) Gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Lys; gln 49 → Asn; tyr 52 → Met; asn 65 → Asp; ser 68 → Ala; leu 70 → Ala; arg 72 → Asp; lys 73 → Asp; asp 77 → Thr; trp 79 → Asp; arg 81 → Trp; cys 87 → Ser; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu; and Lys 134 → Tyr;

(e) gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Lys; gln 49 → Ser; tyr 52 → Met; asn 65 → Asp; ser 68 → Gly; leu 70 → Ser; arg 72 → Asp; lys 73 → Asp; asp 77 → Thr; trp 79 → Ala; arg 81 → Met; cys 87 → Ser; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu; and Lys 134 → Tyr;

(f) gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Lys; gln 49 → Val; tyr 52 → Met; asn 65 → Asp; ser 68 → Gly; leu 70 → Thr; arg 72 → Asp; lys 73 → Asp; asp 77 → Arg; trp 79 → Asp; arg 81 → Ser; cys 87 → Ser; leu 94 → Phe; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu; and Lys 134 → Tyr;

(g) Gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Arg; gln 49 → His; tyr 52 → Met; asn 65 → Asp; ser 68 → Gly; leu 70 → Thr; arg 72 → Asp; lys 73 → Asp; asp 77 → Thr; trp 79 → Ala; arg 81 → Ser; cys 87 → Ser; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu; and Lys 134 → Tyr;

(h) gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Lys; gln 49 → Asn; tyr 52 → Met; asn 65 → Asp; ser 68 → Gly; leu 70 → Thr; arg 72 → Asp; lys 73 → Asp; asp 77 → Thr; trp 79 → Ala; arg 81 → Ser; phe 83 → Leu; cys 87 → Ser; leu 94 → Phe; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu; and Lys 134 → Tyr;

(i) gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Arg; gln 49 → Ser; tyr 52 → Met; asn 65 → Asp; ser 68 → Ala; leu 70 → Thr; arg 72 → Asp; lys 73 → Asp; asp 77 → Asn; trp 79 → Ala; arg 81 → Ser; cys 87 → Ser; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu; and Lys 134 → Tyr;

(j) Leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Ser; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(k) leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Ser; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; lys 98 → Arg; tyr 100 → Asp; pro 101 → Leu; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(l) Asn 25 → Tyr; leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Gly; ser 68 → Asp; leu 70 → Met; phe 71 → Leu; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Gln; phe 92 → Ser; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(m) Leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Gly; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; tyr 78 → His; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(n) Asn 25 → Asp; leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Gly; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(o) Val 33 → Ile; leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Gly; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(p) Gln 20 → Arg; leu 36 → Met; ala 40 → Asn; ile 41 → Leu; glu 44 → Val; gln 49 → His; tyr 52 → Gly; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; phe 122 → Tyr; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(q) Leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Ser; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; ile 80 → Asn; arg 81 → Trp; thr 82 → Pro; asn 96 → Phe; tyr 100 → Asp; pro 101 → Leu; leu 103 → Pro; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(r) Leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Gly; lys 59 → Asn; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly; and

(s) Leu 36 → Met; ala 40 → Asn; ile 41 → Leu; glu 44 → Asp; gln 49 → His; tyr 52 → Ser; ser 68 → Asp; leu 70 → Met; phe 71 → Leu; arg 72 → Leu; lys 73 → Asp; asp 77 → His; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly.

39. The fusion protein of any one of claims 34-38, wherein the amino acid sequence of the lipocalin mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-59 or a fragment or variant thereof.

40. The fusion protein of any one of claims 34-38, wherein the amino acid sequence of the lipocalin mutein hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 41-59 have at least 85% sequence identity.

41. The fusion protein of any one of claims 1-40, wherein one subunit is linked to another subunit by a linker.

42. The fusion protein of any one of claims 1-41, wherein the N-terminus of the second subunit is linked to the N-or C-terminus of each heavy chain constant region (CH) of the first subunit or to the N-or C-terminus of each light chain constant region (CL) of the first subunit by a linker.

43. The fusion protein of any one of claims 1-42, wherein the N-terminus of the third subunit is linked to the N-or C-terminus of each heavy chain constant region (CH) of the first subunit, to the N-or C-terminus of each light chain constant region (CL) of the first subunit, or to the C-terminus of each second subunit by a linker.

44. The fusion protein of any one of claims 41-43, wherein the linker is non-structural (Gly-Gly-Gly-Gly-Ser)₃Linker (SEQ ID NO: 13).

45. The fusion protein of any one of claims 41-43, wherein the linker is a non-structural glycine-serine linker, a polyproline linker, a proline-alanine-serine polymer, or a peptide selected from the group consisting of SEQ ID NOs: 13-23.

46. The fusion protein of any one of claims 1-45, wherein the first subunit is an antibody.

47. The fusion protein of claim 46, wherein the heavy chain variable region of the antibody is selected from the group consisting of SEQ ID NOs: 75-79, and wherein the light chain variable region of said monoclonal antibody is selected from the group consisting of SEQ ID NOs: 80-84.

48. The fusion protein of claim 46, wherein the antibody comprises a heavy chain variable region sequence that is SEQ ID NOs: 85-86, and the heavy chain of any one of SEQ ID NOs: 87, in a pharmaceutically acceptable carrier.

49. The fusion protein of claim 46, wherein the antibody comprises a heavy chain variable region and a light chain variable region, respectively, as follows: SEQ ID NOs: 75 and 80, SEQ ID NOs: 76 and 81, SEQ ID NOs: 77 and 82, SEQ ID NOs: 78 and 83, or SEQ ID NOs: 79 and 84.

50. The fusion protein of claim 46, wherein the antibody comprises heavy and light chains, respectively, as follows: SEQ ID NOs: 85 and 87, and SEQ ID NOs: 86 and 87.

51. The fusion protein of claim 46, wherein the heavy chain of the antibody comprises one of the following sets of CDR sequences:

(a)GFSLSNYD(HCDR1，SEQ ID NO：59)，IWTGGAT(HCDR2，SEQ ID NO：60)，VRDSNYRYDEPFTY(HCDR3；SEQ ID NO：61)；

(b) GFDIKDTY (HCDR1, SEQ ID NO: 65), IDPADGNT (HCDR2, SEQ ID NO: 66), ARGLGAWFAS (HCDR 3; SEQ ID NO: 67); and

(c)GFNIKDTY(HCDR1，SEQ ID NO：70)，IDPANGNT(HCDR2，SEQ ID NO：71)，SRGPPGGIGEYIYAMDY(HCDR3；SEQ ID NO：72)。

52. The fusion protein of claim 46, wherein the light chain of the antibody comprises one of the following sets of CDR sequences:

(a)QSIGTN(LCDR1，SEQ ID NO：63)，YAS(LCDR2)，QQSNSWPYT(LCDR3；SEQ ID NO：64)；

(b) QDITNS (LCDR1, SEQ ID NO: 68), YTS (LCDR2), QQGHTLPPT (LCDR 3; SEQ ID NO: 69); and

(c)SSVSSSY(LCDR1，SEQ ID NO：73)，STS(LCDR2)，HQYHRSPPT(LCDR3；SEQ ID NO：74)。

53. the fusion protein of claim 46, wherein the heavy chain of the antibody comprises the following set of CDR sequences: GFSLSNYD (HCDR1, SEQ ID NO: 59), IWTGGAT (HCDR2, SEQ ID NO: 60) and VRDSNYRYDEPFTY (HCDR 3; SEQ ID NO: 61), and the light chain of the antibody comprises the following set of CDR sequences: QSIGTN (LCDR1, SEQ ID NO: 62), YAS (LCDR2) and QQSNSWPYT (LCDR 3; SEQ ID NO: 63).

54. The fusion protein of claim 46, wherein the monoclonal antibody has an IgG4 backbone.

55. The fusion protein of claim 54, wherein the IgG4 backbone has one or more of the following mutations: S228P, N297A, F234A, L235A, M428L, N434S, M252Y, S254T, and T256E.

56. The fusion protein of any one of claims 1-55, wherein the fusion protein comprises the amino acid sequence of SEQ ID NOs: 86-94, or wherein the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 88-94, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or more.

57. The fusion protein of any one of claims 1-56, wherein the fusion protein comprises the amino acid sequence of SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, or SEQ ID NOs: 90 and 91.

58. The fusion protein of any one of claims 1-56, wherein the fusion protein comprises a sequence identical to any one of SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87 or SEQ ID NOs: 90 and 91, having a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or more.

59. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1-58.

60. The nucleic acid molecule of claim 59, wherein the nucleic acid molecule is operably linked to regulatory sequences that allow expression of the nucleic acid molecule.

61. The nucleic acid molecule of claim 59 or 60, wherein the nucleic acid molecule is comprised in a vector or a phagemid vector.

62. A host cell comprising the nucleic acid molecule of any one of claims 58-60.

63. A method of producing the fusion protein of any one of claims 1-62, wherein the fusion protein is produced starting from a nucleic acid encoding the fusion protein.

64. The method of claim 63, wherein the fusion protein is produced in a bacterial or eukaryotic host organism and isolated from the host organism or culture thereof.

65. Use of a fusion protein according to any one of claims 1-58 or a composition comprising such fusion protein for simultaneously activating the downstream signaling pathway of CD137 and engaging PD-L1-positive tumor cells.

66. A method of simultaneously activating a downstream signaling pathway of CD137 and engaging PD-L1-positive tumor cells, the method comprising applying to a tumor-containing tissue one or more fusion proteins of any one of claims 1-58 or one or more compositions comprising such fusion proteins.

67. A method of co-stimulating T cells and engaging PD-L1-positive tumor cells simultaneously, the method comprising applying to a tumor-containing tissue one or more fusion proteins of any one of claims 1-58 or one or more compositions comprising such fusion proteins.

68. A method of simultaneously inducing lymphocyte activity and engaging PD-L1-positive tumor cells, the method comprising applying to a tumor-containing tissue one or more fusion proteins of any one of claims 1-58 or one or more compositions comprising such fusion proteins.

69. A method of inducing CD137 aggregation and activation on T cells and directing the T cells to PD-L1-positive tumor cells, the method comprising applying to a tumor-containing tissue one or more fusion proteins of any one of claims 1-58 or one or more compositions comprising such fusion proteins.

70. A method of inducing a local lymphocyte response in the vicinity of PD-L1-positive tumor cells, the method comprising applying to a tumor-containing tissue one or more fusion proteins of any one of claims 1-58 or one or more compositions comprising such fusion proteins.

71. A method of inducing increased secretion of IL-2 and/or cytotoxic factors by T cells in the vicinity of PD-L1-positive tumor cells, the method comprising applying to a tumor-containing tissue one or more fusion proteins of any one of claims 1-58 or one or more compositions comprising such fusion proteins.

72. The method of claim 71, wherein said cytotoxic factor is selected from the group consisting of perforin, granzyme B and granzyme A.

73. A method of inducing increased secretion of cytotoxic factors by T cells in the vicinity of PD-L1-positive tumor cells, the method comprising applying to a tumor-containing tissue one or more fusion proteins of any one of claims 1-58 or one or more compositions comprising such fusion proteins.

74. A pharmaceutical composition comprising one or more fusion proteins of any one of claims 1-58.

75. A method of preventing, ameliorating, or treating PD-L1-positive cancer, the method comprising applying to a tumor-containing tissue one or more fusion proteins of any one of claims 1-58 or one or more compositions comprising such fusion proteins.

76. A fusion protein according to any one of claims 1-58 for use in therapy.

77. The fusion protein for use according to claim 70, wherein the use is in the treatment of cancer.

78. Use of a fusion protein according to any one of claims 1-58 in the manufacture of a medicament.

79. The use according to claim 78, wherein the medicament is for cancer treatment.

Background

Programmed death-ligand 1, or PD-L1 (also known as cluster of differentiation 274 or CD274 and B7 homolog 1 or B7-H1), is a single pass type I membrane protein belonging to the B7 family of co-stimulatory/co-inhibitory molecules for antigen presentation. The extracellular portion of PD-L1 contains two domains, an N-terminal IgV-type domain and an IgC-type domain. PD-L1 has a short cytoplasmic domain without any significant signal transduction motifs, which has led to the initial belief that PD-L1 does not have intrinsic signaling as a receptor. However, recent data indicate that the cytoplasmic domain of PD-L1 contains a non-classical conserved signaling motif that inhibits Interferon (IFN) transduction and protects cancer cells from IFN cytotoxicity (Gato-Canas et al, Cell Rep, 2017).

PD-L1 plays a key role in the suppression of the immune system during pregnancy, chronic infections, tissue allografts, autoimmune diseases and cancer. PD-L1 is expressed on a variety of cell types including B cells, T cells, macrophages, myeloid dendritic cells, mast cells, epithelial and vascular endothelial cells. PD-L1 is also expressed in a variety of cancer types including, but not limited to, melanoma, lung, bladder, colon, and breast cancers. High PD-L1 expression levels are associated with increased tumor invasiveness by mediating depletion and anergy of tumor infiltrating T cells, secretion of immunosuppressive cytokines and protection from lysis by cytotoxic T cells.

PD-L1 is a ligand for programmed cell death protein 1(PD-1), and PD-1 is the major immune checkpoint inhibitory receptor that is expressed primarily on activated T cells, but also on other cells of the immune system, including B cells and monocytes. PD-1 is a member of the immunoglobulin family, which contains an IgV-like extracellular domain, a transmembrane domain, and a cytoplasmic tail with an ITIM (immunoreceptor tyrosine-based inhibitory motif) and an ITSM (immunoreceptor tyrosine-based switch motif). The engagement of PD-1 by PD-L1 results in the recruitment of src homolog 2 domain containing tyrosine phosphatases 1 and 2(SHP 1 and 2) to the intracellular transition motif of PD-1 and results in the expression of E3 ubiquitin ligase of the CBL family. These ubiquitin ligases then ubiquitinate and inactivate key TCR signaling mediators, resulting in the removal of TCRs from the cell surface (Karwacz et al EMBO Mol Med, 2011). SHP 1 and SHP 2 phosphatases directly inhibit TCR signaling by terminating ZAP70 phosphorylation with PI 3K. Furthermore, PD-1 conjugated to PD-L1 can cause TCR signaling pathway inhibition by affecting the expression and activity of CK2 and Cyclin Dependent Kinase (CDK) (Arasanz et al, Oncotarget, 2017). Also studies have shown that PD-1 engagement leads to reprogramming of T cell metabolism from increased glycolysis (which is required to generate energy for effector function) to fatty acid beta-oxidation (which is associated with long-lived cells). This also explains the survival and persistence of high PD-1 expressing cells in patients with chronic infection and cancer (Patsoukis et al, Nat Commun, 2015).

Blocking the PD-1/PD-L1 interaction by anti-PD-1 or anti-PD-L1 targeting agents can reverse immune checkpoint function and release the brake (brake) on T cell responses. Three antibodies, PD-L1, currently approved for the treatment of cancer, are trastuzumab (atezolizumab) (TECENTRIQ, MPDL3280A, RG7466), avizumab (avelumab) (BAVENTIO, MSB0010718C) and Dewar lumab (Durvalumab) (IMFINZI, MEDI 4736). Several successful clinical trials with these antibodies showed that they had high objective response rates, response persistence or improved survival in bladder, skin and lung cancer (Xu-Monette et al, Front Immunol, 2017).

Cluster of differentiation 137 or CD137 (also known as 4-1BB or TNFRS9) is a member of the costimulatory immunoreceptor and Tumor Necrosis Factor Receptor (TNFR) superfamily. It is expressed predominantly on activated CD4+ and CD8+ T cells, activated B cells, and Natural Killer (NK) cells, but can also be found on resting monocytes and dendritic cells (Li and Liu, Clin Pharmacol, 2013) or endothelial cells (Snell et al, Immunol Rev, 2011). CD137 plays an important role in modulating immune responses and is therefore a target for cancer immunotherapy. CD137 ligand (CD137L) is the only natural ligand for CD137 known and is constitutively expressed on several antigen presenting cells such as activated B cells, monocytes and splenic dendritic cells, and can also induce CD137 ligand on T lymphocytes.

CD137L is a trimeric protein that exists in membrane-bound form and as a soluble variant. However, the ability of soluble CD137L to activate CD137 on, for example, CD 137-expressing lymphocytes is limited, requiring high concentrations to elicit utility (Wyzgol et al, J Immunol, 2009). The natural method of activating CD137 is by conjugation of CD 137-positive cells with CD 137L-positive cells. It is believed that activation of CD137 is then induced by aggregation of CD137L on the opposing cells, resulting in signaling via TRAF1, 2, and 3 (Yao et al, Nat Rev Drug Discov, 2013; Snell et al, Immunol Rev, 2011) and further concomitant downstream effects in CD 137-positive T cells. In the case of activation of T cells through recognition of their corresponding cognate targets, the effects elicited by co-stimulation of CD137 are further enhanced activation, enhanced survival and proliferation, production of pro-inflammatory cytokines, and improved killing ability.

The benefit of CD137 co-stimulation to eliminate cancer cells has been demonstrated in a number of in vivo models. Over-expression of CD137L on tumors (forced) leads to, for example, tumor rejection (Melero et al, Eur J Immunol, 1998). Similarly, overexpression of anti-CD 137 scFv on tumors resulted in CD4 ⁺T-cell and NK-cell dependent tumor elimination (Yang et al, Cancer Res, 2007; Zhang et al, Mol Cancer Ther, 2006; Ye et al, Nat Med, 2002). Studies have also demonstrated that systemic administration of anti-CD 137 antibodies results in tumor growth retardation (Martinet et al, Gene Ther, 2002).

Studies also indicate that CD137 is an excellent marker for tumor-reactive T cells naturally present in human tumors (Ye et al, Clin Cancer Res, 2014), and that anti-CD 137 antibodies can be used to improve CD8⁺Expansion and activity of melanoma-infiltrating lymphocytes for use in adoptive T cell therapy (Chacon et al, PLoS One, 2013).

Preclinical demonstration of potential therapeutic benefits of CD137 co-stimulation prompted the development of therapeutic antibodies targeting CD137, including BMS-663513 (described in U.S. patent No.7,288,638) and PF-05082566(Fisher et al, Cancer Immunol, 2012).

The present invention provides, among other things, a novel means of simultaneously conjugating CD137 and PD-L1 via one or more fusion proteins having the properties of binding specificity for CD137 and binding specificity for PD-L1.

Definition of

The following list defines terms, phrases and abbreviations used in this specification. All terms listed and defined herein are intended to encompass all grammatical forms.

As used herein, "CD 137" means human CD137(huCD137), unless otherwise specified. Human CD137 means the full-length protein defined by UniProt Q07011, a fragment thereof, or a variant thereof. CD137 is also known as 4-1BB or as tumor necrosis factor receptor superfamily member 9(TNFRSF9) and lymphocyte activation Induced (ILA). In some particular embodiments, CD137 of a non-human species is used, such as cynomolgus monkey (cynomolgus) CD137 and mouse CD 137.

As used herein, "programmed cell death 1 ligand 1" or "PD-L1" means, unless otherwise specified, human PD-L1 (huPD-L1). Human PD-L1 means the full-length protein defined by UniProt Q9NZQ7, a fragment thereof, or a variant thereof. Human PD-L1 is encoded by the CD274 gene. PD-L1 is also known as Cluster of differentiation 274(CD274) or B7 homolog 1 (B7-H1). In some particular embodiments, non-human species of PD-L1, such as cynomolgus PD-L1 and mouse PD-L1, are employed.

As used herein, "binding affinity" describes the ability of a biomolecule of the invention (e.g., a polypeptide or protein, such as a lipocalin mutein, an antibody, a fusion protein or any other peptide or protein) to bind to a selected target and form a complex. Binding affinity can be measured by a variety of methods known to those skilled in the art, including, but not limited to, fluorescence titration, enzyme-linked immunosorbent assay (ELISA) based assays (including direct and competitive ELISA), calorimetry such as Isothermal Titration Calorimetry (ITC), and Surface Plasmon Resonance (SPR). These methods are well known in the art, and some examples of which are described further below. Thus, binding affinity is expressed as the dissociation constant (K) measured using these methods _D) Half maximal Effective Concentration (EC)₅₀) Or half maximal Inhibitory Concentration (IC)₅₀) The value of (c). Lower K_D、EC₅₀Or IC₅₀The values reflect better (higher) binding capacity (affinity). Thus, the binding affinity of two biomolecules to a selected target can be measured and compared. The terms "substantially the same", "substantially the same" or "substantially similar" when comparing the binding affinities of two biomolecules for a selected target means that one biomolecule has a K expressed as the same or similar to another molecule within the experimental variation of the binding affinity measurement (variabilty)_D、EC₅₀Or IC₅₀Binding affinity of the values. The experimental variation of the binding affinity measurement depends on the specific method employed and is known to those skilled in the art.

As used herein, the term "substantially" may also refer to a qualitative condition that exhibits all or nearly all of the range or extent of a characteristic or property of interest. Those of ordinary skill in the biological arts will appreciate that little, if any, biological and chemical phenomena proceeds toward completion and/or progression to completion or to achieve or avoid absolute results. Thus, the term "substantially" as used herein is used to capture the potentially lack of integrity inherent in many biological and chemical phenomena.

As used herein, the term "detect/detection/detecting" or "detectable" is understood to mean at the quantitative and qualitative level as well as combinations thereof. Thus it includes quantitative, semi-quantitative and qualitative measurements performed on the biomolecules of the invention.

As used herein, "detectable affinity" generally means that the binding capacity between a biomolecule and its target is at most about 10^-5M or less, which is represented by K_D、EC₅₀Or IC₅₀The value is obtained. Generally no longer can be measured by conventional methods such as ELISA and SPR to more than 10^-5Binding affinity of M (denoted as K)_D、EC₅₀Or IC₅₀Value) and thus it is secondary.

It is noted that the formation of a complex between a biomolecule of the invention and its target is influenced by a number of different factors, such as the concentration of the respective target, the presence of competitors, the pH and ionic strength of the buffer system employed, the experimental methods used to determine binding affinity (e.g. fluorescence titration, competition ELISA and surface plasmon resonance), and even mathematical algorithms used to evaluate experimental data. Thus, it is also clear to the skilled person that K is the expression_D、EC₅₀Or IC₅₀The binding affinity of the values may vary within a certain experimental range, depending on the method and experimental setup. This means that the measured K _D、EC₅₀Or IC₅₀The values may have slight deviations orThe tolerance range, for example, depends on whether the value is determined by ELISA (including direct or competitive ELISA), by SPR or another method.

As used herein, "specific for", "specific binding", "specifically binding" or "binding specificity" relates to the ability of a biomolecule to distinguish a desired target (e.g., CD137 and PD-L1) from one or more reference targets (e.g., cellular receptors for neutrophil gelatinase-associated lipocalin). It is understood that such specificity is not an absolute property, but a relative property, and can be determined, for example, by SPR, western blot, ELISA, Fluorescence Activated Cell Sorting (FACS), Radioimmunoassay (RIA), Electrochemiluminescence (ECL), immunoradiometric assay (IRMA), Immunohistochemistry (IHC), and peptide scanning.

The terms "specific for", "specific binding", "specifically binds" or "binding specificity" when used herein in the context of a fusion protein of the invention that binds to CD137 and PD-L1, mean that the fusion protein as described herein binds to CD137 and PD-L1, reacts with CD137 and PD-L1 or is directed against CD137 and PD-L1, but does not substantially bind to another protein. The term "another protein" includes any protein that is not CD137 or PD-L1 or a protein closely related or homologous to CD137 or PD-L1. However, fragments and/or variants of CD137 or PD-L1 and CD137 or PD-L1 from species other than human are not excluded by the term "another protein". The term "substantially not binding" means that the fusion protein of the invention binds to another protein with a lower binding affinity than CD137 and/or PD-L1, i.e. shows a cross-reactivity of less than 30%, preferably 20%, more preferably 10%, particularly preferably less than 9%, 8%, 7%, 6% or 5%. Whether the fusion protein as defined above reacts specifically can easily be tested, in particular by comparing the reaction of the fusion protein of the invention with CD137 and/or PD-L1 and the reaction of the fusion protein with other (further) proteins.

As used herein, the term "lipocalin" refers to a monomeric protein of about 18-20kDa in weight, having a cylindrical beta-pleated sheet supersecondary structural region, the structure of whichA region comprises a plurality of β -strands (preferably 8 β -strands, labelled a to H) joined pair-wise by a plurality (preferably 4) loops at one end to thereby comprise a ligand binding pocket and an entrance defining the ligand binding pocket. Preferably, the loops comprising the ligand binding pockets used in the present invention are loops connecting the open ends of the β -strands a and B, C and D, E and F, and G and H, and are labeled as AB, CD, EF and GH loops. It is well recognized that in members of the lipocalin family, it is this diversity of the loops in the otherwise rigid lipocalin backbone that leads to a variety of different binding patterns, each of which can accommodate targets of different size, shape and chemical characteristics (e.g. reviewed in Skerra, Biochim Biophys Acta, 2000; Flower et al, Biochim Biophys Acta, 2000; Flower, Biochem J, 1996). It will be appreciated that the lipocalin family of proteins has been naturally evolved to bind a wide range of ligands, with a very low level of overall sequence conservation (typically with less than 20% sequence identity), but retaining a highly conserved overall folding pattern. The correspondence between positions in different lipocalins is also well known to the person skilled in the art (see e.g. U.S. Pat. No.7,250,297). Proteins falling within the definition of "lipocalin" as used herein include, but are not limited to, human lipocalin including tear lipocalin (Tlc, Lcn1), lipocalin-2 (Lcn2) or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein d (apod), apolipoprotein M, alpha 1-acid glycoprotein 1, alpha 1-acid glycoprotein (apod) ₁Acid glycoprotein 2, α 1-microglobulin, complement component 8 γ, Retinol Binding Protein (RBP), epididymis retinoic acid binding protein, glycodellin, odour binding protein IIa, odour binding protein IIb, lipocalin-15 (Lcn15) and prostaglandin D synthase.

As used herein, "tear lipocalin" refers to human tear lipocalin (hTlc), and further refers to mature human tear lipocalin, unless otherwise specified. The term "mature" when used to characterize a protein means that the protein is substantially free of signal peptide. "mature hTalc" of the present invention refers to the mature form of human tear lipocalin, which does not have a signal peptide. Mature hTlc is depicted as residues 19-176 of the sequence with SWISS-PROT database accession number P31025, whose amino acids are shown in SEQ ID NO: 1.

as used herein, "lipocalin-2" or "neutrophil gelatinase-associated lipocalin" refers to human lipocalin-2 (hTalc 2) or human neutrophil gelatinase-associated lipocalin (hNGAL), and further refers to mature human lipocalin-2 or mature human neutrophil gelatinase-associated lipocalin. The term "mature" when used to characterize a protein means that the protein is substantially free of signal peptide. The invention of the "mature hNGAL" refers to the human neutrophil gelatinase associated lipocalin mature form, which does not have a signal peptide. Mature hNGAL is described as residues 21-198 of the sequence with SWISS-PROT database accession number P80188, the amino acids of which are shown in SEQ ID NO: 2.

As used herein, "native sequence" refers to a protein or polypeptide having a sequence that occurs in nature or having a wild-type sequence, regardless of the manner in which such protein or polypeptide is made. Such native sequence proteins or polypeptides may be isolated from nature or may be produced by other means, such as recombinant or synthetic methods.

"native sequence lipocalin" refers to a lipocalin having the same amino acid sequence as a corresponding polypeptide derived from nature. Thus, a native sequence lipocalin may have the amino acid sequence of a corresponding naturally occurring (wild-type) lipocalin from any organism, in particular a mammal. The term "native sequence" when used in the context of a lipocalin specifically encompasses naturally occurring truncated or secreted forms, naturally occurring variant forms (e.g. alternatively spliced forms and naturally occurring allelic variants of a lipocalin) of the lipocalin. The term "native sequence lipocalin" is used interchangeably herein with "wild-type lipocalin".

As used herein, "mutein", "mutated" entity (protein or nucleic acid) or "mutant" refers to the exchange, deletion or insertion of one or more amino acids or nucleotides compared to a naturally occurring (wild-type) protein or nucleic acid. The term also includes fragments of the muteins as described herein. The present invention specifically encompasses a lipocalin mutein as described herein, such mutein having a cylindrical β -sheet supersecondary structural region comprising 8 β -strands connected pairwise by 4 loops at one end to thereby comprise a ligand binding pocket and an entrance defining the ligand binding pocket, wherein at least one amino acid of each of at least three of the 4 loops has been mutated as compared to the native sequence lipocalin. Preferably, the lipocalin muteins of the invention have the function of binding CD137 as described herein.

As used herein, the term "fragment" in relation to a lipocalin mutein of the invention refers to a protein or polypeptide derived from full length mature hTLc or hNGAL or a lipocalin mutein which is N-terminally and/or C-terminally truncated (i.e.lacks at least one of the N-terminal and/or C-terminal amino acids). Such fragments may comprise at least 10 or more, such as 20 or 30 or more, consecutive amino acids of the primary sequence of the mature hTalc or hNGAL or lipocalin mutein from which they are derived, and are typically detectable in an immunoassay for mature hTalc or hNGAL. Such fragments may lack up to 2, up to 3, up to 4, up to 5, up to 10, up to 15, up to 20, up to 25 or up to 30 (including all numbers in between) of the N-terminal and/or C-terminal amino acids. As an illustrative example, such a fragment may lack 1, 2, 3, or 4 of the N-terminal amino acids (His-Leu) and/or 1 or 2 of the C-terminal amino acids (Ser-Asp) of mature hTlc. It will be appreciated that the fragment is preferably a functional fragment of the mature hTalc or hNGAL or lipocalin mutein from which it is derived, meaning that the fragment preferably retains the preferred binding affinity for CD137 of the mature hTalc/hNGAL or lipocalin mutein from which it is derived. As an illustrative example, such a functional fragment may comprise at least the amino acid at position 5-153, 5-150, 9-148, 12-140, 20-135, or 26-133 corresponding to the linear polypeptide sequence of mature hTalc. As another illustrative example, such a functional fragment can at least correspond to the mature hNGAL linear polypeptide sequence of the position 13-157, 15-150, 18-141, 20-134, 25-134 or 28-134 amino acids.

The term "fragment" with respect to the corresponding target CD137 or PD-L1 of the fusion protein of the invention refers to the protein domain of CD137 or PD-L1 or CD137 or PD-L1 truncated at the N-terminus and/or C-terminus. The fragments of CD137 or PD-L1 described herein retain the ability of full-length CD137 or PD-L1 to be recognized and/or bound by the fusion proteins of the invention. As an illustrative example, the fragment may be the extracellular domain of CD137 or PD-L1. As an illustrative example, such an extracellular domain may comprise amino acids of the extracellular subdomain of CD137, such as the amino acid sequences of Domain 1 (residues 24-45 of UniProt Q07011), Domain 2 (residues 46-86), Domain 3(87-118) and Domain 4 (residues 119-159), alone or in combination. As another illustrative example, such an extracellular domain may comprise amino acid residues 19-238 of UniProt Q9NZQ 7.

As used herein, the term "variant" relates to a derivative of a protein or polypeptide comprising a mutation, for example by substitution, deletion, insertion and/or chemical modification of the amino acid sequence or nucleotide sequence. In some embodiments, such mutations and/or chemical modifications do not reduce the function of the protein or peptide. Such substitutions may be conservative, i.e., an amino acid residue is replaced with a chemically similar amino acid residue. Examples of conservative substitutions are substitutions between members of the following groups: 1) alanine, serine, threonine, and valine; 2) aspartic acid, glutamic acid, glutamine, asparagine, and histidine; 3) arginine, lysine, glutamine, asparagine, and histidine; 4) isoleucine, leucine, methionine, valine, alanine, phenylalanine, threonine, and proline; and 5) isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Such variants include proteins or polypeptides in which one or more amino acids are replaced by their respective D-stereoisomers or amino acids other than the naturally occurring 20 amino acids (such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, pentanine). Such variants also include, for example, proteins or polypeptides having one or more amino acid residues added or deleted at the N-and/or C-terminus. In general, a variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or at least about 98% amino acid sequence identity to a native sequence protein or polypeptide. The variant preferably retains biological activity, such as binding to the same target of the protein or polypeptide from which it is derived.

The term "variant" as used herein in relation to the corresponding protein ligand CD137 or PD-L1 of the fusion protein of the invention relates to CD137 or PD-L1 or a fragment thereof having one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 60, 70, 80 or more) amino acid substitutions, deletions and/or insertions compared to CD137 or PD-L1 (wild-type CD137 or PD-L1, such as CD137 preserved with UniProt Q07011 or PD-L1 preserved with UniProt Q9NZQ7, as described herein), respectively. The CD137 or PD-L1 variant preferably has at least 50%, 60%, 70%, 80%, 85%, 90% or 95% amino acid identity with wild type CD137 or PD-L1, respectively. The CD137 variants or PD-L1 variants described herein retain the ability to bind to the fusion proteins disclosed herein that are specific for CD137 and PD-L1.

The term "variant" as used herein in relation to a lipocalin mutein relates to a lipocalin mutein of the invention or a fragment thereof, wherein the sequence thereof has mutations and/or chemical modifications including substitutions, deletions and insertions. The variants of the lipocalin muteins described herein retain biological activity, such as the CD 137-binding biological activity of the lipocalin mutein from which the variant is derived. In general, a variant of a lipocalin mutein has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% amino acid sequence identity to the lipocalin mutein from which it is derived.

As used herein, the term "mutagenesis" refers to the introduction of a mutation into a polynucleotide or amino acid sequence. Preferably, mutations are introduced under experimental conditions such that the naturally occurring amino acid at a given position of a protein or polypeptide sequence may be altered, e.g., replaced by at least one amino acid. The term "mutagenesis" also includes (additionally) changing the length of a sequence segment by deletion or insertion of one or more amino acids. Therefore, the following is within the scope of the invention: for example, one amino acid at a selected sequence position is replaced by an extension of three amino acids (stretch), resulting in the addition of two amino acid residues compared to the length of the corresponding segment of the native protein or polypeptide amino acid sequence. Such insertions or deletions can be introduced independently of one another into any sequence segment which can be subjected to mutagenesis in the context of the invention. In an exemplary embodiment of the invention, the insertion may be introduced into a segment of the amino acid sequence corresponding to the AB loop of a native sequence lipocalin (see international patent publication No. wo 2005/019256, incorporated herein by reference in its entirety).

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

As used herein, the term "sequence identity" or "identity" is a sequence property that measures the similarity or relationship of sequences. The term "sequence identity" or "identity" as used herein means the percentage of paired identical residues after alignment of the protein or polypeptide sequence of the invention with the sequence in question (homology), relative to the number of residues in the longer of the two sequences. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the result by 100.

As used herein, the term "sequence homology" or "homology" is its ordinary meaning, and homologous amino acids include identical amino acids as well as amino acids that are considered conservative substitutions at equivalent positions in the linear amino acid sequence of a protein or polypeptide of the invention (e.g., any fusion protein or lipocalin mutein of the invention).

The skilled worker can identify available computer programs, for example BLAST (Altschul et al, NucleicAcids Res, 1997), BLAST2(Altschul et al, J Mol Biol, 1990) and Smith-Waterman (Smith and Waterman, J Mol Biol, 1981) to determine sequence homology or sequence identity using standard parameters. Sequence homology or percent sequence identity may be determined herein using, for example, the program BLASTP, version 2.2.5 (2002, 16.11) (Altschul et al, Nucleic Acids Res, 1997). In this embodiment, the percentage homology is based on an alignment of the complete protein or polypeptide sequence including the propeptide sequence (matrix: BLOSUM 62; gap penalty: 11.1; cutoff set to 10 ^-3) Preferably, the wild-type protein scaffold is used as a reference in the pairwise comparison. It is calculated as the number of "positives" (homologous amino acids) expressed as the result of the BLASTP program output divided by the percentage of the total number of amino acids selected by the program for alignment.

In particular, to determine whether the amino acid residue of a lipocalin mutein amino acid sequence differs from the wild type lipocalin corresponding to a certain position in the wild type lipocalin amino acid sequence, the skilled person may use means and methods well known in the art, such as aligning manually or by using a computer program such as BLAST 2.0 (which represents a basic local alignment search tool) or ClustalW or any other suitable program suitable for generating a sequence alignment. Thus, the wild-type sequence of a lipocalin may serve as the "test sequence" or "reference sequence", while the amino acid sequence of a lipocalin mutein different from the wild-type lipocalin described herein serves as the "query sequence". The terms "wild-type sequence", "reference sequence" and "test sequence" are used interchangeably herein. Preferably, the wild type sequence of the lipocalin is the sequence shown in SEQ ID NO: 1 or the hTlc sequence shown in SEQ ID NO: 2, or a variant thereof.

A "gap" is the space in the alignment resulting from the addition or deletion of amino acids. Thus, two identical sequences have 100% identity, but sequences that are less highly conserved and have deletions, additions or substitutions may have a lower degree of sequence identity.

As used herein, the term "position" means the position of an amino acid in an amino acid sequence described herein or the position of a nucleotide in a nucleic acid sequence described herein. It is to be understood that the term "corresponding" or "corresponding" as used herein in the context of one or more lipocalin mutein amino acid sequence positions, a corresponding position is not only determined by the number of preceding nucleotides or amino acids. Thus, the absolute position of a given amino acid according to the invention may differ from the corresponding position due to amino acid deletions or additions at other positions of the (mutant or wild-type) lipocalin. Similarly, the absolute position of a given nucleotide according to the invention may differ from the corresponding position due to nucleotide deletions or additions at other positions in the mutein or wild type lipocalin 5' -untranslated region (UTR), including the promoter and/or any other regulatory sequences or genes, including exons and introns.

A "corresponding position" according to the present invention may be a sequence position that matches its corresponding sequence position in a pair or multiple sequence alignment according to the present invention. With respect to "corresponding positions" according to the present invention, it is preferably understood that the absolute position of a nucleotide or amino acid may be different from the adjacent nucleotide or amino acid, but that said same "corresponding position or positions" may still comprise said adjacent nucleotides or amino acids which may have been exchanged, deleted or added.

Furthermore, for the corresponding positions in the lipocalin mutein based on the reference sequence according to the invention, it is preferably understood that the positions of the nucleotides or amino acids of the lipocalin mutein may correspond in structure to positions at other positions in the reference lipocalin (wild-type lipocalin) or in another lipocalin mutein, although these positions may differ in absolute position numbers, as understood by the skilled person in view of the highly conserved overall folded form between lipocalins.

The terms "conjugate/conjugation", "fusion" or "linkage", as used interchangeably herein, refer to the joining together of two or more subunits via all forms of covalent or non-covalent bonds (links) by means including, but not limited to, gene fusion, chemical conjugation, coupling by linker or crosslinking agent, and non-covalent association.

As used herein, "fusion polypeptide" or "fusion protein" refers to a polypeptide or protein comprising two or more subunits. In some embodiments, a fusion protein described herein comprises two or more subunits, at least one of which is capable of specifically binding CD137, and another subunit is capable of specifically binding PD-L1. In the fusion protein, the subunits may be linked by covalent or non-covalent bonds. Preferably, the fusion protein is a translational fusion between two or more subunits. The translational fusion can be produced by genetically engineering the coding sequence of one subunit in frame with the coding sequence of the other subunit. The two subunits are interspersed with nucleotide sequences encoding linkers (interpersed). However, the subunits of the fusion protein of the present invention may also be linked by chemical conjugation. Typically, the subunits forming the fusion protein are linked to each other, the C-terminus of one subunit being linked to the N-terminus of another subunit, or the C-terminus of one subunit being linked to the C-terminus of another subunit, or the N-terminus of one subunit being linked to the N-terminus of another subunit, or the N-terminus of one subunit being linked to the C-terminus of another subunit. The subunits of the fusion protein can be linked in any order and can include more than one of any of the constituent subunits. The term "fusion protein" may also refer to a protein comprising the fused sequence as well as all other polypeptide chains of the protein (complex), if one or more subunits are part of a protein (complex) consisting of more than one polypeptide chain. As an illustrative example, where a full-length immunoglobulin is fused to a lipocalin mutein via the heavy or light chain of the immunoglobulin, the term "fusion protein" may refer to a single polypeptide chain comprising the lipocalin mutein and the heavy or light chain of the immunoglobulin. The term "fusion protein" may also refer to intact immunoglobulins (light and heavy chains) and lipocalin muteins fused to one or both of their heavy and/or light chains.

As used herein, the term "subunit" of a fusion protein disclosed herein refers to a single protein or an independent polypeptide chain, which itself can form a stable folded structure and define a unique function that provides a binding motif to a target. In some embodiments, preferred subunits of the invention are lipocalin muteins. In some other embodiments, preferred subunits of the invention are full-length immunoglobulins or antigen-binding domains thereof.

Two or more subunits of a fusion protein as described herein may be joined by a "linker" comprised by the fusion protein of the invention. The bond (linkage) may be covalent or non-covalent. One preferred covalent bond (linkage) is through a peptide bond, such as a peptide bond between amino acids. One preferred linker is a peptide linker. Thus, in preferred embodiments, the linker comprises one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. Preferred peptide linkers are described herein, including glycine-serine (GS) linkers, glycosylated GS linkers, and proline-alanine-serine Polymer (PAS) linkers. In some preferred embodiments, the GS linker is as set forth in SEQ ID NO: 13 (G) of ₄S)₃Which are used to link together the subunits of the fusion protein. Other preferred linkers include chemical linkers.

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

As used herein, the term "organic molecule" or "small organic molecule" means an organic molecule comprising at least two carbon atoms, but preferably comprising no more than 7 or 12 rotatable carbon bonds, having a molecular weight in the range of 100 to 2,000 daltons, preferably 100 to 1,000 daltons, and optionally comprising one or two metal atoms.

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

A "subject" is a vertebrate, preferably a mammal, more preferably a human. The term "mammal" as used herein refers to any animal classified as a mammal, including but not limited to humans, domestic and agricultural animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as macaque (cynomolgous monkey), to name a few illustrative examples. Preferably, a "mammal" as used herein is a human.

An "effective amount" is an amount sufficient to produce a beneficial or desired result. An effective amount may be administered in one or more administrations or administrations.

As used herein, "antibody" includes whole antibodies or any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. Whole antibodies refer to glycoproteins comprising at least two Heavy Chains (HC) and two Light Chains (LC) that are linked to each other by disulfide bonds. Each heavy chain is composed of a heavy chain variable domain (V)_HOr HCVR) and heavy chain constant region (C)_H) And (4) forming. The heavy chain constant region is composed of three domains C_H1、C_H2And C_H3And (4) forming. Each light chain is composed of a light chain variable domain (V)_LOr LCVR) and light chain constant region (C)_L) And (4) forming. The light chain constant region consists of a domain C_LAnd (4) forming. V_HAnd V_LThe regions can be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each V_HAnd V_LConsists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen (e.g., PD-L1). The constant region of the antibody optionally mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the typical complement system (C1 q).

As used herein, an "antigen-binding fragment" of an antibody refers to one or more fragments that retain the ability of the antibody to specifically bind an antigen (e.g., PD-L1). The research shows that the antigen binding function of the antibody can be improved through fullFragments of long antibodies. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a polypeptide consisting of V_H、V_L、C_LAnd C_H1A Fab fragment consisting of the domain; (ii) f (ab') comprising two Fab fragments linked by a disulfide bridge at the hinge region₂A fragment thereof; (iii) from V_H、V_L、C_LAnd C_H1Domains and C_H1And C_H2Fab' fragments consisting of the region between the domains; (iv) from V_HAnd C_H1Domain-forming Fd fragments; (v) v with one arm consisting of antibody_HAnd V_LA single chain Fv fragment consisting of a domain; (vi) from V_HdAb fragments consisting of domains (Ward et al, Nature, 1989); and (vii) an isolated Complementarity Determining Region (CDR) or a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker; (viii) comprising V linked in the same polypeptide chain using short linkers_HAnd V_L"diabodies" (see, e.g., patent documents EP 404, 097; WO 93/11161; and Holliger et al, Proc Natl Acad Sci USA, 1993); (ix) containing only V _HOr V_LThe "domain antibody fragment" of (a), wherein in some cases, two or more V_HThe regions are covalently linked.

The antibody may be polyclonal or monoclonal; xenogeneic, allogeneic or syngeneic; or modified forms thereof (e.g., humanized, chimeric, or multispecific). Antibodies may also be fully human.

As used herein, "framework" or "FR" refers to variable domain residues and not hypervariable region (CDR) residues.

"fragment crystallizable region" or "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of immunoglobulin heavy chains may vary, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or from the amino acid residue at Pro230 (numbering according to the EU index of Kabat) to its carboxy terminus (Johnson and Wu, Nucleic Acids Res, 2000). The C-terminal lysine of the Fc region (numbering according to the EU index of Kabat, residue 447) may be removed, for example, during production or purification of the antibody or by recombinantly engineering a nucleic acid encoding the heavy chain of the antibody. Thus, a composition of intact antibodies may comprise a population of antibodies with all K447 residues removed, a population of antibodies without K447 residues removed, and a population of antibodies with a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for use in the antibodies of the invention include human IgG1, IgG2(IgG2A, IgG2B), IgG3 and IgG 4.

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

As used herein, "isolated antibody" refers to an antibody that is substantially free of its natural environment. For example, an isolated antibody is substantially free of cellular material and other proteins from the cell or tissue source from which it is derived. An "isolated antibody" further refers to an antibody that is substantially free of other antibodies having different antigenic specificities. Herein, an isolated antibody that specifically binds PD-L1 is substantially free of antibodies that specifically bind antigens other than PD-L1. However, an isolated antibody that specifically binds PD-L1 may have cross-reactivity with other antigens, such as PD-L1 molecules from other species.

As used herein, "monoclonal antibody" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

As used herein, "humanized antibody" refers to an antibody composed of CDRs of an antibody derived from a mammal other than a human and a human antibody or FR regions and constant regions derived from a human antibody. In some embodiments, humanized antibodies comprise variable domains having variable region amino acid sequences that are more closely related to human than to other species as assessed using the immunological Information System (IMGT) DomainGapAlign tool as described in Ehrenmann et al, (2010). In some embodiments, the humanized antibody may be an effective ingredient in a therapeutic agent due to reduced antigenicity. The term "therapeutic agent" or "therapeutically active agent" as used herein refers to an agent that is therapeutically useful. The therapeutic agent can be any agent useful for the prevention, amelioration, or treatment of a disease, physiological state, symptom, or for the assessment or diagnosis of a disease, physiological state, symptom.

As used herein, "human antibody" includes antibodies having variable regions in which both framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the invention may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) are grafted onto human framework sequences.

Description of the drawings

FIG. 1: a general summary of the design of representative fusion proteins described in this application that are bispecific to the targets CD137 and PD-L1 is provided. Representative fusion proteins are prepared based on an antibody specific for PD-L1 (e.g., an antibody whose heavy chain is provided by SEQ ID NO: 86, or comprises the heavy chain variable domain of SEQ ID NO: 77, or comprises the CDR sequences of GFSLSNYD (HCDR1, SEQ ID NO: 60), IWTGGAT (HCDR2, SEQ ID NO: 61) and VRDSNYRYDEPFTY (HCDR 3; SEQ ID NO: 62), and whose light chain is provided by SEQ ID NO: 87, or comprises the heavy chain variable domain of SEQ ID NO: 82, or comprises the CDR sequences of QSIGTN (LCDR1, SEQ ID NO: 63), YAS (LCDR2) and QQSNSWPYT (LCDR 3; SEQ ID NO: 64)) and one or more lipocalin muteins specific for CD137 (e.g., the lipocalin mutein of SEQ ID NO: 42). As shown in fig. 1A-1I, one or more lipocalin muteins were genetically fused to the C-and/or N-terminus of the heavy or light chain of a PD-L1-specific antibody, resulting in a fusion protein, as shown in SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87 and SEQ ID NOs: 90 and 91. The resulting fusion protein can be bivalent to CD137 (as shown in FIGS. 1A-1D), or tetravalent to CD137 (as shown in FIGS. 1E-1H), or even more valent to CD137 (as shown in FIG. 1I). Additional monospecific fusion proteins were generated by fusing one or more CD 137-specific lipocalin muteins (as shown in figures 1J-1K) via a peptide linker to the C-terminus of the Fc region of an antibody provided as described herein. As shown in SEQ ID NO: 88 and SEQ ID NO: 89 provides the resulting monospecific fusion protein.

FIG. 2: the results of an ELISA assay are shown in which the binding of a representative fusion protein to PD-L1 or CD137 was determined as described in example 4. PD-L1 or CD137 (with a C-terminal His or Fc tag) was coated on a microtiter plate and titration of the tested reagents was started with the highest concentration of 100 nM. The bound reagents in the study were detected by anti-human IgG Fc-HRP or anti-NGAL-HRP, respectively. The data were fitted using a 1: 1 binding model with EC50 values and maximum signal as free parameters and the slope fixed at 1. The obtained EC₅₀The values are provided in table 4.

FIG. 3: the results of an ELISA assay are shown in which the ability of a representative fusion protein to bind both targets PD-L1 and CD137 was determined as described in example 5. Recombinant huPD-L1-His or huCD137-His was coated onto microtiter plates and the fusion protein was titrated starting with the highest concentration of 100 nM. Subsequently, a constant concentration of biotinylated huCD137-His or biotinylated huPD-L1-His, respectively, was added, which was detected by alkaline phosphatase-Peroxidase (Extravidin-Peroxidase).

FIG. 4: shown are the results of the assessment of target binding of the fusion proteins by flow cytometry using Flp-In-CHO cells expressing human or cynomolgus CD137 (fig. 4A-4B) and human or cynomolgus PD-L1 (fig. 4C-4D) as described In example 6. When mock (mock) transfected Flp-In-CHO cells were used, no binding was observed (FIG. 4E). EC was calculated using non-linear regression (shared below, slope 1) as the geometric mean of fluorescence intensity ₅₀The value is obtained. EC is provided in Table 6₅₀The value is obtained.

FIG. 5: binding of the fusion protein to PD-L1-positive tumor cells is shown, which was assessed by flow cytometry by incubating RKO cells and fusion proteins as described in example 7.

FIG. 6: an example of a multiplex SPR-based assay is provided, which was designed to investigate whether the interaction of the fusion protein with CD137 was affected by the binding of CD137L to CD137, as described in example 8. Evaluation was performed by generating complexes of huCD137 (C-terminal Fc fusion) with huCD137L (with a C-terminal His tag) on SPR sensor chips and checking whether the fusion protein still bound to the complexes of huCD137 and CD 137L. As a reference, huCD137 was also incubated with the tested fusion proteins in the absence of huCD 137L. SPR traces of the respective fusion proteins bound to huCD137 alone were marked with solid stem arrows. SPR traces of binding of the respective fusion proteins to huCD137 which had been saturated with huCD137L were marked with a hollow-stem arrow. As a control, a blank injection without fusion protein was used. This experiment shows that all the fusion proteins tested were able to bind CD137 in the presence of CD 137L.

FIG. 7: the fusion protein is shown to compete with PD-L1 for binding to PD-1, as depicted in the competition ELISA study described in example 9. A constant concentration of huPD-1-His was coated onto the microtiter plates, and then a mixture of different concentrations of test molecules with a fixed concentration of tracer huPD-L1-Fc was added. Bound tracer was detected with HRP labeled anti-IgG Fc antibody. Dose-dependent inhibition of binding of huPD-L1-Fc to PD-1 was observed with either the CD137 and PD-L1 bispecific fusion protein or the PD-L1 specific antibody.

FIG. 8: the potential of representative fusion proteins to co-stimulate T cell activation in a PD-L1 target-dependent manner was assessed using CD137 bioassay. NF-. kappa.B-luc 2/CD137 Jurkat cells were co-cultured with the tumor cell line RKO expressing PD-L1 in the presence of various concentrations of fusion proteins or controls. After 4 hours, Luciferase (Luciferase) assay reagent was added and the luminescence signal was measured. Using GraphPadFour parameter logistic curve analysis was performed to calculate EC₅₀Values (see table 9). The fusion proteins costimulated T cell activation only in the presence of PD-L1 (fig. 8A and 8C), but not in the absence of PD-L1 (fig. 8B and 8D). In contrast, reference antibodies in the presence and absence of PD-L1 positive RKO cellsThe CD137 mAbs (SEQ ID NOs: 28 and 29) displayed similar activation.

FIG. 9: results of representative experiments are shown in which the ability of selected fusion proteins to induce T cell activation was studied. PD-L1 antibodies, including the corresponding PD-L1 antibody building block (building block), CD137 binding lipocalin muteins as Fc fusion proteins and anti-CD 137 benchmark antibodies were tested alone and in combination as an anti-PD-L1/anti-CD 137 mixture (cocktail). In this experiment, human Peripheral Blood Mononuclear Cells (PBMC) were incubated with fusion proteins, antibodies, lipocalin mutein Fc fusion protein, cocktail (cocktail) or control in the presence of 1ng/mL Staphylococcal Enterotoxin B (SEB). The level of secreted interleukin 2(IL-2), representative of T cell activation, was determined as a readout for T cell activation by an electrochemiluminescence-based assay and normalized to the level of the corresponding IgG4 control, as described in example 11. All fusion proteins were able to induce T cell activation and the activation was stronger or at least comparable to a single building block or a benchmark anti-PD-L1/anti-CD 137 antibody mixture.

FIG. 10: the ability of representative fusion proteins to co-stimulate T cell activation in a PD-L1 target-dependent manner is shown. PD-L1 antibody (including the corresponding PD-L1 antibody building block), CD137 binding lipocalin mutein as Fc fusion protein and anti-CD 137 benchmark antibody can be tested alone and in combination as an anti-PD-L1/anti-CD 137 mixture (cocktail). Different tumor cell lines expressing different PD-L1 levels (high: RKO; medium: HCC 827; negative: HepG) were inoculated into plates coated with anti-human CD 3. Pan T cells and different concentrations of fusion protein and single building blocks were added and incubated for 3 days. The level of secreted IL-2 was determined by electrochemiluminescence-based assays as described in example 12. All fusion proteins increased IL-2 secretion in a PD-L1-dependent manner.

FIG. 11: the storage stability of the fusion protein after incubation at 1mg/mL or 20mg/mL concentration in PBS or 25mM histidine, 60mM NaCl, 200mM arginine pH 6 at 37 ℃ or 40 ℃ for 1, 2, 3 or 4 weeks is shown. Stability was assessed by recovering monomers from analytical size exclusion or by recovering functional proteins from quantitative ELISA as described in example 13.

FIG. 12: representative fusion proteins (SEQ ID NOs: 90 and 87) are shown using CD4 ⁺Ability to stimulate IL-2 secretion in the Mixed Lymphocyte Reaction (MLR) of T cells. Fusion proteins, PD-L1 antibody building blocks (SEQ ID NOs: 86 and 87), CD137 binding lipocalin mutein (SEQ ID NO: 89) as Fc fusion protein and anti-CD 137 or anti-PD-L1 reference antibody were tested at equimolar concentrations, alone or in combination as anti-PD-L1/anti-CD 137 mixture (cocktail), as described in example 14. In total human CD4 from a different healthy donor⁺IL-2 secretion in supernatants was determined after 6 days of incubation of T cells and monocyte derived dendritic cells (modCs). FIG. 12A shows that the fusion proteins SEQ ID NOs: 90 and 87 showed a significant increase in IL-2 secretion. Data from 8 independent experiments are shown. Fig. 12B shows fusion proteins SEQ ID NOs: concentrations of 90 and 87 in the range of 0.001 to 20. mu.g/mL induced dose-dependent IL-2 secretion. The fusion protein induced higher levels of IL-2 compared to equimolar concentrations of a mixture of reference anti-PD-L1 antibodies (SEQ ID NOs: 26 and 27) and reference anti-CD 137 antibodies (SEQ ID NOs: 28 and 29). Data from representative donors are shown.

FIG. 13: exemplary fusion proteins (SEQ ID NOs: 90 and 87) are shown to induce CD8⁺The ability of T cell effector molecules to be secreted. Fusion proteins were treated with mocC and CD8 from mismatched (mismatch) healthy donors as described in example 15⁺T cells were cultured for 6 days, followed by quantification of IL-2 and CD8 in the supernatant using a Luminex assay⁺Secretion of T cell effector molecules. In contrast to the reference anti-PD-L1 antibodies (SEQ ID NOs: 26 and 27) and the reference anti-CD 137 antibodies (SEQ ID NOs: 28 and 29), when used alone or as a mixture, the fusion proteins SEQ ID NOs: 90 and 87 at 10. mu.g/mL showed IL-2 and cytotoxic factors (perforin, granzyme B and granzyme B)A) The secretion of (2) is increased.

FIG. 14: the fusion protein is shown to bind overlapping epitopes with the clinically active CD137 antibody (SEQ ID NOs: 28 and 29), as shown in the competition ELISA study described in example 17. Constant concentrations of SEQ ID NOs: 28 and 29 were coated on microtiter plates and a mixture of different concentrations of test molecule and fixed concentrations of biotinylated huCD137-Fc tracer was subsequently added. The bound tracer was detected via alkaline phosphatase-Peroxidase (ExtrAvidin-Peroxidase). The fusion protein competes with the CD137 antibody for binding of CD 137.

FIG. 15: the potential of representative fusion proteins to block the inhibitory signal mediated by the PD-1/PD-L1 interaction is shown, and this potential was assessed using the PD-1/PD-L1 blocking bioassay as described in example 18. PD-1-NFAT-luc Jurkat T cells (Jurkat cell line expressing PD-1 and an NFAT-mediated luciferase gene under the control of an NFAT promoter) were co-cultured with PD-Ll aAPC/CHO-K1 cells in the presence of varying concentrations of test molecules. After 6 hours, luciferase assay reagent was added and the luminescent signal was measured. The background signal was obtained by co-culturing PD-1-NFAT-luc Jurkat T cells with only PD-L1 aAPC/CHO-K1 cells. SEQ ID NOs: 90 and 87 block the PD-1/PD-L1 pathway, which is comparable to the PD-L1 antibody tested, which PD-L1 antibody comprises the amino acid sequence shown in SEQ ID NOs: 86 and 87 and the PD-L1 antibody set forth in SEQ ID NOs: reference PD-L1 antibody to 26 and 27.

FIG. 16: the ability of representative fusion proteins to induce T cell activation is shown. PD-L1 antibody building blocks and the reference CD137 antibody (when used alone and in combination with PD-L1 antibody) were also tested. In the experiment, human PBMCs were incubated with fusion proteins, antibodies, mixtures (cocktails) or controls in the presence of 0.1ng/mL SEB. Secreted IL-2 levels were determined as a readout of T cell activation by electrochemiluminescence-based assays as described in example 19 and shown in figure 16A. Figure 16B shows that the level of IL-2 secretion induced by the test molecule was doubled when compared to the background level of IL-2 secretion (PBMC stimulated with 0.1ng/mL SEB and without any test molecule). The fusion protein resulted in a dose-dependent increase in IL-2 secretion that was stronger than either PD-L1 antibody or CD137 antibody alone or in combination.

FIG. 17: the ability of representative fusion proteins to co-stimulate T cell activation in the presence of PD-L1 was demonstrated. A mixture of PD-L1 antibody building blocks, reference CD137 antibody, CD137 antibody and reference PD-L1 antibody was tested in parallel. CHO cells transfected with human PD-L1 (fig. 17A) or mock-transfected CHO cells (human PD-L1 negative, fig. 17B) were seeded into human anti-CD 3-coated plates. Pan T cells were added along with different concentrations of test molecules and incubated for 2 days. The level of secreted IL-2 in the supernatant was determined by an electrochemiluminescence-based assay as described in example 20. IL-2 secretion levels were normalized to background levels (Pan T cells + anti-CD 3+ CHO cells) to demonstrate a fold increase in IL-2 secretion in the presence of human PD-L1 expressing CHO cells (FIG. 17C) or mock transfected CHO cells (FIG. 17D). The fusion protein induced a strong dose-dependent increase in IL-2 secretion in the presence of PD-L1 only, which was stronger than the reference CD137 antibody alone or in combination with the reference PD-L1 antibody.

FIG. 18: results of a pharmacokinetic analysis of the bispecific fusion protein in mice and the building block PD-L1 antibody (SEQ ID NOs: 86 and 87) are provided, as described in example 21. Male CD-1 mice (3 mice per time point) were injected intravenously with the fusion protein at a dose of 10 mg/kg. Drug levels were detected using a sandwich ELISA that detects the intact molecule by the targets PD-L1 and CD 137. anti-PD-L1 antibody plasma levels were determined using a sandwich ELISA with the targets PD-L1 and human Fc.

FIG. 19: the results of a pharmacokinetic analysis of representative fusion proteins (SEQ ID NOs: 90 and 87) in mice compared to two previously described CD 137-and PD-L1-binding fusion proteins (SEQ ID NO: 147 and SEQ ID NO: 148) are provided, as described in example 22. Male CD-1 mice (2 mice per time point) were injected intravenously with test molecules at a dose of 2 mg/kg. Drug levels were measured using ELISA at the indicated time points. Data are plotted as time vs. concentration. The SEQ ID NOs: 90 and 87, but not SEQ ID NO: 147 or SEQ ID NO: 148, show good pharmacokinetic profiles or antibody-like pharmacokinetics.

Description of the invention

As described herein, the present invention encompasses the recognition that bivalent CD137 binding agents (such as antibodies) may not be sufficient on their own to aggregate CD137 on T cells or NK cells and result in potent activation, similar to the lack of activity of trivalent soluble CD 137L. In the latest releases using preclinical mouse models, in vivo evidence suggests that the mode of action of other anti-TNFR antibodies requires that the antibody interact via its Fc portion with an Fc- γ -receptor on Fc- γ receptor expressing cells (Bulliard et al, Immunol Cell Biol, 2014; Bulliard et al, J Exp Med, 2013). Depending on the presence of Fc-gamma receptor expressing cells, the mode of action of these anti-TNFR antibodies may be dominated by non-targeted aggregation via Fc-gamma receptors, which do not need to be overexpressed in the targeted tumor microenvironment compared to normal tissue.

Thus, there is an unmet need for generating therapies that aggregate and activate CD137 with specific, tumor-targeted modes of action.

To meet this unmet need, the present invention provides, among other things, a novel method for simultaneously conjugating CD137 and PD-L1 via one or more fusion proteins having both CD137 binding specificity and PD-L1 binding specificity. The fusion proteins provided are designed to promote CD137 aggregation by bridging CD 137-positive T cells with PD-L1 expressed in the tumor microenvironment. Such bispecific molecules can combine CD 137-induced T cell activation and expansion with anti-PD-L1-mediated immune checkpoint blockade, which can overcome certain limitations of monotherapy and provide benefits to patients who are, for example, drug resistant or non-responsive. The fusion proteins are also designed to provide the potential for combination therapy in one molecule while allowing local induction of antigen-specific T cells in the tumor microenvironment, potentially reducing peripheral toxicity.

In some aspects, the invention provides fusion proteins that bind to CD137 and PD-L1, as well as methods and useful applications thereof. The invention also provides methods of making fusion proteins that bind to CD137 and PD-L1 as described herein and compositions comprising such proteins. The fusion proteins of the invention that bind to CD137 and PD-L1 and compositions thereof can be used in methods of detecting CD137 and/or PD-L1 in a sample, in methods of binding to CD137 and/or PD-L1 in a subject, or in methods of modulating an immune response in a subject. Such fusion proteins having these characteristics relevant to the uses provided by the present invention have not been previously described. In contrast to the fusion proteins provided herein, previously known fusion proteins targeting CD137 and PD-L1 are affected by one or more of the following: poor pharmacokinetics, unacceptable levels of off-target binding, reduced or degraded ability to bind to one or both targets of a particular fusion protein (e.g., PD-L1 and/or CD137), and/or unacceptable levels of nonspecific (e.g., PD-L1 independent) activation of the immune system, for example.

A. Exemplary fusion proteins of the invention specific for CD137 and PD-L1.

In some embodiments, provided fusion proteins contain at least two subunits in any order: (1) a first subunit comprising a full-length immunoglobulin or antigen-binding domain thereof specific for PD-L1, and (2) a second subunit comprising a lipocalin mutein specific for CD 137.

In some embodiments, provided fusion proteins further comprise at least one additional subunit, e.g., a third subunit. For example, the fusion protein may contain a third subunit specific for CD 137. In some embodiments, the third subunit can be or comprise a lipocalin mutein specific for CD 137. For example, two lipocalin muteins can be fused to a first immunoglobulin subunit, one at the C-terminus and one at the N-terminus of the immunoglobulin. In some embodiments, the lipocalin mutein may be fused to the heavy or light chain of an immunoglobulin.

In some embodiments, provided fusion proteins can comprise one or more additional subunits (e.g., a fourth, fifth, or sixth subunit).

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

In some embodiments, at least one subunit may be linked to another subunit via a linker. In some further embodiments, the linker is a peptide linker, for exampleSuch as a non-structural glycine-serine (GS) linker, a glycosylated GS linker or a proline-alanine-serine Polymer (PAS) linker. In some embodiments, the GS linker is as set forth in SEQ ID NO: 13 (Gly)₄Ser)₃Linker ((G)₄S)₃). Other exemplary linkers are shown in SEQ ID NOs: 14-23. In some embodiments, the peptide linker may have 1 to 50 amino acids, such as 1, 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids. For example, when the first subunit comprises a full-length immunoglobulin, the second subunit can be linked via a peptide linker between the N-terminus of the second subunit and the C-terminus of the heavy chain constant region (CH) of the immunoglobulin. In some other embodiments, the third subunit can be linked via a peptide linker between the N-terminus of the third subunit and the C-terminus of the light chain constant region (CL) of the immunoglobulin.

In some embodiments, one subunit may be linked to another subunit substantially as shown in figure 1. In general, one subunit can be fused to another at its N-terminus and/or its C-terminus. For example, in some embodiments, the lipocalin mutein subunit may be fused at its N-terminus and/or its C-terminus to an immunoglobulin subunit. By way of further example, a lipocalin mutein may preferably be linked via a peptide bond to the C-terminus of an immunoglobulin heavy chain domain (HC), the N-terminus of a HC, the C-terminus of an immunoglobulin Light Chain (LC) and/or the N-terminus of a LC (fig. 1A-1D).

In some embodiments, the lipocalin mutein subunit may be fused at its N-terminus and/or its C-terminus to an immunoglobulin fragment. For example, in some embodiments, the lipocalin muteins may preferably be linked via a peptide linker at the C-terminus of the heavy chain constant region (CH) or the C-terminus of the light chain constant region (CL) of the immunoglobulin.

In some embodiments, when one subunit comprises a full-length immunoglobulin, a second subunit can be linked between the N-terminus of the second subunit and the C-terminus of the heavy chain constant region (CH) of the immunoglobulin.

In some embodiments, a third subunit can be linked between the N-terminus of the third subunit and the C-terminus of the light chain constant region (CL) of the immunoglobulin.

In some embodiments, for fusion proteins of the invention in which at least one subunit may be or comprise a full-length immunoglobulin, Fc function of the Fc region of the full-length immunoglobulin on Fc receptor positive cells may be simultaneously retained when the fusion protein simultaneously engages CD137 and PD-L1.

In some embodiments, where at least one subunit of a provided fusion protein can be or comprise a full-length immunoglobulin, Fc function of the Fc region of the full-length immunoglobulin on Fc receptor positive cells can be reduced or completely inhibited by protein engineering, while the fusion protein simultaneously engages CD137 and PD-L1. In some embodiments, this can be achieved by, for example, converting the IgG1 backbone to IgG4, since IgG4 is known to exhibit reduced Fc-gamma receptor interaction compared to IgG 1. In some embodiments, to further reduce residual binding to Fc-gamma receptors, mutations such as F234A and L235A may be introduced into the IgG4 backbone. In some embodiments, the S228P mutation may also be introduced into the IgG4 backbone to reduce IgG4 half-antibody exchange (Silva et al, J Biol Chem, 2015). In some embodiments, F234A and L235A mutations may be introduced to reduce ADCC and ADCP (Glaesener et al, Diabetes Metab Res Rev, 2010) and/or M428L and N434S mutations or M252Y, S254T and T256E mutations may be introduced to extend serum half-life (Dall' Acqua et al, J Biol Chem, 2006; Zalevsky et al, Nat Biotechnol, 2010). In some embodiments, an additional N297A mutation may be present in the immunoglobulin heavy chain of the fusion protein in order to remove the native glycosylation motif.

In some embodiments, the Fc portion of an immunoglobulin included in a fusion protein of the invention can help maintain serum levels of the fusion protein. For example, when the Fc moiety binds to Fc receptors on endothelial cells and phagocytes, the fusion protein may become internalized and circulate back into the bloodstream, thereby increasing its half-life in vivo.

In one aspect, the fusion proteins of the invention bind CD137 with high affinity. In another aspect, the provided fusion proteins bind to PD-L1 with high affinity. In some preferred embodiments, the provided fusion proteins bind both CD137 and PD-L1. In some embodiments, simultaneous binding to CD137 and PD-L1 allows the provided fusion proteins to exhibit a durable anti-tumor or anti-infective response.

In some embodiments, a fusion protein of the invention can have a K of up to about 2nM or even lower, such as about 1.5nM or lower, about 1nM or lower, about 0.6nM or lower, or about 0.4nM or lower_DValues were combined with PD-L1. In some embodiments, the fusion proteins of the invention are capable of modulating the K of an immunoglobulin specific for PD-L1 (such as an antibody having heavy and light chains provided by SEQ ID NOs: 86 and 87) contained in such fusion proteins _DK of comparable or lower value_DValues were combined with PD-L1. The K of the provided fusion protein may be measured, for example, in a Surface Plasmon Resonance (SPR) assay, such as the SPR assay substantially as described in example 3_DThe value is obtained.

In some embodiments, a fusion protein of the invention can have a K of up to about 10nM or even lower, such as about 7nM, about 6nM, about 5nM, about 4nM, about 3nM, about 2nM or even lower_DValues bind to CD 137. In some embodiments, the fusion proteins of the invention are capable of binding to the lipocalin mutein specific for CD137 (e.g., SEQ ID NO: 42) contained in a particular fusion protein or to the lipocalin mutein of the Fc region of an antibody (e.g., SEQ ID NO: 89) with a K_DK of comparable or lower value_DValues bind to CD 137. The K of the provided fusion protein may be measured, for example, in an SPR assay, such as the SPR assay essentially described in example 3_DThe value is obtained.

In some embodiments, a fusion protein of the invention can have an EC of up to about 0.5nM or even lower, such as about 0.3nM or lower, about 0.2nM or lower, about 0.15nM or lower, or about 0.1nM or lower₅₀Values were combined with PD-L1. In some embodiments, the fusion proteins of the invention can be conjugated to immunoglobulins (such as those having a sequence represented by SEQ ID NOs) specific for PD-L1 contained in a particular fusion protein : 86 and 87 heavy and light chain antibodies)₅₀EC with equivalent or lower value₅₀Values were combined with PD-L1. The EC of the provided fusion protein can be measured, for example, in an enzyme-linked immunosorbent assay (ELISA) assay, such as an ELISA assay substantially as described in example 4₅₀The value is obtained.

In some embodiments, a fusion protein of the invention can have an EC of up to about 0.6nM or even lower, such as about 0.5nM or lower, about 0.2nM or lower, about 0.15nM or lower, or about 0.1nM or lower₅₀Values bind to CD 137. In some embodiments, the fusion proteins of the invention are capable of binding to the EC of a lipocalin mutein specific for CD137 (e.g., SEQ ID NO: 42) or a lipocalin mutein fused to the Fc region of an antibody (e.g., SEQ ID NO: 89) comprised in a particular fusion protein₅₀EC with equivalent or lower value₅₀Values bind to CD 137. The EC of a provided fusion protein may be measured, for example, in an ELISA assay, such as an ELISA assay substantially as described in example 4₅₀The value is obtained.

In some embodiments, the fusion protein of the invention is cross-reactive with cynomolgus monkey PD-L1. In some embodiments, provided fusion proteins can have an EC of up to about 0.5nM or even lower, such as about 0.2nM or lower, about 0.1nM or lower, or about 0.05nM or lower ₅₀Values were combined with cynomolgus monkey PD-L1. The EC of a provided fusion protein may be measured, for example, in an ELISA assay, such as an ELISA assay substantially as described in example 4₅₀The value is obtained.

In some embodiments, the fusion proteins of the invention cross-react with cynomolgus CD 137. In some embodiments, provided fusion proteins can have an EC of up to about 15nM or even lower, such as about 10nM or lower, about 8nM or lower, about 6nM or lower, about 3nM or lower, about 1nM or lower, about 0.5nM or lower, about 3nM or lower, or about 0.1nM or lower₅₀Values bind to cynomolgus CD 137. The EC of a provided fusion protein may be measured, for example, in an ELISA assay, such as an ELISA assay substantially as described in example 4₅₀The value is obtained. In some embodiments, the affinity effect (a) may be by way ofA vitamin effect) enhances the binding of the provided fusion protein to cynomolgus CD 137.

In some embodiments, the fusion protein of the invention is capable of binding to both CD137 and PD-L1. In some embodiments, provided fusion proteins can have an EC of up to about 1nM or even lower, such as 0.8nM or lower, 0.6nM or lower, or 0.4nM or lower₅₀Values bind both CD137 and PD-L1. In some other embodiments, provided fusion proteins can have an EC of up to about 10nM or even lower, such as 8nM or lower, 6nM or lower, 3nM or lower, or 2nM or lower ₅₀Values bind both CD137 and PD-L1. The simultaneous binding may be determined, for example, in an ELISA assay, such as an ELISA assay substantially as described in example 5.

In some embodiments, a fusion protein of the invention can have an EC of up to about 60nM or even lower, such as about 50nM or even lower, about 40nM or even lower, about 30nM or lower, about 10nM or lower, about 7nM or lower, about 5nM or lower, about 3nM or lower, or about 1nM or even lower₅₀Values bind to CD137 expressed on the cells. The EC of a provided fusion protein can be measured, for example, in a flow cytometry assay substantially as described in example 6₅₀The value is obtained. The CD137 expressing cells may be, for example, CHO cells transfected with human CD137 or cynomolgus CD 137.

In some embodiments, a fusion protein of the invention can have an EC of up to about 10nM or even lower, such as about 8nM or lower, about 6nM or lower, about 4nM or lower, about 2nM or lower, or about 1 or even lower₅₀Values bind to PD-L1 expressed on the cells. The EC of a provided fusion protein can be measured, for example, in a flow cytometry assay substantially as described in example 6₅₀The value is obtained. The cell expressing PD-L1 can be, for example, a CHO cell transfected with human PD-L1 or cynomolgus PD-L1.

In some embodiments, the fusion protein of the invention is capable of binding PD-L1 expressed on tumor cells. In some embodiments, provided fusion proteins can have an E of up to about 2nM or even lower, such as about 1.5nM or lower, about 1nM or lower, about 0.6nM or lower, or about 0.3nM or lowerC₅₀Values bind to PD-L1 expressed on tumor cells. EC of a fusion protein that binds to tumor cells expressing PD-L1 can be measured, for example, in a flow cytometry assay essentially as described in example 7₅₀The value is obtained. The PD-L1-expressing tumor cell can be, for example, an RKO cell.

In some embodiments, the fusion protein of the invention does not substantially affect the binding of CD137 to CD 137L. In some embodiments, the fusion protein of the invention is capable of binding CD137 when formed into a complex with CD 137L. In some embodiments, the fusion protein of the invention is capable of hybridizing to a polypeptide having an amino acid sequence represented by SEQ ID NO: 28 and 29 bind to CD137 in a similar fashion to the heavy and light chain anti-CD 137 antibodies provided. The binding pattern of the fusion protein to CD137 can be determined, for example, by an SPR assay, such as the SPR assay essentially described in example 8.

In some embodiments, the fusion protein of the invention is capable of competing with PD-1 for binding to PD-L1. In some embodiments, provided fusion proteins can have an IC of up to about 5nM or even lower, such as about 3nM or lower, about 2nM or lower, or about 1 or even lower ₅₀Values compete with PD-1 for binding to PD-L1. The inhibitory mode of action may be determined, for example, by an ELISA assay, such as an ELISA assay substantially as described in example 9.

In some embodiments, the fusion proteins of the invention are capable of hybridizing to the sequences shown in SEQ ID NOs: 28 and 29 compete for binding to CD 137. Such competition can be assessed by an ELISA assay essentially as described in example 17. In some embodiments, the provided fusion proteins can be compared to the fusion proteins shown in SEQ ID NOs: 28 and 29 have overlapping epitopes.

In some embodiments, the fusion proteins of the invention are capable of co-stimulating T cell responses. In some embodiments, the provided fusion proteins result in comparable or stronger T cell activation compared to a PD-L1 antibody (such as the building block PD-L1 antibody of seq id nos: 86 and 87 or the reference PD-L1 antibody of seq id nos: 26 and 27) or a CD137 antibody (such as the reference antibody of seq id nos: 28 and 29). In some embodiments, the provided fusion protein results in T cell activation with comparable or better efficacy compared to a combination of an anti-PD-L1 antibody and a molecule targeting CD137 (such as an anti-CD 137 antibody or a previously known CD 137-specific lipocalin mutein). Stimulated T cell responses or T cell activation may be measured, for example, in a CD137 bioassay as substantially described in example 10, in a PD-1/PD-L1 blocking bioassay as substantially described in example 18, or in a functional T cell activation assay as substantially described in example 11, example 12, example 19 and example 20.

In some embodiments, the fusion proteins of the invention are capable of inducing increased IL-2 secretion. In some preferred embodiments, the fusion proteins provided are capable of inducing concentration-dependent IL-2 secretion, and/or exhibit a tendency to induce enhanced IL-2 secretion at higher concentrations, preferably coating concentrations. In some embodiments, the provided fusion protein results in increased IL-2 secretion with comparable or better efficacy as compared to a combination of an anti-PD-L1 antibody and a molecule targeting CD137 (such as an anti-CD 137 antibody or a previously known CD 137-specific lipocalin mutein). IL-2 secretion can be measured, for example, in a functional T cell activation assay essentially as described in example 11 and example 19.

In some embodiments, the fusion proteins of the invention are capable of co-stimulating T cell responses in a PD-L1-dependent manner. In some embodiments, the provided fusion protein can result in local induction of IL-2 production by T cells in the vicinity of PD-L1 positive cells (such as PD-L1 transfected cells or PD-L1 positive tumor cells). As used herein, "in the vicinity of PD-L1 positive cells" means that T cells and PD-L1 positive cells are brought into proximity with each other via a provided fusion protein that simultaneously binds CD137 and PD-L1. The PD-L1 dependent T cell activation of the provided fusion protein may be determined, for example, in a CD137 bioassay as substantially described in example 10, in a PD-1/PD-L1 blocking bioassay as substantially described in example 18, or in a functional T cell activation assay as substantially described in example 12 and example 20.

In some preferred embodiments, the fusion proteins provided are capable of co-stimulating T cells in the presence of PD-L1-expressing tumor cells and/or in a tumor microenvironmentAnd (6) responding. In some embodiments, provided fusion proteins are capable of an EC of about 1nM or less, about 0.5nM or less, about 0.3nM or less, about 0.1nM or less, or about 0.05nM or less in the presence of a PD-L1 positive tumor₅₀Values co-stimulate T cell responses. T cell activation of a fusion protein provided in the presence of PD-L1-expressing tumor cells and/or in a tumor microenvironment can be assessed, for example, in a CD137 bioassay as substantially described in example 10 or in a functional T cell activation assay as substantially described in example 12.

In some embodiments, provided fusion proteins are incapable of co-stimulating a T cell response in the absence of PD-L1. In some embodiments, the provided fusion proteins are incapable of co-stimulating a T cell response in the absence of a cell expressing PD-L1. In some embodiments, the provided fusion proteins are capable of recognizing the presence of PD-L1 and result in a protein that is more abundant than the protein shown in SEQ ID NOs: CD137 antibodies of 28 and 29 better respond to T cell activation. The PD-L1 dependent effect of the fusion protein may be determined, for example, in a CD137 bioassay as substantially described in example 10, in a PD-1/PD-L1 blocking bioassay as substantially described in example 18, or in a functional T cell activation assay as substantially described in example 12 and example 20.

In some embodiments, provided fusion proteins are capable of blocking an inhibitory signal mediated by PD-1 binding to PD-L1. In some embodiments, the provided fusion proteins are capable of releasing the inhibition of T cell activation (brake) or resulting in successful T cell activation by blocking the PD-1/PD-L1 interaction. The blockade of the PD-1 inhibitory signal can be measured, for example, in a PD-1/PD-L1 blockade bioassay as substantially described in example 18.

In some embodiments, the fusion proteins of the invention are capable of stimulating T cell proliferation and/or activation. In some embodiments, the fusion protein provided is capable of stimulating CD4⁺T cell proliferation and/or activation. In some embodiments, the provided fusion proteins are capable of inducing IL-2 secretion, preferably dose-dependent IL-2 secretion. In some embodiments, with anti-PD-L1 antibodies and molecules targeting CD137 (such as anti-beta-gamma)CD137 antibody or a previously known CD 137-specific lipocalin mutein) is able to induce higher IL-2 secretion. IL-2 secretion as used herein can be a measure of T cell activation. Fusion protein provided stimulated CD4 can be assessed by, for example, a Mixed Lymphocyte Reaction (MLR) assay as substantially described in example 14 ⁺T cell proliferation and/or activation.

In some embodiments, the fusion proteins of the invention are capable of stimulating CD8⁺T cell proliferation and/or activation. In some embodiments, provided fusion proteins are capable of inducing production of IL-2 and effector molecules, such as perforin, granzyme a and granzyme B. In some embodiments, the provided fusion proteins are capable of inducing increased production of IL-2 and cytotoxic factors (perforin, granzyme B and granzyme a) as compared to a combination of an anti-PD-L1 antibody and a molecule targeting CD137, such as an anti-CD 137 antibody or a previously known CD 137-specific lipocalin mutein. The fusion protein provided can be assessed for stimulated CD8 by, e.g., an MLR assay as substantially described in example 15⁺T cell proliferation and/or activation.

In some embodiments, provided fusion proteins have good stability and pharmacokinetic characteristics. In some embodiments, provided fusion proteins have a sequence identical to the building block antibody SEQ ID NOs: 86 and 87 are comparable in pharmacokinetic profile. In some embodiments, the provided fusion protein has antibody-like pharmacokinetics. In some embodiments, the provided fusion protein has a terminal half-life of about 200 hours or more, about 250 hours or more, about 300 hours or more, about 350 hours or more, about 400 hours or more, or even more. In some embodiments, provided fusion proteins have greater than SEQ ID NO: 147 better pharmacokinetic profile. In some embodiments, provided fusion proteins have greater than SEQ ID NO: 148 better pharmacokinetic profile. The pharmacokinetic profile of the fusion proteins provided can be analyzed as described in example 21 and example 22. In some embodiments, if after 336h, C _maxThe percentage (%) of (C) is 10%In the above, it is considered that good pharmacokinetic characteristics or antibody-like pharmacokinetics are achieved.

In some embodiments, provided fusion proteins comprise SEQ ID NOs: 88-94, or a pharmaceutically acceptable salt thereof.

In some embodiments, provided fusion proteins comprise a polypeptide that differs from SEQ ID NOs: 88-94, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or even higher sequence identity.

In some embodiments, provided fusion proteins comprise SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87 or SEQ ID NOs: 90 and 91.

In some embodiments, provided fusion proteins comprise a polypeptide that differs from SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87 or SEQ ID NOs: 90 and 91 has an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or even higher sequence identity.

B. Exemplary immunoglobulins contained in fusion proteins

In some embodiments, for fusion proteins provided, the first subunit can be or comprise a full-length immunoglobulin or antigen-binding domain thereof specific for PD-L1. In some embodiments, the immunoglobulin may be, for example, IgG1, IgG2, or IgG 4. In some embodiments, the immunoglobulin is or comprises IgG 4. In some embodiments, the immunoglobulin is a monoclonal antibody directed against PD-L1.

Illustrative examples of antibodies of the invention that bind PD-L1 can comprise an antigen binding region that cross-blocks or binds to the same epitope as a PD-L1-binding antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) of a known antibodyAntibodies such as trastuzumab (atezolizumab) (also known as MPDL3280A or RG7446, trade name)) Avelumab (also known as MSB0010718C, trade name)) Dewar (Durvalumab) (formerly MEDI4736, trade name)) And BMS-936559 (also known as MDX-1105), 5C10 (including humanized 5C10), 5F10 (including humanized 5F10), and 9F6 (including humanized 9F 6). In some embodiments, the PD-L1-binding antibody of the invention may comprise an antigen binding region, such as any of three heavy chain Complementarity Determining Regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), from an antibody selected from the group consisting of trastuzumab, avizumab, desvacizumab, BMS-936559, 5C10, 5F10, and 9F 6.

In some embodiments, provided PD-L1 antibodies or antigen binding domains thereof can have an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-79 and/or a Heavy Chain Variable Region (HCVR) selected from SEQ ID NOs: 80-84 Light Chain Variable Region (LCVR).

In some embodiments, provided PD-L1 antibodies or antigen binding domains thereof can have an amino acid sequence that is SEQ ID NOs: 85-86, and/or is SEQ ID NO: 87, in a pharmaceutically acceptable carrier.

In some embodiments, a PD-L1 antibody or antigen-binding domain thereof is provided wherein the heavy and light chain pairs are or comprise HCVR and LCVR, respectively, as follows: SEQ ID NOs: 75 and 80, SEQ ID NOs: 76 and 81, SEQ ID NOs: 77 and 82, SEQ ID NOs: 78 and 83 or SEQ ID NOs: 79 and 84.

In some embodiments, provided pairs of heavy and light chains of PD-L1 antibodies are or comprise SEQ ID NOs: 85 and 87 or SEQ ID NO: 86 and 87, or a pharmaceutically acceptable salt thereof.

In some embodiments, provided PD-L1 antibodies or antigen binding domains thereof can have an amino acid sequence identical to a sequence selected from SEQ ID NOs: 75-79, and/or a HCVR having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or even higher sequence identity to an amino acid sequence selected from SEQ ID NOs: 80-84 has an LCVR of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or even higher sequence identity. In other embodiments, provided PD-L1 antibodies or antigen binding domains thereof can have an amino acid sequence identical to a sequence selected from SEQ ID NOs: 85-86, and/or a heavy chain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or even higher sequence identity to the amino acid sequence of SEQ ID NO: 87 having a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or even higher.

In some embodiments, the heavy chain variable region of a provided PD-L1 antibody or antigen binding domain thereof can have three CDRs having the following sequences: GFSLSNYD (HCDR1, SEQ ID NO: 60), IWTGGAT (HCDR2, SEQ ID NO: 61), VRDSNYRYDEPFTY (HCDR 3; SEQ ID NO: 62). In some embodiments, the heavy chain variable region of a provided PD-L1 antibody or antigen binding domain thereof can have three CDRs having the following sequences: GFDIKDTY (HCDR1, SEQ ID NO: 65), IDPADGNT (HCDR2, SEQ ID NO: 66), ARGLGAWFAS (HCDR 3; SEQ ID NO: 67). In some embodiments, the heavy chain variable region of a provided PD-L1 antibody or antigen binding domain thereof can have three CDRs having the following sequences: GFNIKDTY (HCDR1, SEQ ID NO: 70), IDPANGNT (HCDR2, SEQ ID NO: 71), SRGPPGGIGEYIYAMDY (HCDR 3; SEQ ID NO: 72).

In some embodiments, the light chain variable region of a provided PD-L1 antibody or antigen binding domain thereof can have three CDRs having the following sequences: QSIGTN (LCDR1, SEQ ID NO: 63), YAS (LCDR2), QQSNSWPYT (LCDR 3; SEQ ID NO: 64). In some embodiments, the light chain variable region of a provided PD-L1 antibody or antigen binding domain thereof can have three CDRs having the following sequences: QDITNS (LCDR1, SEQ ID NO: 68), YTS (LCDR2), QQGHTLPPT (LCDR 3; SEQ ID NO: 69). In some embodiments, the light chain variable region of a provided PD-L1 antibody or antigen binding domain thereof can have three CDRs having the following sequences: SSVSSSY (LCDR1, SEQ ID NO: 73), STS (LCDR2), HQYHRSPPT (LCDR 3; SEQ ID NO: 74).

In some embodiments, provided PD-L1 antibodies or antigen binding domains thereof comprise a heavy chain variable region having three CDRs with the following sequences: GFSLSNYD (HCDR1, SEQ ID NO: 60), IWTGGAT (HCDR2, SEQ ID NO: 61), VRDSNYRYDEPFTY (HCDR 3; SEQ ID NO: 62), the light chain variable region having three CDRs with the following sequences: QSIGTN (LCDR1, SEQ ID NO: 63), YAS (LCDR2), QQSNSWPYT (LCDR 3; SEQ ID NO: 64). In some embodiments, provided PD-L1 antibodies or antigen binding domains thereof comprise a heavy chain variable region having three CDRs with the following sequences: GFDIKDTY (HCDR1, SEQ ID NO: 65), IDPADGNT (HCDR2, SEQ ID NO: 66), ARGLGAWFAS (HCDR 3; SEQ ID NO: 67), the light chain variable region having three CDRs with the following sequences: QDITNS (LCDR1, SEQ ID NO: 68), YTS (LCDR2), QQGHTLPPT (LCDR 3; SEQ ID NO: 69). In some embodiments, provided PD-L1 antibodies or antigen binding domains thereof comprise a heavy chain variable region having three CDRs with the following sequences: GFNIKDTY (HCDR1, SEQ ID NO: 70), IDPANGNT (HCDR2, SEQ ID NO: 71), SRGPPGGIGEYIYAMDY (HCDR 3; SEQ ID NO: 72), the light chain variable region having three CDRs with the following sequences: SSVSSSY (LCDR1, SEQ ID NO: 73), STS (LCDR2), HQYHRSPPT (LCDR 3; SEQ ID NO: 74).

Unless otherwise indicated, all CDR sequences disclosed herein are defined according to The IMGT method described in Lefranc, m. -p., The Immunologist, 7, 132-136 (1999). The CDR1 consists of positions 27 to 38, the CDR2 of positions 56 to 65, the CDR3 of the germline (germline) V-gene of positions 105 to 116, the CDR3 of the rearranged V-J-gene or V-D-J-gene of positions 105 to 117 (positions before the J-PHE or J-TRP 118) with gaps at the loop top for the rearranged CDR3-IMGT with less than 13 amino acids or with further positions 112.1, 111.1, 112.2, 111.2 etc. for the rearranged CDR3-IMGT with more than 13 amino acids. The positions given in this paragraph are numbered according to IMGT as described in Lefranc, m. -p., The Immunologist, 7, 132-.

An antibody specifically binding to PD-1 comprised in a fusion protein of the invention may comprise an Fc portion which allows to extend the in vivo half-life of the bispecific binding molecule of the invention. In some embodiments, such Fc portion is preferably from a human source, more preferably a human Fc portion of an IgG1 or IgG4 antibody, even more preferably an engineered human Fc portion of IgG1 or IgG4 with activated or silenced effector functions. In some embodiments, a silent effector function may be preferred over an activated effector function. In some embodiments, this Fc portion is engineered to have a silent effector function with mutations at positions 234 and/or 235 numbered according to the EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). In some embodiments, mutations can be introduced at positions F234 and L235 of provided anti-PD-1 antibodies to silence effector function. In other embodiments, mutations can be introduced at positions D265 and P329 of provided anti-PD-1 antibodies to silence effector function. The numbering of both sets of these potential mutations is according to the EU index of Kabat (Shields et al, J Biol Chem, 2001).

Various techniques for producing antibodies and fragments thereof are well known in the art and are described, for example, in Altsuhler et al (2010). Thus, for example, polyclonal antibodies can be obtained from the blood of an animal after immunization with an antigen mixed with additives and adjuvants, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous cell line cultures. Examples of such techniques are described, for example, in Harlow and Lane (1999), (1988), and include the techniques originally invented byAnd the hybridoma technology described by Milstein, 1975, the trioma technology, the human B-cell hybridoma technology (see, e.g., Li et al, Proc Natl Acad Sci USA, 2006; Kozbor and Roder, Immunol Today, 1983) and the EBV-hybridoma technology to produce human monoclonal antibodies (Cole et al, Cancer Res, 1984). In addition, recombinant antibodies can be obtained from monoclonal antibodies or can be prepared de novo by various display methods (such as phage, ribosome, mRNA or cell display). In some embodiments, suitable systems for expressing recombinant (humanized) antibodies or fragments thereof can be selected from, for example, bacteria, yeast, insects, mammalian cell lines, or transgenic animals or plants (see, e.g., U.S. Pat. No.6,080,560; Holliger and Hudson, Nat Biotechnol, 2005). In addition, the described techniques for generating single chain antibodies (see, inter alia, U.S. Pat. No.4,946,778) can be adapted to generate single chain antibodies specific for the targets of the invention. Surface plasmon resonance applied in the BIAcore system can be used to increase the efficiency of phage antibodies.

C. Exemplary lipocalin muteins of the invention

Lipocalins are proteinaceous binding molecules that have evolved naturally into binding ligands. Lipocalins are present in many organisms, including vertebrates, insects, plants and bacteria. Members of the lipocalin family (Pervaiz and Brew, FASEB J, 1987) are generally small secreted proteins and have a single polypeptide chain. They are characterized by a range of different molecular recognition properties: they bind a variety of small molecules that are predominantly hydrophobic (e.g., retinoids, fatty acids, cholesterol, prostaglandins, biliverdin, pheromones, sweeteners (tastant), and olfactants (odorants)); and their formation of complexes that bind to specific cell surface receptors and their macromolecules. Although they were primarily classified as transporters in the past, it is now clear that lipocalins fulfill a variety of physiological functions. These functions include roles in retinol transport, olfaction, pheromone signaling, and prostaglandin synthesis. Lipocalins are also involved in the regulation of immune responses and in the mediation of cellular homeostasis (for example, reviewed in Flower et al, Biochim Biophys Acta, 2000; Flower, Biochem J, 1996).

The level of full sequence conservation between lipocalins is very low, typically with less than 20% sequence identity. In strong contrast, their overall folding pattern is highly conserved. The central part of the lipocalin structure consists of a single eight-stranded antiparallel beta-sheet that closes upon itself to form a continuous hydrogen-bonded beta-barrel. The beta barrel forms a central cavity. One end of the barrel is sterically blocked by an N-terminal peptide stretch across its bottom and three peptide loops connecting the β -strands. The other end of the β -barrel is open to the solvent and contains a target binding site formed by four flexible peptide loops (AB, CD, EF, and GH). It is this diversity of the loops in the otherwise rigid lipocalin backbone that leads to a variety of different binding patterns, each capable of accommodating targets of different sizes, shapes and chemical characteristics (e.g., reviewed in Skerra, Biochim Biophys Acta, 2000; Flower et al, Biochim Biophys Acta, 2000; Flower, Biochem J, 1996).

The lipocalin mutein according to the invention may be a mutein of any lipocalin. Examples of suitable lipocalins (also sometimes referred to as "reference lipocalin", "wild-type lipocalin", "reference protein scaffold" or simply "scaffold") for which muteins may be used include, but are not limited to, tear lipocalin (lipocalin-1, Tlc or von Ebner's gland protein), retinol binding protein, neutrophil lipocalin-type prostaglandin D synthase, beta lactoglobulin, Bile Binding Protein (BBP), apolipoprotein D (apod), neutrophil gelatinase-associated lipocalin (NGAL), α 2 microglobulin-associated protein (A2m), 24p 3/uterine calpain (24p3), von Ebner's glandins 1(VEGP 1), von Ebner's glandins 2(VEGP 2), and major allergen Can f 1 (ALL-1). In a related embodiment, the lipocalin mutein is derived from the group of lipocalins consisting of human tear lipocalin (hTIc), human neutrophil gelatinase-associated lipocalin (hNGAL), human apolipoprotein d (hapod), and the bile-binding protein of european Pieris (Pieris brassicae).

The amino acid sequence of a lipocalin mutein according to the invention may have a high sequence identity compared to the sequence identity to another lipocalin (see also above) with the reference (or wild-type) lipocalin from which it is derived (e.g. hTlc or hNGAL). In this general case, the amino acid sequence of a lipocalin mutein according to the invention is at least substantially similar to the amino acid sequence of the corresponding reference (wild-type) lipocalin, provided that there may be gaps (as defined herein) in the alignment due to the addition or deletion of amino acids. A corresponding sequence of a lipocalin mutein of the invention that is substantially similar to the sequence of a corresponding reference (wild-type) lipocalin, in some embodiments has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90% identity, including at least 95% identity, to the sequence of the corresponding lipocalin. In this aspect, the lipocalin muteins of the invention may of course comprise a substitution as described herein, which enables the lipocalin mutein to bind CD 137.

Typically, a lipocalin mutein contains one or more mutated amino acid residues in four loops (see above) comprising the ligand binding pocket and defining the open end of the ligand binding pocket entrance relative to the amino acid sequence of a wild-type or reference lipocalin (e.g. hTlc or hNGAL). As described above, these regions are critical in determining the binding specificity of the lipocalin mutein for the desired target. In some embodiments, the lipocalin muteins of the invention may also contain mutated amino acid residues in regions outside the four loops. In some embodiments, the lipocalin muteins of the invention may contain one or more mutated amino acid residues in one or more of the three peptide loops (designated BC, DE and FG) connecting the β -chain at the blocking end of the lipocalin. In some embodiments, a mutein derived from tear lipocalin, NGAL lipocalin, or a homologue thereof may have 1, 2, 3, 4, or more mutated amino acid residues at any sequence position in the three peptide loops BC, DE, and FG arranged at the N-terminal region and/or the end of the β -barrel structure located opposite the natural lipocalin binding pocket. In some embodiments, a mutein derived from tear lipocalin, NGAL lipocalin, or a homologue thereof may have no mutated amino acid residues in the peptide loop DE arranged at the end of the β -barrel structure compared to the wild-type sequence of tear lipocalin.

In some embodiments, a lipocalin mutein according to the invention may comprise one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even more) mutated amino acid residues compared to the corresponding reference (wild-type) lipocalin, with the proviso that such lipocalin mutein should be able to bind CD 137. In some embodiments, a lipocalin mutein of the invention comprises at least two (including 2, 3, 4, 5 or even more) mutated amino acid residues, wherein the natural amino acid residue of the corresponding reference (wild-type) lipocalin is replaced by an arginine residue.

Any type and number of mutations (including substitutions, deletions and insertions) are contemplated as long as the lipocalin mutein provided retains its ability to bind CD137 and/or has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more sequence identity to the amino acid sequence of a reference (wild-type) lipocalin (e.g., mature hTlc or mature hNGAL).

In some embodiments, the substitutions are conservative substitutions. In some embodiments, the substitutions are non-conservative substitutions or one or more from the exemplary substitutions listed below.

In particular, to determine whether an amino acid residue of a lipocalin mutein amino acid sequence differs from a reference (wild-type) lipocalin corresponding to a certain position in a reference (wild-type) lipocalin amino acid sequence, the skilled person may use means and methods well known in the art, such as aligning manually or by using a computer program, such as BLAST 2.0 (which stands for basic local alignment search tool) or ClustalW or any other suitable program suitable for generating sequence alignments. Thus, the amino acid sequence of a reference (wild-type) lipocalin may serve as "test sequence" or "reference sequence", while the amino acid sequence of a lipocalin mutein serves as "query sequence" (see also above).

Conservative substitutions are generally the following substitutions, each of which is followed by one or more substitutions that may be conservative, according to the amino acid list to be mutated: ala → Ser, Thr or Val; arg → Lys, Gln, Asn, or His; asn → Gln, Glu, Asp or His; asp → Glu, Gln, Asn or His; gln → Asn, Asp, Glu or His; glu → Asp, Asn, Gln or His; his → Arg, Lys, Asn, Gln, Asp or Glu; ile → Thr, Leu, Met, Phe, Val, Trp, Tyr, Ala or Pro; leu → Thr, Ile, Val, Met, Ala, Phe, Pro, Tyr or Trp; lys → Arg, His, Gln, or Asn; met → Thr, Leu, Tyr, Ile, Phe, Val, Ala, Pro or Trp; phe → Thr, Met, Leu, Tyr, Ile, Pro, Trp, Val or Ala; ser → Thr, Ala or Val; thr → Ser, Ala, Val, Ile, Met, Val, Phe, Pro or Leu; trp → Tyr, Phe, Met, Ile or Leu; tyr → Trp, Phe, Ile, Leu or Met; val → Thr, Ile, Leu, Met, Phe, Ala, Ser or Pro. Other substitutions are also permissible and can be determined empirically or based on other known conservative or non-conservative substitutions. As another orientation, each of the following groups contains amino acids that can be commonly used to define conservative substitutions for each other:

(a) Alanine (Ala), serine (Ser), threonine (Thr), valine (Val)

(b) Aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), asparagine (Asn), histidine (His)

(d) Isoleucine (Ile), leucine (Leu), methionine (Met), valine (Val), alanine (Ala), phenylalanine (Phe), threonine (Thr), proline (Pro)

(e) Isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp)

If such conservative substitutions result in a change in biological activity, more substantial changes may be introduced (as described below or further below with respect to amino acid classes) and the products screened for the desired characteristics. Examples of such more substantial changes are: ala → Leu or Phe; arg → Glu; asn → Ile, Val or Trp; asp → Met; cys → Pro; gln → Phe; glu → Arg; his → Gly; ile → Lys, Glu or Gln; leu → Lys or Ser; lys → Tyr; met → Glu; phe → Glu, Gln or Asp; trp → Cys; tyr → Glu or Asp; val → Lys, Arg, His.

In some embodiments, substantial modifications in the physical and biological properties of lipocalins (muteins) are accomplished by selecting substitutions that differ significantly in their effectiveness in maintaining: (a) the structure of the polypeptide backbone in the replacement region, e.g., sheet (sheet) or helical conformation; (b) the charge or hydrophobicity of the molecule at the target; or (c) the volume of the side chain (bulk).

Naturally occurring residues are classified into the following groups based on general side chain properties: (1) hydrophobicity: methionine, alanine, valine, leucine, isoleucine; (2) neutral hydrophilicity: cysteine, serine, threonine, asparagine, glutamine; (3) acidity: aspartic acid, glutamic acid; (4) alkalinity: histidine, lysine, arginine; (5) residues that influence chain orientation: glycine, proline; and (6) aromatic: tryptophan, tyrosine, phenylalanine. In some embodiments, the permutation may entail exchanging members of one of these classes for another.

Any cysteine residues not involved in maintaining the correct conformation of the corresponding lipocalin can also be replaced with serine in general to improve the oxidative stability of the molecule and to prevent abnormal cross-linking. Instead, cysteine bonds may be added to the lipocalin to increase its stability.

D. Exemplary CD 137-specific lipocalin muteins of the invention

As mentioned above, a lipocalin is a polypeptide defined by its supersecondary structure, i.e. a cylindrical β -pleated sheet supersecondary structure region comprising 8 β -strands connected pair by 4 loops at one end to thereby define a binding pocket. The present invention is not limited to the lipocalin muteins specifically disclosed herein. In this aspect, the invention relates to a lipocalin mutein having a cylindrical β -sheet supersecondary structural region comprising 8 β -strands connected pair by pair through 4 loops at one end to thereby define a binding pocket, wherein at least one amino acid of each of at least 3 of said 4 loops has been mutated, and wherein said lipocalin protein effectively binds CD137 with detectable affinity.

In some embodiments, a lipocalin mutein disclosed herein may be or comprise a mutein of mature human tear lipocalin (hTlc). The mutein of mature hTlc is designated herein as the "hTlc mutein". In some other embodiments, the lipocalin muteins disclosed herein are muteins of mature human neutrophil gelatinase-associated lipocalin (hNGAL). The mature hNGAL mutant protein is named "hNGAL mutant protein".

In one aspect, the invention includes any number of lipocalin muteins derived from a reference (wild-type) lipocalin (preferably derived from mature hTlc or mature hNGAL) that bind CD137 with detectable affinity. In a related aspect, the invention comprises a plurality of lipocalin muteins capable of activating a signaling pathway downstream of CD137 by binding to CD 137. In this sense, CD137 may be considered as a non-natural target of a reference (wild-type) lipocalin, preferably hTlc or hNGAL, wherein "non-natural target" refers to a substance that does not bind to the reference (wild-type) lipocalin under physiological conditions. By engineering the reference (wild-type) lipocalin with one or more mutations at certain sequence positions, the inventors of the present invention have demonstrated that high affinity and high specificity for the non-natural target, CD137, is possible. In some embodiments, based on the goal of generating lipocalin muteins capable of binding CD137, random mutagenesis can be performed by substitution with a subset of nucleotide triplets (subset) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even more nucleotide triplets at positions encoding certain sequence positions on the wild-type lipocalin.

In some embodiments, the lipocalin muteins of the invention may have mutated (including substituted, deleted or inserted) amino acid residues at one or more sequence positions corresponding to the linear polypeptide sequence of a reference lipocalin (preferably hTlc or hNGAL). In some embodiments, the number of mutated amino acid residues of a lipocalin mutein of the invention compared to the amino acid sequence of a reference lipocalin (preferably hTlc or hNGAL) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more (such as 25, 30, 35, 40, 45 or 50), wherein preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, more preferably 9, 10 or 11. However, preferably the lipocalin muteins of the invention are still capable of binding to CD 137.

In some embodiments, a lipocalin mutein of the invention may have 1, 2, 3, 4 or more amino acids deleted from its N-terminus and/or 1, 2 or more amino acids deleted from its C-terminus compared to the corresponding reference (wild-type) lipocalin; for example, SEQ ID NOs: 34-40. In some embodiments, the invention encompasses hTalc muteins as defined above, wherein the first 4, 1, 2 or 3N-terminal amino acid residues (His-His-Leu-Leu; positions 1-4) of mature hTalc and/or the last 1 or 2C-terminal amino acid residues (Ser-Asp; positions 157-158) of the linear polypeptide sequence of mature hTalc are deleted (e.g.SEQ ID NOs: 34-40). In some embodiments, the invention covers the definition of hNGAL mutant protein, wherein the mature hNGAL linear polypeptide sequence of amino acid residues (Lys-Asp-Pro; position 46-48) deletion (SEQ ID NO: 45). Furthermore, in addition to the mutated amino acid sequence position, the lipocalin muteins of the invention may comprise the wild-type (native) amino acid sequence of a reference (wild-type) lipocalin, preferably hTlc or hNGAL.

In some embodiments, the introduction of one or more mutated amino acid residues into a lipocalin mutein of the invention does not substantially hinder or interfere with the binding activity and folding of the mutein with the intended target. Such mutations (including substitutions, deletions and insertions) can be carried out at the DNA level using established standard methods (Sambrook and Russell, 2001, Molecular cloning: a laboratory manual). In some embodiments, at one or more sequence positions corresponding to a linear polypeptide sequence of a reference (wild-type) lipocalin (preferably hTlc or hNGAL), mutated amino acid residues are introduced by random mutagenesis by replacing one or more nucleotide triplets encoding the corresponding sequence position of the reference lipocalin with a subset of nucleotide triplets.

In some embodiments, provided lipocalin muteins that bind CD137 with detectable affinity may comprise at least one amino acid substitution that replaces the native cysteine residue with another amino acid (e.g., a serine residue). In some embodiments, with detectable affinity binding CD137 lipocalin muteins can comprise one or more non-native cysteine residues replacing one or more amino acids of a reference (wild-type) lipocalin (preferably hTalc or hNGAL). In some embodiments, the lipocalin muteins according to the invention comprise at least two amino acid substitutions replacing the natural amino acid with a cysteine residue, thereby forming one or more cysteine bridges. In some embodiments, the cysteine bridge may link at least two loop regions. The definitions of these regions are used herein in terms of (Biochim Biophys Acta, 2000), Flower (1996) and Breustedt et al, (2005).

In general, the lipocalin muteins of the invention have at least about 70%, including at least about 80%, such as at least about 85% amino acid sequence identity with the amino acid sequence of mature hTalc (SEQ ID NO: 1) or mature hNGAL (SEQ ID NO: 2).

In some aspects, the invention provides an hTlc mutein that binds CD 137. In this aspect, the invention provides one or more hTalc muteins capable of binding to hTalc protein in a molecule capable ofK of 0nM or less_DThe measured affinity binds to CD 137. In some embodiments, the provided hTlc muteins are capable of an EC of about 250nM, 150nM, 100nM, 50nM, 20nM, or even lower₅₀Values bind to CD 137. In some embodiments, a hTlc mutein that binds CD137 can cross-react with cynomolgus CD137(cyCD 137).

In some embodiments, the hTlc muteins of the present invention may interfere with the binding of CD137L to CD 137.

In some embodiments, provided hTalc muteins can comprise mutated amino acid residues at one or more of positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104, 106, 108, 111, 114, 121, 133, 148, 150, and 153, corresponding to the linear polypeptide sequence of mature hTalc (SEQ ID NO: 1).

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

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

In some embodiments, provided hTalc muteins can comprise mutated amino acid residues at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or even more at positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequence of mature hTalc (SEQ ID NO: 1). In some preferred embodiments, provided hTlc muteins are capable of binding to CD137, in particular human CD 137.

In some embodiments, provided hTalc muteins comprise mutated amino acid residues at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even more positions corresponding to positions 26-34, 55-58, 60-61, 65, 104-106 and 108 of the linear polypeptide sequence of mature hTalc (SEQ ID NO: 1). In some preferred embodiments, provided hTlc muteins are capable of binding to CD137, in particular human CD 137.

In some embodiments, the lipocalin muteins according to the invention may comprise at least one amino acid substitution replacing the native cysteine residue with, for example, a serine residue. In some embodiments, the hTalc muteins according to the present invention comprise an amino acid substitution that replaces the native cysteine residue at a position corresponding to position 61 and/or 153 of the linear polypeptide sequence of mature hTalc (SEQ ID NO: 1) with another amino acid, such as a serine residue. It should be noted in this context that it has been found (in the corresponding native context)At the level of the nucleic acid library) the structural disulfide bond of wild-type hTlc formed by cysteine residues 61 and 153 (see breustdet et al, J Biol Chem, 2005) can provide hTlc muteins that not only stably fold, but also bind a given non-natural target with high affinity. In some embodiments, the elimination of structural disulfide bonds may provide the further advantage of allowing the generation or intentional introduction of non-native disulfide bonds into the muteins of the present invention, thereby increasing the stability of the muteins. However, a hTlc mutein that binds CD137 and has a disulfide bridge formed between Cys 61 and Cys 153 is also part of the present invention.

In some particular embodiments, the hTlc muteins of the present invention may comprise one or more of the following amino acid substitutions at positions corresponding to positions 61 and/or 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1): cys 61 → Ala, Phe, Lys, Arg, Thr, Asn, Gly, Gln, Asp, Asn, Leu, Tyr, Met, Ser, Pro or Trp and/or Cys 153 → Ser or Ala.

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

In some embodiments, a mutein according to the present invention comprises an amino acid substitution in which the natural amino acid at the position corresponding to position 28 or 105 of the linear polypeptide sequence of mature hTalc (SEQ ID NO: 1) is replaced by a cysteine residue. Furthermore, in some embodiments, the mutein according to the present invention comprises an amino acid substitution of the native arginine residue at the position corresponding to position 111 of the linear polypeptide sequence of mature hTalc (SEQ ID NO: 1) with a proline residue. In addition, in some embodiments, the mutein according to the present invention comprises the amino acid substitution of the native lysine residue at the position corresponding to position 114 of the linear polypeptide sequence of mature hTalc (SEQ ID NO: 1) by a tryptophan residue or a glutamic acid.

In some embodiments, provided hTalc muteins that bind CD137 may comprise one or more mutated amino acid residues at one or more of positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153, corresponding to the linear polypeptide sequence of mature hTalc (SEQ ID NO: 1): ala 5 → Val or Thr; arg 26 → Glu; glu 27 → Gly; phe 28 → Cys; pro 29 → Arg; glu 30 → Pro; met 31 → Trp; leu 33 → Ile; glu 34 → Phe; thr 42 → Ser; gly 46 → Asp; lys 52 → Glu; leu 56 → Ala; ser 58 → Asp; arg 60 → Pro; cys 61 → Ala; lys 65 → Arg or Asn; thr 71 → Ala; val 85 → Asp; lys 94 → Arg or Glu; cys 101 → Ser; glu 104 → Val; leu 105 → Cys; his 106 → Asp; lys 108 → Ser; arg 111 → Pro; lys 114 → Trp; lys 121 → Glu; ala 133 → Thr; arg 148 → Ser; ser 150 → Ile; and Cys 153 → Ser. In some embodiments, the hTalc muteins of the invention comprise two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more or even all mutated amino acid residues at these sequence positions of mature hTalc (SEQ ID NO: 1).

In some embodiments, provided hTalc muteins that bind CD137 may comprise one of the following sets of mutated amino acid residues, as compared to the linear polypeptide sequence of mature hTalc (SEQ ID NO: 1):

In some embodiments, the remaining region of the hTalc muteins of the present invention, i.e., the region differing from the positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequence of mature hTalc (SEQ ID NO: 1), may comprise the wild-type (native) amino acid sequence of the linear polypeptide sequence of mature hTalc outside the mutated amino acid sequence positions.

In some embodiments, the hTalc muteins of the present invention have at least 70% sequence identity or at least 70% sequence homology with the sequence of mature hTalc (SEQ ID NO: 1). As an illustrative example, SEQ ID NO: 34 has approximately 84% amino acid sequence identity or sequence homology to the amino acid sequence of mature hTlc.

In some embodiments, the hTlc muteins of the present invention comprise a sequence as set forth in SEQ ID NOs: 34-40 or a fragment or variant thereof.

In some embodiments, the hTlc muteins of the present invention are substantially identical to those selected from the group consisting of SEQ ID NOs: 34-40 have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more sequence identity.

The invention also comprises polypeptides having amino acid sequences selected from the group consisting of SEQ ID NOs: 34-40, said structural homologue having greater than about 60%, preferably greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 92%, and most preferably greater than 95% amino acid sequence homology or sequence identity to said hTlc mutein.

In some aspects, the invention provides binding to CD137 hNGAL mutant protein. In this aspect, the invention provides one or more hNGAL mutant protein, which can pass about 800nM, 700nM, 200nM, 140nM, 100nM or lower, preferably about 70nM, 50nM, 30nM, 10nM, 5nM, 2nM or even lower K_DThe measured affinity binds to CD 137. In some embodiments, the hNGAL mutant protein can be provided with about 1000nM, 500nM, 100nM, 80nM, 50nM, 25nM, 18nM, 15nM, 10nM, 5nM or lower EC ₅₀Values bind to CD 137.

In some embodiments, provided to bind to CD137 hNGAL mutant protein can and macaque CD137 cross-reactive. In some embodiments, the hNGAL mutant protein provided can pass through about 50nM, 20nM, 10nM, 5nM, 2nM or even lower K_DThe measured affinity binds cynomolgus CD 137. In some embodiments, the hNGAL mutant protein can be provided with about 100nM, 80nM, 50nM, 30nM or even lower EC₅₀Values bind to cynomolgus CD 137.

In some embodiments, the invention of the hNGAL mutant protein can interfere with or compete with CD137 binding with CD 137L. In some other embodiments, the invention of the hNGAL mutant protein in the presence of CD137L and/or CD137/CD137L binding complexes capable of binding to CD 137.

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

In some embodiments, the hNGAL mutant protein provided can comprise a mutated amino acid residue at positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or even more positions. In some preferred embodiments, the hNGAL mutant protein can bind to CD137, especially human CD 137.

In some embodiments, the hNGAL mutant protein provided can include a mutated amino acid residue at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 87, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the hNGAL mutant protein can bind to CD137, especially human CD 137.

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

In some other embodiments, the hNGAL mutant protein provided can include a mutation of amino acid residues at one or more of the positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

In other embodiments, the hNGAL mutant protein provided can comprise mutated amino acid residues at one or more positions corresponding to positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) and at one or more positions corresponding to positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 87, 92, 98, 101 and 122 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

In some embodiments, the lipocalin muteins according to the invention may comprise at least one amino acid substitution replacing the native cysteine residue with, for example, a serine residue. In some embodiments, according to the invention of the hNGAL mutant protein can include another amino acid (such as serine residues) replaced natural cysteine residues in the corresponding to mature hNGAL linear polypeptide sequence (SEQ ID NO: 2) position 76 and/or 175 amino acid substitution. It should be noted in this context that it has been found (in the corresponding native context)Nucleic acid library level) removal of the cysteine residues 76 and 175 formed by wild type hNGAL structure disulfide bond (see Breustdet et al, J Biol Chem, 2005) can provide hNGAL mutant protein, not only stable folding, but also with high affinity binding to a given non-natural target. In some embodiments, the elimination of structural disulfide bonds may provide the further advantage of allowing the generation or intentional introduction of non-native disulfide bonds into the muteins of the present invention, thereby increasing the stability of the muteins. However, combined with CD137 and Cys 76 and Cys 175 between the disulfide bridge of hNGAL mutant protein is also part of the invention.

In some embodiments, provided in the CD137 binding hNGAL mutant protein in the corresponding to mature hNGAL linear polypeptide sequence (SEQ ID NO: 2) in the position 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 one or more of the following mutations in the amino acid residues: gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Arg or Lys; gln 49 → Val, Ile, His, Ser or Asn; tyr 52 → Met; asn 65 → Asp; ser 68 → Met, Ala or Gly; leu 70 → Ala, Lys, Ser or Thr; arg 72 → Asp; lys 73 → Asp; asp 77 → Met, Arg, Thr or Asn; trp 79 → Ala or Asp; arg 81 → Met, Trp or Ser; phe 83 → Leu; cys 87 → Ser; leu 94 → Phe; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu and Lys 134 → Tyr. In some embodiments, the hNGAL mutant protein in the invention in mature hNGAL (SEQ ID NO: 2) at these sequence positions contains two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, even more such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or all mutation of the amino acid residues.

In some embodiments, provided by the binding of CD137 hNGAL mutant protein in the corresponding to mature hNGAL linear polypeptide sequence (SEQ ID NO: 2) in the position 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 one or more of the following mutations in the amino acid residues: gln 20 → Arg; asn 25 → Tyr or Asp; gln 28 → His; val 33 → Ile; leu 36 → Met; ala 40 → Asn; ile 41 → Leu; glu 44 → Val or Asp; gln 49 → His; tyr 52 → Ser or Gly; lys 59 → Asn; ser 68 → Asp; leu 70 → Met; phe 71 → Leu; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln or His; tyr 78 → His; trp 79 → Ile; ile 80 → Asn; arg 81 → Trp or Gln; thr 82 → Pro; cys 87 → Ser; phe 92 → Leu or Ser; asn 96 → Phe; lys 98 → Arg; tyr 100 → Asp; pro 101 → Leu; leu 103 → His or Pro; phe 122 → Tyr; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly.

In some embodiments, the provided hNGAL mutant proteins that bind CD137 can comprise one or more of the following mutated amino acid residues at one or more positions corresponding to positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Ser or Gly; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln or His; trp 79 → Ile; arg 81 → Trp or Gln; asn 96 → Phe; tyr 100 → Asp; leu 103 → His or Pro; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly. In some embodiments, the provided hNGAL mutant proteins that bind CD137 can further comprise one or more of the following mutated amino acid residues at one or more positions corresponding to positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 87, 92, 98, 101 and 122 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): gln 20 → Arg; asn 25 → Tyr or Asp; val 33 → Ile; glu 44 → Val or Asp; lys 59 → Asn; phe 71 → Leu; tyr 78 → His; ile 80 → Asn; thr 82 → Pro; phe 92 → Leu or Ser; lys 98 → Arg; pro 101 → Leu; and Phe 122 → Tyr.

In some embodiments, and the mature hNGAL linear polypeptide sequence (SEQ ID NO: 2), the provided combination of CD137 hNGAL mutant protein can contain the following mutations of the amino acid residues of one of the group:

(a) gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Lys; gln 49 → Asn; tyr 52 → Met; ser 68 → Gly; leu 70 → Thr; arg 72 → Asp; lys 73 → Asp; asp 77 → Thr; trp 79 → Ala; arg 81 → Ser; cys 87 → Ser; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu; and Lys 134 → Tyr;

(i) Gln 28 → His; leu 36 → Gln; ala 40 → Ile; ile 41 → Arg; gln 49 → Ser; tyr 52 → Met; asn 65 → Asp; ser 68 → Ala; leu 70 → Thr; arg 72 → Asp; lys 73 → Asp; asp 77 → Asn; trp 79 → Ala; arg 81 → Ser; cys 87 → Ser; asn 96 → Lys; tyr 100 → Phe; leu 103 → His; tyr 106 → Ser; lys 125 → Phe; ser 127 → Phe; tyr 132 → Glu; and Lys 134 → Tyr.

In some other embodiments, in the remaining region, i.e. with the mature hNGAL linear polypeptide sequence (SEQ ID NO: 2) position 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 different region, in the mutation of the amino acid sequence position, the hNGAL mutant protein can contain mature hNGAL wild-type (natural) amino acid sequence.

In some other embodiments, and mature hNGAL linear polypeptide sequence (SEQ ID NO: 2), the hNGAL binding to CD137 mutant protein can contain the following mutations of the amino acid residues of one of the groups:

(a) leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Ser; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(b) leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Ser; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; lys 98 → Arg; tyr 100 → Asp; pro 101 → Leu; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(c) Asn 25 → Tyr; leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Gly; ser 68 → Asp; leu 70 → Met; phe 71 → Leu; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Gln; phe 92 → Ser; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(d) leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Gly; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; tyr 78 → His; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(e) asn 25 → Asp; leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Gly; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(f) val 33 → Ile; leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Gly; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(g) Gln 20 → Arg; leu 36 → Met; ala 40 → Asn; ile 41 → Leu; glu 44 → Val; gln 49 → His; tyr 52 → Gly; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; phe 122 → Tyr; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(h) leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Ser; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; ile 80 → Asn; arg 81 → Trp; thr 82 → Pro; asn 96 → Phe; tyr 100 → Asp; pro 101 → Leu; leu 103 → Pro; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly;

(i) leu 36 → Met; ala 40 → Asn; ile 41 → Leu; gln 49 → His; tyr 52 → Gly; lys 59 → Asn; ser 68 → Asp; leu 70 → Met; arg 72 → Leu; lys 73 → Asp; asp 77 → Gln; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly; and

(j) Leu 36 → Met; ala 40 → Asn; ile 41 → Leu; glu 44 → Asp; gln 49 → His; tyr 52 → Ser; ser 68 → Asp; leu 70 → Met; phe 71 → Leu; arg 72 → Leu; lys 73 → Asp; asp 77 → His; trp 79 → Ile; arg 81 → Trp; phe 92 → Leu; asn 96 → Phe; tyr 100 → Asp; leu 103 → His; lys 125 → Ser; ser 127 → Ile; tyr 132 → Trp; and Lys 134 → Gly.

In some embodiments, in the invention of the hNGAL mutant protein of the remaining region, i.e., and mature hNGAL linear polypeptide sequence (SEQ ID NO: 2) position 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 different region, in the mutation of the amino acid sequence position, can contain mature hNGAL wild type (natural) amino acid sequence.

In some embodiments, the invention of the hNGAL mutant protein and mature hNGAL sequence (SEQ ID NO: 2) has at least 70% sequence identity or at least 70% sequence homology. As an illustrative example, SEQ ID NO: 42 and mature hNGAL amino acid sequence has approximately 87% of the amino acid sequence identity or sequence homology.

In some embodiments, the invention of the hNGAL mutant protein contains such as SEQ ID NOs: 41-59 or a fragment or variant thereof.

In some embodiments, the invention of the hNGAL mutant protein and selected from the group consisting of SEQ ID NOs: 41-59 have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity.

The invention also comprises polypeptides having amino acid sequences selected from the group consisting of SEQ ID NOs: 41-59, the structural homolog has greater than about 60%, preferably greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 92%, and most preferably greater than 95% amino acid sequence homology or sequence identity to the hNGAL mutein.

In some embodiments, the invention provides a K of about 5nM or less_DA lipocalin mutein that binds CD137 with a measured affinity, wherein the lipocalin mutein binds to SEQ ID NO: 42 has a sequence identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more.

In some embodiments, the lipocalin muteins of the invention may comprise a heterologous amino acid sequence, such as a Strep II tag (SEQ ID NO: 12) or a cleavage site sequence of certain restriction enzymes, at their N-or C-terminus, preferably at their C-terminus, without affecting the biological activity of the lipocalin mutein (binding to its target, such as CD 137).

In some embodiments, other modifications of the lipocalin muteins can be introduced to modulate certain characteristics of the muteins, such as increasing folding stability, serum stability, protein resistance or water solubility, or to reduce aggregation tendency, or to introduce new characteristics into the muteins. In some embodiments, the modification can result in modulation of two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) features of the provided mutein.

For example, one or more amino acid sequence positions of a lipocalin mutein may also be mutated to introduce new reactive groups, e.g. for conjugation with other compounds such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins, or for formation of non-naturally occurring disulfide bonds (links). In some cases, conjugated compounds (e.g., PEG and HES) are capable of increasing the serum half-life of the corresponding lipocalin muteins.

In some embodiments, the reactive group of the lipocalin mutein may naturally occur in its amino acid sequence, such as a cysteine residue naturally occurring in said amino acid sequence. In some other embodiments, such reactive groups may be introduced by mutagenesis. In case such reactive groups are introduced by mutagenesis, one possibility is to mutate the amino acid in the appropriate position to a cysteine residue. Exemplary possibilities for such mutations to introduce a cysteine residue into the amino acid sequence of the hTalc mutein include the substitutions Thr 40 → Cys, Glu 73 → Cys, Arg 90 → Cys, Asp 95 → Cys, and Glu 131 → Cys of the wild type sequence of hTalc (SEQ ID NO: 1). An exemplary possibility of such mutations that introduce cysteine residues into the amino acid sequence of the hNGAL mutein includes the introduction of cysteine residues at one or more of the sequence positions corresponding to sequence position 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wild-type sequence of hNGAL (SEQ ID NO: 2). The thiol moieties generated can be used to pegylate or HES the mutein, e.g., in order to increase the serum half-life of the corresponding lipocalin mutein.

In some embodiments, artificial amino acids may be introduced into the amino acid sequence of the lipocalin mutein in order to provide suitable amino acid side chains as new reactive groups for conjugating one of the above-described compounds to the lipocalin mutein. In general, these artificial amino acids are designed to be more reactive, thereby facilitating conjugation with the desired compound. Such an artificial amino acid that can be introduced by mutagenesis (e.g., using an artificial tRNA) is para-acetyl-phenylalanine.

In some embodiments, the lipocalin muteins of the invention are fused at their N-terminus or their C-terminus to a protein, protein domain or peptide, e.g., an antibody, a signal sequence and/or an affinity tag. In some other embodiments, the lipocalin muteins of the invention are conjugated with their N-terminus or their C-terminus to a partner, which is a protein, protein domain or peptide, such as an antibody, signal sequence and/or affinity tag.

Examples of suitable fusion partners are affinity tags such as Strep-tag or Strep-tag II (Schmidt et al, J Mol Biol, 1996), c-myc-tag, FLAG-tag, His-tag or HA-tag or proteins such as glutathione-S-transferase, which allow easy detection and/or purification of the recombinant protein. Proteins with chromogenic or fluorescent properties, such as Green Fluorescent Protein (GFP) or Yellow Fluorescent Protein (YFP), are also suitable fusion partners for the lipocalin muteins of the invention. In general, the lipocalin muteins of the invention may be labeled with any suitable chemical substance or enzyme that directly or indirectly generates a detectable compound or signal in a chemical, physical, optical or enzymatic reaction. For example, a fluorescent or radioactive label can be conjugated to the lipocalin mutein to generate fluorescence or X-rays as a detectable signal. Alkaline phosphatase, horseradish peroxidase and beta-galactosidase are examples of enzyme labels (and at the same time optical labels) that catalyze the formation of a chromogenic reaction product. In general, all labels commonly used for antibodies (except those specifically used with the carbohydrate moiety in the Fc portion of an immunoglobulin) can also be used for conjugation to the lipocalin muteins of the invention.

In some embodiments, the lipocalin muteins of the invention can be fused or conjugated to a moiety that extends the serum half-life of the mutein (see also international patent publication No. wo 2006/056464, where such fusion or conjugation strategy is described with reference to a mutein of human neutrophil gelatinase-associated lipocalin (hNGAL) with CTLA-4 binding affinity). The moiety that extends serum half-life may be a PEG molecule, a HES molecule, a fatty acid such as palmitic acid (Vajo and Duckworth, Pharmacol Rev, 2000), an Fc portion of an immunoglobulin, a C of an immunoglobulin_H3 Domain, C of immunoglobulin_H4 domain, albumin binding peptide or albumin binding protein or transferrin, onlyTo name a few.

In some embodiments, if PEG is used as a conjugation partner, the PEG molecule may be substituted, unsubstituted, linear, or branched. It may also be an activated polyalkylene derivative. Examples of suitable compounds are PEG molecules as described in International patent publication No. WO 1999/64016, U.S. Pat. No.6,177,074 or U.S. Pat. No.6,403,564 for interferons, or PEG molecules as described for other proteins such as PEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA) or PEG-superoxide dismutase (Fuertges and Abuchowski, Journal of Controlled Release, 1990). Such polymers, such as polyethylene glycol, can have a molecular weight of from about 300 to about 70,000 daltons, including, for example, polyethylene glycol having a molecular weight of about 10,000, about 20,000, about 30,000, or about 40,000 daltons. In addition, carbohydrate oligomers and polymers such as HES can be conjugated to the muteins of the present invention for the purpose of extending serum half-life, as described, for example, in U.S. patent No.6,500,930 or 6,620,413.

In some embodiments, if the Fc portion of an immunoglobulin is used for the purpose of extending the serum half-life of the lipocalin muteins of the invention, a SynFusion commercially available from Syntonix Pharmaceuticals, Inc^TMProvided is a technique. The use of this Fc-fusion technology allows the production of longer lasting biopharmaceuticals and may for example consist of two copies of the said mutein linked to the Fc region of an antibody to improve pharmacokinetics, solubility and production efficiency.

Examples of albumin binding peptides that can be used to extend the serum half-life of lipocalin muteins are those having Cys-Xaa, for example as described in U.S. patent publication No.20030069395 or Dennis et al (2002)₁-Xaa₂-Xaa₃-Xaa₄-those of Cys consensus sequence, where Xaa₁Asp, Asn, Ser, Thr or Trp; xaa₂Is Asn, Gln, His, Ile, Leu or Lys; xaa₃Is Ala, Asp, Phe, Trp or Tyr; and Xaa₄Asp, Gly, Leu, Phe, Ser or Thr. Fusion or conjugation to lipocalin muteins to prolongThe long serum half-life albumin binding peptides may be bacterial albumin binding proteins, antibodies, antibody fragments comprising domain antibodies (see, e.g., U.S. Pat. No. 6,696,245) or lipocalin muteins with albumin binding activity. Examples of bacterial albumin binding proteins include streptococcal protein G ((Konig and Skerra, J Immunol Methods, 1998).

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

In some embodiments, albumin itself (Osborne et al, J Pharmacol Exp Ther, 2002) or biologically active fragments of albumin may be used as a partner for the lipocalin muteins of the invention to increase serum half-life. The term "albumin" includes all mammalian albumins, such as human serum albumin or bovine serum albumin or rat albumin. The albumin or fragments thereof may be produced recombinantly as described in us patent No.5,728,553 or european patent publications nos. ep0330451 and ep 0361991. Accordingly, recombinant human albumin (e.g., from Novozymes Delta Ltd., Nottingham, UK) can be used) Conjugated or fused to the lipocalin muteins of the invention.

In some embodiments, if transferrin is used as a partner to extend the serum half-life of a lipocalin mutein of the invention, the mutein may be genetically fused to the N-or C-terminus or both of non-glycosylated transferrin. Non-glycosylated transferrin has a half-life of 14-17 days, and transferrin fusion proteins will similarly have an extended half-life. The transferrin carrier also provides high bioavailability, biodistribution and circulatory stability. This technique is commercially available from BioRexis (BioRexis Pharmaceutical Corporation, PA, USA) And (4) obtaining. Recombinant human transferrin (DeltaFerrin) for use as a protein stabilizer/half-life extending partner^TM) Commercially available from Novozymes Delta Ltd. (Nottingham, UK).

Another option for extending the half-life of the lipocalin muteins of the invention is to fuse a long, non-structural, flexible glycine-rich sequence (e.g.polyglycine having about 20 to 80 consecutive glycine residues) to the N-or C-terminus of the mutein. Such a method is disclosed in international patent publication No. wo2007/038619, for example also referred to as "rPEG" (recombinant PEG).

E. Exemplary uses and applications of fusion proteins specific for CD137 and PD-L1

In some embodiments, the fusion proteins of the present invention may produce a synergistic effect by dual-targeting CD137 and PD-L1. In some embodiments, the fusion proteins of the invention may produce a synergistic effect through CD137 co-stimulation and PD-1/PD-L1 pathway blockade. In some embodiments, the fusion proteins of the invention can produce a local anti-tumor effect by dual targeting of CD137 and PD-L1. Thus, there are many possible applications of the fusion proteins of the present invention in medicine.

In some embodiments, the invention encompasses the use of one or more fusion proteins disclosed herein or one or more compositions comprising such fusion proteins for simultaneously binding CD137 and PD-L1.

The invention also relates to the use of one or more of said fusion proteins for forming a complex with CD137 and/or PD-L1.

Thus, in one aspect of the invention, the fusion proteins provided are useful for detecting CD137 and PD-L1. Such use may include the steps of: contacting one or more of said fusion proteins with a sample suspected of containing CD137 and/or PD-L1 under suitable conditions, thereby allowing a complex to form between the fusion protein and CD137 and/or PD-L1, and detecting the complex by a suitable signal. The detectable signal may be generated by a label as described above, or by a change in a physical property due to binding (i.e. complex formation) itself. One example is surface plasmon resonance, the magnitude of which changes during binding of binding partners, one of which is immobilized on a surface, e.g. gold foil.

The fusion proteins of the invention may also be used to isolate CD137 and/or PD-L1. Such use may include the steps of: contacting one or more of said fusion proteins with a sample suspected of containing CD137 and/or PD-L1 under suitable conditions, thereby allowing a complex to form between said fusion protein and CD137 and/or PD-L1, and isolating said complex from said sample.

In some aspects, the invention provides diagnostic and/or analytical kits comprising one or more fusion proteins according to the invention.

In addition to its use in diagnosis, in a further aspect, the invention contemplates a pharmaceutical composition comprising one or more fusion proteins of the invention and a pharmaceutically acceptable excipient.

Furthermore, in some embodiments, the invention provides fusion proteins that simultaneously bind CD137 and/or PD-L1 for use as, for example, anticancer and/or anti-infective agents and immunomodulators. In some embodiments, the fusion proteins of the present invention are contemplated for use in methods of preventing, ameliorating or treating human diseases such as various cancers, including PD-L1 positive cancers. Accordingly, also provided are methods of preventing, ameliorating or treating human diseases such as various cancers, including PD-L1 positive cancers, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more fusion proteins of the invention.

Examples of cancers that can be treated with the fusion proteins of the present invention include: liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes (carcinoma), carcinoma of the endometrium (carcinoma), carcinoma of the cervix (carcinoma), carcinoma of the vagina (carcinoma), carcinoma of the vulva (carcinoma), non-hodgkin lymphoma, carcinoma of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of children, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis (carcinosa), tumors of the Central Nervous System (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary tumor, Kaposi's (Kaposi) sarcoma, epidermoid carcinoma, Squamous cell carcinoma, environmentally induced cancers including those induced by asbestos, hematological malignancies including, for example, multiple myeloma, B-cell lymphoma, hodgkin's lymphoma/primary mediastinal B-cell lymphoma, non-hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, outer membrane (mantle) cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides (mycosis fungoides), anaplastic large cell lymphoma, T-cell lymphoma, precursor T-lymphoblastic lymphoma, and any combination of said cancers. In some embodiments, the invention is also useful for treating metastatic cancer.

In some embodiments, the fusion proteins of the present invention can simultaneously target tumor cells in which PD-L1 is expressed and activate lymphocytes of the host immune system in the vicinity of such tumor cells. In some embodiments, the fusion proteins of the invention can increase targeted anti-tumor T cell activity, enhance anti-tumor immunity, and/or have a direct inhibitory effect on tumor growth, thereby producing a synergistic anti-tumor outcome. In some embodiments, the fusion proteins of the invention can activate an immune response in the tumor microenvironment. In some embodiments, the fusion proteins of the invention can reduce the side effects of effector lymphocytes on healthy cells, i.e., off-target toxicity, for example, by locally inhibiting oncogene activity and inducing lymphocyte activation.

In some embodiments, the invention encompasses the use of a fusion protein of the invention or a composition comprising a provided fusion protein for inducing a local lymphocyte response in the vicinity of a PD-L1 positive tumor cell. Thus, in some embodiments, the invention provides methods of inducing a local lymphocyte response in the vicinity of a PD-L1 positive tumor cell, the method comprising applying one or more fusion proteins of the invention or one or more compositions comprising such fusion proteins. By "local" is meant that upon simultaneous binding of T cells via CD137 and engagement with PD-L1 positive tumor cells, T cells produce cytokines, particularly IL-2 and/or IFN γ, in the vicinity of PD-L1 positive cells. Such cytokines reflect activation of T cells and can subsequently kill PD-L1 positive cells directly or indirectly by attracting other killer cells (such as T cells or NK cells).

In some embodiments, the invention encompasses the use of the fusion proteins of the invention or compositions comprising such fusion proteins for co-stimulating T cells and/or activating CD137 downstream signaling pathways. Preferably, the provided fusion proteins co-stimulate T cells and/or activate CD137 downstream signaling pathways when engaging tumor cells in which PD-L1 is expressed. Accordingly, the present invention provides a method of inducing T lymphocyte proliferation and/or activating a signaling pathway downstream of CD137, preferably when conjugating a tumor cell in which PD-L1 is expressed, comprising the use of one or more fusion proteins of the invention and/or one or more compositions comprising such fusion proteins.

In some embodiments, the invention encompasses the use of the fusion proteins of the invention or compositions comprising such fusion proteins for inducing CD137 aggregation and activation on T cells and directing such T cells to tumor cells in which PD-L1 is expressed.

Further objects, advantages and features of the present invention will become apparent to those skilled in the art upon examination of the following examples and figures thereof, which are not intended to be limiting. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the disclosure herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

F. Production of exemplary fusion proteins specific for CD137 and PD-L1

In some embodiments, the invention provides nucleic acid molecules (DNA and RNA) comprising nucleotide sequences encoding the provided fusion proteins. In some embodiments, the invention encompasses host cells containing the provided nucleic acid molecules. Since the degeneracy of the genetic code allows certain codons to be substituted with other codons specifying the same amino acid, the present invention is not limited to a particular nucleic acid molecule encoding a fusion protein as described herein, but encompasses all nucleic acid molecules comprising a nucleotide sequence encoding a functional fusion protein. In this regard, the invention also relates to nucleotide sequences encoding the provided fusion proteins.

A nucleic acid molecule, e.g. DNA, is said to be "capable of expressing a nucleic acid molecule" or "capable of allowing the expression of a nucleotide sequence" if it comprises sequence elements that contain information about the regulation of transcription and/or translation, and such a sequence is "operably linked" to the nucleotide sequence encoding the protein. The operable connection is such that: in this connection, the regulatory sequence elements and the sequence to be expressed are connected in such a way that gene expression is possible. The exact nature of the regulatory regions necessary for gene expression may vary from species to species, but generally these regions include promoters which contain in prokaryotes the promoter itself (i.e., the DNA elements which direct the initiation of transcription) as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation. Such promoter regions typically include 5 'non-coding sequences involved in transcription and translation initiation, such as the-35/-10 box and Shine-Dalgamo elements in prokaryotes or the TATA box, CAAT sequences and 5' -capping elements in eukaryotes. These regions may also include enhancer or repressor elements as well as translation signals and leader sequences for targeting the native protein to specific compartments of the host cell.

In addition, the 3' non-coding sequence may contain regulatory elements involved in transcription termination, polyadenylation, and the like. However, if these termination sequences are not satisfactorily functional in a particular host cell, they may be replaced by signals functional in that cell.

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

In some embodiments, a nucleic acid molecule encoding a lipocalin mutein disclosed in the present application may be "operably linked" to another nucleic acid molecule encoding an immunoglobulin of the invention to allow for expression of the fusion protein disclosed herein.

In some embodiments, provided methods can include subjecting at least one nucleic acid molecule encoding mature hTalc to mutagenesis at nucleotide triplets encoding one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104 and 106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of hTalc (SEQ ID NO: 1) to obtain a lipocalin mutein comprised in the provided fusion protein. In some embodiments, the method provided can include at least one encoding mature hNGAL nucleic acid molecules at nucleotide triplets encoding one or more corresponding to hNGAL linear polypeptide sequence (SEQ ID NO: 2) position 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 position mutation, to obtain included in the fusion protein provided. In some embodiments, the method provided can include at least one encoding mature hNGAL nucleic acid molecules at nucleotide triplets encoding one or more corresponding to hNGAL linear polypeptide sequence (SEQ ID NO: 2) of position 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 position to obtain included in the fusion protein provided lipocalin muteins.

In addition, for the invention of the hTalc mutein or hNGAL mutein included in the fusion protein, in some embodiments, can remove Cys 61 and Cys 153 or Cys 76 and Cys 175 between the naturally occurring disulfide bond. Thus, such muteins can be produced in a cellular compartment with a reduced redox environment (redox milieu), for example in the cytoplasm of gram-negative bacteria.

Further to the fusion protein containing the invention provides hTalc mutant protein or hNGAL mutant protein, the invention also includes encoding such mutant protein nucleic acid molecules, in some embodiments, it can experimental mutagenesis in the designated sequence position outside of one or more additional mutations. Such mutations are often tolerable or even prove advantageous, for example, if they contribute to an increase in folding efficiency, serum stability, thermostability or ligand binding affinity of the lipocalin mutein and/or the fusion protein.

In some embodiments, the provided nucleic acid molecule may also be part of a vector or any other kind of cloning vector (vechicle), such as a plasmid, phagemid, phage, baculovirus, cosmid or artificial chromosome.

In some embodiments, the provided nucleic acid molecules can be contained in a phagemid. As used in this context, a phagemid vector refers to a vector encoding the intergenic region of a temperate phage (such as M13 or f1), or a functional portion thereof fused to a cDNA of interest. For example, in some embodiments, upon superinfection of a bacterial host cell via such provided phagemid vectors and appropriate helper phage (e.g., M13K07, VCS-M13, or R408), intact phage particles are produced, thereby enabling the encoded heterologous cDNA to be physically coupled to the polypeptide displayed on the surface of its corresponding phage (Lowman, Annu Rev Biophys Biomol Struct, 1997; Rodi and Makowski, Curr Opin Biotechnol, 1999).

According to various embodiments, in addition to the regulatory sequences described above and the nucleic acid sequence encoding the fusion protein as described herein, the cloning vector may comprise replication and control sequences derived from a species compatible with the host cell used for expression, and a selectable marker conferring a selectable phenotype on the transformed or transfected cell. A large number of suitable cloning vectors are known in the art and are commercially available.

In some embodiments, the invention also relates to methods of producing the fusion proteins of the invention using genetic engineering methods, starting from nucleic acids encoding the fusion protein or any subunit thereof. In some embodiments, the method may be performed in vivo, wherein the provided fusion protein may be produced, for example, in a bacterial or eukaryotic host organism, and then isolated from the host organism or culture thereof. The fusion proteins of the invention can also be produced in vitro, for example, using an in vitro translation system.

When fusion proteins are produced in vivo, nucleic acids encoding such proteins may be introduced into a suitable bacterial or eukaryotic host organism using recombinant DNA techniques well known in the art. In some embodiments, DNA molecules encoding fusion proteins as described herein (e.g., SEQ ID NOs: 138-144), particularly cloning vectors containing the coding sequence of such fusion proteins, can be transformed into host cells capable of expressing the gene. Transformation can be carried out using standard methods. Thus, the invention also relates to host cells containing the nucleic acid molecules described herein.

In some embodiments, the transformed host cell may be cultured under conditions suitable for expression of the nucleotide sequence encoding the fusion protein of the invention. In some embodiments, the host cell may be prokaryotic such as e.coli (e.coli) or Bacillus subtilis, or eukaryotic such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia pastoris (Pichia pastoris), SF9 or High5 insect cells, immortalized mammalian cell lines such as HeLa cells or CHO cells, or primary mammalian cells.

In some embodiments, wherein the lipocalin muteins of the invention (including comprised in the fusion proteins disclosed herein) comprise intramolecular disulfide bonds, the nascent protein may preferably be directed to a cellular compartment having a redox environment using an appropriate signal sequence. Such an oxidizing environment may be provided by the periplasm of gram-negative bacteria (such as E.coli), in the extracellular environment of gram-positive bacteria, or in the lumen of the endoplasmic reticulum of eukaryotic cells, and generally favors the formation of structural disulfide bonds.

In some embodiments, the fusion protein of the invention may also be produced in the cytosol of a host cell, preferably E.coli. In this case, the fusion protein provided can be obtained directly in the soluble and folded state or recovered in the form of inclusion bodies and then renatured in vitro. Another option is to use a specific host strain that has an oxidizing intracellular environment, which thus allows the formation of disulfide bonds in the cytosol (Venturi et al, J Mol Biol, 2002).

In some embodiments, the fusion proteins of the invention as described herein are not necessarily produced or produced in whole or in part by the use of genetic engineering. Rather, such proteins may also be obtained by any of a number of conventional and well-known techniques, such as general organic synthesis strategies, solid-phase-related synthesis techniques, commercially available automated synthesizers, or by in vitro transcription or translation. For example, it is possible to identify promising fusion proteins or lipocalin muteins comprised in such fusion proteins using molecular modeling, synthesize in vitro, and study the binding activity to a target of interest. Methods for solid and/or solution phase synthesis of proteins are well known in the art (see, e.g., Bruckdorfer et al, Curr Pharm Biotechnol, 2004).

In some embodiments, the fusion proteins of the invention can be produced by in vitro transcription/translation using well-established methods known to those skilled in the art.

In some further embodiments, the fusion proteins described herein can also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques.

Furthermore, in some embodiments, the fusion protein according to the present invention may be obtained by conjugating separate subunits (e.g. immunoglobulins) together with a mutein comprised in the fusion protein. Such conjugation can be achieved, for example, by all forms of covalent or non-covalent attachment using conventional methods.

One skilled in the art will appreciate methods that may be used to prepare fusion proteins contemplated by the present invention, but whose protein or nucleic acid sequences are not explicitly disclosed herein. By way of overview, such modifications of the amino acid sequence include, for example, directed mutagenesis of individual amino acid positions to simplify subcloning of protein genes or portions thereof by introducing cleavage sites for certain restriction enzymes. In addition, these mutations can also be introduced to further increase the affinity of the fusion protein for its target (e.g., CD137 and PD-L1). Furthermore, mutations may be introduced to modulate one or more characteristics of the protein, if desired, for example, to improve folding stability, serum stability, protein resistance or aqueous solubility or to reduce aggregation propensity.

V. examples

Example 1: expression and analysis of representative fusion proteins

In this example, a PD-L1 specific antibody was conjugated to SEQ ID NO: 42 via a linker (such as the non-structural (G) of SEQ ID NO: 13₄S)₃Linker) to simultaneously ligate PD-L1 and CD137, resulting in a representative antibody-lipocalin mutein fusion protein, the PD-L1 specific antibody having the amino acid sequence of SEQ ID NO: 86, or a light chain variable domain comprising SEQ ID NO: 77, or comprises the CDRs of GFSLSNYND (HCDR1, SEQ ID NO: 60), IWTGGAT (HCDR2, SEQ ID NO: 61), VRDSNYRYDEPFTY (HCDR 3; SEQ ID NO: 62), and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 87, or a light chain comprising SEQ ID NO: 82, or comprises the CDRs of QSIGTN (LCDR1, SEQ ID NO: 63), YAS (LCDR2), QQSNSWPYT (LCDR 3; SEQ ID NO: 64). The resulting different forms are shown in fig. 1. For example, by converting one or more of SEQ ID NOs: 42 with one or more of the four ends of an antibody to produce a lipocalin mutein such as SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, and SEQ ID NOs: 90 and 91, said antibody comprising SEQ ID NO: 86, or a light chain comprising SEQ ID NO: 77, or comprises the CDRs of GFSLSNYND (HCDR1, SEQ ID NO: 60), IWTGGAT (HCDR2, SEQ ID NO: 61), VRDSNYRYDEPFTY (HCDR 3; SEQ ID NO: 62), and SEQ ID NO: 87, or a light chain comprising SEQ ID NO: 82, or comprises the CDRs of QSIGTN (LCDR1, SEQ ID NO: 63), YAS (LCDR2), QQSNSWPYT (LCDR 3; SEQ ID NO: 64). The resulting fusion protein can be bivalent to CD137 (e.g., as shown in FIGS. 1A-1D), tetravalent to CD137 (e.g., as shown in FIGS. 1E-1H), or even more valent to CD137 (e.g., as shown in FIG. 1I).

The PD-L1 specific antibodies and the whole antibody lipocalin mutein fusion proteins described in this example have an engineered IgG4 backbone comprising the S228P mutation to minimize IgG4 half-antibody exchange in vitro and in vivo (Silva et al, J Biol Chem, 2015). Additional mutations in the IgG4 backbone may also be present in all of the antibodies and fusion proteins described herein, including any one or more of the mutations F234A, L235A, M428L, N434S, M252Y, S254T, and T256E. F234A and L235A mutations may also be introduced to reduce ADCC and ADCP (Glaesener et al, Diabetes Metab Res Rev, 2010). M428L and N434S mutations, or M252Y, S254T and T256E mutations, may also be introduced to extend serum half-life (Dall' Acqua et al, J Biol Chem, 2006; Zalevsky et al, Nat Biotechnol, 2010). All antibody expressions were free of carboxy-terminal lysines to avoid heterogeneity.

Furthermore, as shown in fig. 1J-1K, the sequence is determined by comparing one or more of SEQ ID NOs: 42 via a linker (non-structural as SEQ ID NO: 13 (G)₄S)₃Linker) to SEQ ID NO: 30 to obtain a monospecific lipocalin mutein Fc fusion protein. SEQ ID NOs: 88-89, the resulting constructs are provided.

The invention also encompasses asymmetric antibody-lipocalin mutein fusion forms, wherein, for example, one light chain of the antibody may be fused to the lipocalin mutein while the other light chain is not fused.

Constructs of the fusion proteins were produced by gene synthesis and cloning into mammalian expression vectors. Constructs of the fusion proteins were then transiently expressed in Expi293FTM cells (Life Technologies). By application ofProtein A affinity chromatography columns (Applied Biosystems) HPLC (Agilent technologies) measure the concentration of the fusion protein in the cell culture medium. The titers of the fusion proteins are summarized in table 1.

The fusion protein was purified by protein a chromatography followed by Size Exclusion Chromatography (SEC) in Phosphate Buffered Saline (PBS). After SEC purification, fractions containing monomeric protein were pooled and re-analyzed using analytical-grade SEC.

Table 1: instantaneous expression titer

Example 2: expression of fusion proteins

Constructs of exemplary fusion proteins were produced by gene synthesis (including codon optimization) and cloning into mammalian expression vectors. The construct of the fusion protein was then stably expressed in Chinese Hamster Ovary (CHO) cells. The concentration of the fusion protein in the cell culture medium was measured by Octet (ForteBio, Pall Corp.) with a protein-a sensor and quantified using a human IgG1 standard. The titers of the fusion proteins are summarized in table 2. The data indicate that the geometry (geometry) of the fusion protein may have an effect on product yield and cell productivity.

Table 2: stable expression titer

Example 3: determination of the binding of the fusion protein to PD-L1 or CD137 by Surface Plasmon Resonance (SPR)

The binding kinetics and affinity of exemplary fusion proteins to huPD-L1-His or huCD137-His (human PD-L1 or human CD137, R & D Systems with C-terminal polyhistidine tags) were determined by Surface Plasmon Resonance (SPR) using Biacore 8K or Biacore T200(GE Healthcare).

Anti-human IgG Fc antibody (GE Healthcare) was immobilized on CM5 sensor chip using standard amine chemistry: carboxyl groups on the chip were activated using 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Subsequently, an anti-human IgG Fc antibody solution (GE Healthcare) at a concentration of 25. mu.g/mL in 10mM sodium acetate (pH 5.0) was applied at a flow rate of 5. mu.L/min until a fixed level of 6000-10000 Resonance Units (RU) was reached. The remaining unreacted NHS-ester was blocked by passing a 1M ethanolamine solution across the surface. The reference channel is treated in a similar manner. Then, the test fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, SEQ ID NOs: 90 and 91, SEQ ID NO: 88 and SEQ ID NO: 89) present in HBS-EP + buffer at 0.25. mu.g/mL or 0.5. mu.g/mL were captured with an anti-human IgG Fc antibody on the chip surface for 180 s. After each capture step, the needle (needle) was washed. As controls, anti-PD-L1 antibodies, including reference antibodies (SEQ ID NOs: 26 and 27) and antibodies contained in the fusion protein (SEQ ID NOs: 86 and 87), and reference anti-CD 137 antibodies (SEQ ID NOs: 28 and 29) were also tested.

For affinity assays, dilutions of huPD-L1-His (10nM, 5nM and 2.5nM) or huCD137-His (900nM, 300nM and 100nM) were prepared in HBS-EP + buffer and applied to the prepared chip surface. The binding assay was performed with a contact time of 180s, a dissociation time of 900s and a flow rate of 30. mu.L/min. All measurements were performed at 25 ℃. By injecting 3M MgCl₂Up to 120s to achieve regeneration of the chip surface. Before protein measurement, three priming cycles were performed for conditioning purposes. Evaluation of softness with Biacore T200Piece (V2.0) or Biacore 8K evaluation software (V1.1.1) evaluated the data. The raw data was fitted using a double reference and using a 1: 1 binding model.

K of representative fusion proteins to be determined_on、k_offAnd the resulting equilibrium dissociation constant (K)_D) The results are summarized in Table 3. All bispecific fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, and SEQ ID NOs: 90 and 91) bind PD-L1 and CD137 with sub-nanomolar to low nanomolar affinities. Monospecific CD 137-specific lipocalin mutein-Fc fusion proteins (SEQ ID NO: 88 and SEQ ID NO: 89) bind CD137 only with low nanomolar affinity.

Table 3: kinetic constants and affinities of fusion proteins determined by SPR

Example 4: binding of the fusion protein to PD-L1 or CD137 in an enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) was used to determine the binding potency of the exemplary fusion proteins to human PD-L1 and cynomolgus PD-L1.

Recombinant hupD-L1-His or cyPD-L1-His (human or cynomolgus PD-L1 with a C-terminal polyhistidine tag, R & D Systems or Sino Biologics) at a concentration of 1. mu.g/mL in PBS was coated on microtiter plates overnight at 4 ℃. After washing with PBS-0.05% T (PBS supplemented with 0.05% (v/v) Tween 20), plates were blocked with 2% BSA (w/v) in PBS-0.1% T (PBS supplemented with 0.1% (v/v) Tween 20) for 1 hour at room temperature. After 5 washes with 100 μ L PBS-0.05% T, different concentrations of the exemplary fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, SEQ ID NOs: 90 and 91), CD 137-specific lipocalin mutein Fc fusion protein (SEQ ID NOs: 88 and SEQ ID NO: 89) and anti-PD-L1 antibodies (SEQ ID NOs: 26 and 27, SEQ ID NOs: 86 and 87) were added to the wells and incubated at room temperature for 1h, followed by another wash step. Bound molecules in the study were detected by incubation with anti-human IgG Fc-HRP (Jackson laboratory) diluted 1: 5000 in PBS-0.1% T-2% BSA. After additional washing steps, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescent microplate reader.

The binding potency of the fusion protein to CD137 was also measured using the same ELISA setup, in which huCD137-His (human CD137 with a C-terminal polyhistidine tag, R & D Systems) or cyCD137-Fc (cynomolgus CD 137C-terminally fused to Fc) was coated on microtiter plates. The test reagents and the bound reagents were similarly titrated via anti-NGAL-HRP detection.

The results of exemplary experiments, and fitted curves resulting from 1: 1 in combination with sigmoid fitting, are depicted in FIGS. 2A-2D, where EC₅₀The value and maximum signal are free parameters and the slope is fixed to unity. The obtained EC₅₀The values are provided in table 4.

The EC observed for the provided fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, SEQ ID NOs: 90 and 91, SEQ ID NO: 88, and SEQ ID NO: 89) for two human targets₅₀The values are very similar or comparable to the tested PD-L1 antibody (reference PD-L1 antibody of SEQ ID NOs: 26 and 27 and PD-L1 antibody of SEQ ID NOs: 86 and 87 comprised in the fusion protein) and/or the CD 137-specific lipocalin mutein (SEQ ID NO: 42) comprised in the fusion protein.

All tested fusion proteins showed EC comparable to the reference PD-L1 antibody (SEQ ID NOs: 26 and 27) or the PD-L1 antibody contained in the fusion protein (SEQ ID NOs: 86 and 87) ₅₀The values are cross-reactive with cynomolgus PD-L1. The tetravalent-only fusion protein to CD137 (SEQ ID NOs: 94 and 87, SEQ ID NOs: 90 and 91, and SEQ ID NO: 88) showed cross-reactivity with cynomolgus CD137 at a level comparable to that of human CD137, i.e., at an EC corresponding to that of human CD137₅₀EC in the same range₅₀Values bind to cynomolgus CD 137.

Table 4: ELISA data for PD-L1 or CD137 binding

Example 5: fusion proteins bind to both PD-L1 and CD137 in ELISA

To demonstrate that the exemplary fusion protein binds both PD-L1 and CD137, a dual binding ELISA format was used.

Recombinant huPD-L1-His (R & D Systems) (1. mu.g/mL) in PBS was coated overnight on microtiter plates at 4 ℃. After each incubation step, plates were washed 5 times with 100 μ L PBS-0.05% T. The plates were blocked with 2% BSA in PBS-0.1% T (w/v) for 1 hour at room temperature and then washed again. Different concentrations of the test fusion protein were added to the wells and incubated at room temperature for 1 hour, followed by a washing step. Subsequently, biotinylated huCD137-His (huCD137-His-Bio, Sino Biological) was added at a constant concentration of 1. mu.g/mL in PBS-0.1% T-2% BSA for 1 hour. After washing, a 1: 5000 dilution of Extravavidin-HRP (Sigma-Aldrich) in PBS-0.1% T-2% BSA was added to the wells and incubated for 1 h. After additional washing steps, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescent microplate reader.

The dual binding of the exemplary fusion proteins was also tested in a reverse setup, in which recombinant 1 μ g/ml huCD137-His (R & D Systems) was coated on microtiter plates and bound fusion proteins were detected by addition of biotinylated huPD-L1-His (R & D Systems).

The dual binding data for the fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, and SEQ ID NOs: 90 and 91) and the fitted curves generated by 1: 1 binding sigmoid fitting (sigmoidal binding fit) are shown in FIGS. 3A and 3B, where EC is₅₀The value and the maximum signal are selfThe slope is fixed to unity by a parameter. EC (EC)₅₀The values are summarized in Table 5. All bispecific fusion proteins showed a clear binding signal, indicating that the fusion protein is capable of simultaneously engaging PD-L1 and CD 137. The data further indicate that it may be more advantageous to fuse a CD 137-specific lipocalin mutein to the C-terminus of a PD-L1-specific antibody than to the N-terminus.

Table 5: ELISA data for simultaneous target binding of PD-L1 and CD37

Example 6: flow cytometry analysis of fusion proteins binding to cells expressing human and cynomolgus CD137 and PD-L1

Target-specific binding of the fusion proteins to cells expressing human and cynomolgus PD-L1 and cells expressing human and cynomolgus CD137 was assessed by flow cytometry.

CHO cells were stably transfected with human PD-L1, cynomolgus PD-L1, human CD137, cynomolgus CD137 or mock controls using the Flp-In system (Life technologies) according to the manufacturer's instructions.

Transfected CHO cells were maintained in Ham's F12 medium (Life technologies) supplemented with 10% fetal bovine serum (Biochrom) and 500. mu.g/ml hygromycin B (Roth). According to the manufacturer's instructions (37 ℃, 5% CO)₂Atmosphere), cells were cultured in cell culture flasks.

For flow cytometry analysis, the corresponding cell lines were incubated with the fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, SEQ ID NOs: 90 and 91, SEQ ID NO: 88, and SEQ ID NO: 89) and detected using fluorescently labeled anti-human IgG antibodies in FACS analysis as described below:

5X 104 cells per well were incubated for 1h in ice-cold PBS (PBS-FCS) containing 5% fetal calf serum. Dilution series of fusion protein and control antibody were added to the cells and incubated on ice for 1 h. Cells were washed twice with PBS and then incubated with goat anti-hIgG Alexa647 labeled antibody on ice for 30 min. The cells were subsequently washed and analyzed with an iQue flow cytometer (Intellicy Screen). The mean geometric fluorescence signal was plotted and fitted using Graphpad software using non-linear regression (shared below, slope 1).

Figure 4 depicts the ability of the fusion protein to bind human and cynomolgus PD-L1 and CD 137. Binding affinities (EC) of bispecific fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, and SEQ ID NOs: 90 and 91) to cells expressing human and cynomolgus PD-L1 (see, e.g., FIGS.)₅₀) In the one-digit nanomolar range, complete macaque (cyno) -cross-reactivity was demonstrated (summarized in table 6). The binding affinity of the fusion protein to human CD 137-expressing cells is in the low nanomolar range. The tested fusion proteins were completely cross-reactive with cynomolgus CD137(SEQ ID NOs: 94 and 87, SEQ ID NOs: 90 and 91, and SEQ ID NO: 88) to bind cynomolgus CD137 with an affinity that was reduced by 6-13 fold compared to the binding affinity of human CD137(SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, and SEQ ID NO: 89), or to non-cynomolgus CD137(SEQ ID NOs: 92 and 87 and 86 and 93), respectively. No fusion protein bound to mock transfected cells.

Table 6: binding affinity of fusion proteins to cells expressing human and cynomolgus PD-L1 or CD137

Example 7: binding affinity of fusion proteins to PD-L1 positive tumor cells

The binding of the fusion protein to tumor cells expressing PD-L1 was assessed by flow cytometry.

The colorectal cancer cell line RKO expressing PD-L1 was maintained in RPMI1640(Life technologies) supplemented with 10% FCS at 37 ℃ under a humidified 5% CO2 atmosphere.

For flow cytometry analysis, RKO cells were incubated with the fusion protein and detected as described in example 6 using fluorescently labeled anti-human IgG antibodies.

FIG. 5 depicts fusion proteins (SEQ ID NOs: 90 and 87,SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, and SEQ ID NOs: 90 and 91) ability to bind to PD-L1 positive tumor cells and the corresponding binding affinity (EC)₅₀) The results are summarized in Table 7. The binding affinity of the fusion protein to RKO cells expressing PD-L1 was in the low nanomolar or sub-nanomolar range, comparable to that of the PD-L1 antibodies (SEQ ID NOs: 86 and 87) contained in the fusion protein.

Table 7: binding affinity of fusion proteins to PD-L1 positive tumor cells

Example 8: competition between CD137L and fusion protein in binding to CD137 by SPR

The competition of human CD137L (huCD137L-His, R & D Systems) with exemplary fusion proteins for human CD137 was investigated using SPR assays. The competition assay was performed on a Biacore T200 instrument (GE Healthcare) at 25 ℃.

The BiotinCAPture reagent (GE Healthcare) was immobilized on the CAP sensor chip at a concentration of 50. mu.g/ml and a flow rate of 2. mu.L/min for 300 s. The reference channel is treated in a similar manner. Biotinylated huCD137-Fc (R & D systems) was captured on the chip surface for 300s in another channel at a concentration of 1. mu.g/mL and a flow rate of 5. mu.L/min.

To analyze whether the test fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 90 and 91, SEQ ID NO: 88, and SEQ ID NO: 89) competed with CD137L for binding to CD137, a running buffer (HBS-EP + buffer) or 500nM huCD137L-His was applied to the chip surface at a flow rate of 30. mu.L/min for 180 s. Subsequently, the test fusion protein was applied to the prepared chip surface at a fixed concentration of 1 μ M in HBS-EP + buffer. The binding assay was performed with a contact time of 180s, a dissociation time of 15s and a flow rate of 30. mu.L/min. As a control, buffer was injected with the same parameters. 120s by injecting 6M Gua-HCl, 0, 25M NaOH at a flow rate of 10. mu.L/min, followed by H₂Additional washing step of O (12)0s, 10 mul/min) to achieve regeneration of the chip surface.

The fusion proteins SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 90 and 91, SEQ ID NO: 88. and SEQ ID NO: 89 representative examples of relevant segments of the resulting sensorgrams. The SPR trace of the binding of the corresponding fusion protein to huCD137-Fc alone was marked with a solid-stem arrow (arrow with a solid stem). The SPR trace of the binding of the fusion protein to huCD137-Fc which had been saturated with huCD137L-His was marked with a dashed stem arrow (arrow with a broken stem). The data indicate that all fusion proteins bound huCD137 in the presence of huCD137L, but had slightly reduced signals compared to their binding to CD137 in the absence of CD 137L. This suggests that the fusion protein tested may be sterically hindered by the binding of CD137L to CD 137. This binding behavior of the fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 90 and 91, SEQ ID NO: 88 and SEQ ID NO: 89) was compared to the binding behavior of the anti-CD 137 antibody SEQ ID NOs: the binding behavior of 28 and 29 is similar.

Example 9: determination of the Competition of the fusion protein with PD-L1 in binding to PD-1 by ELISA

To demonstrate the ability of the fusion protein to inhibit the interaction between PD-1 and PD-L1, a competitive ELISA format (format) was used.

Recombinant huPD-1-His (Acrobiosystems) (1. mu.g/mL) in PBS was coated overnight on microtiter plates at 4 ℃. After each incubation step, plates were washed 5 times with 100 μ L PBS-0.05% T (PBS supplemented with 0.05% (v/v) tween 20). Plates were blocked with 2% BSA (w/v) in PBS-0.1% T (PBS supplemented with 0.1% (v/v) Tween 20) for 1 hour at room temperature, then washed again. Different concentrations of the fusion protein were mixed with 15nM recombinant huPD-L1-Fc (R & D systems) as tracer and incubated for 1 hour at room temperature. The mixture of fusion protein and tracer was added to the plate and incubated at room temperature for 20min, followed by 5 wash steps with 100 μ L PBS-0.05% T. A1: 5000 dilution of goat-anti-human IgG-Fc HRP (Jackson) was then added to the wells and incubated for 1 h. After additional washing steps, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescent microplate reader.

The competition data for exemplary fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, and SEQ ID NOs: 90 and 91) are shown in FIG. 7, along with the fitted curves resulting from the 1: 1 binding sigmoid fit, where IC is ₅₀The value and maximum signal are free parameters and the slope is fixed to unity. IC (integrated circuit)₅₀The values are summarized in Table 9. All bispecific fusion proteins were expressed with IC's comparable to the antibody constructs (SEQ ID NOs: 86 and 87) and the reference PD-L1 antibody (SEQ ID NOs: 26 and 27)₅₀Values clearly inhibit the PD-1/PD-L1 interaction.

Table 8: the fusion protein competes with PD-L1 for binding to PD-1

SEQ ID NO	IC₅₀[nM]
		90 and 87	2.3
86 and 91	3.0
		92 and 87	3.4
86 and 93	3.2
		94 and 87	2.4
90 and 91	2.8
		86 and 87	3.5
26 and 27	3.8

Example 10: PD-L1 dependent T cell costimulation using CD137 bioassay

The potential of selected fusion proteins to induce activation of the CD137 signaling pathway in the presence of PD-L1 was evaluated using a commercially available double stably transfected Jurkat cell line expressing both the CD137 and luc2 genes (a humanized version of firefly luciferin), in which the NF κ B response element drives luc2 expression. In this bioassay, CD137 engagement causes CD137 intracellular signaling leading to nfkb-mediated luminescence.

The colorectal cancer cell line RKO expressing PD-L1 was cultured as described in example 7. The day before assay, RKO cells were plated at 1.25X 10 per well⁴Individual cells were plated and incubated at 37 ℃ in humidified 5% CO₂Adhere to the wall overnight in an atmosphere.

The following day, 3.75X 10⁴Individual NF-kB-Luc2/CD137 Jurkat cells were added to each well followed by different concentrations (typically ranging from 0.001nM to 5nM) of the fusion protein or reference CD137 antibody (SEQ ID NOs: 28 and 29). The plates were covered with a gas permeable sealing film and humidified 5% CO at 37 ℃ ₂Incubation in the atmosphere. After 4h, 30. mu.L of Bio-Glo^TMReagents were added to each well and bioluminescent signals were quantified using a luminometer (PHERAstar). Using GraphPadFour parameter logistic curve analysis was performed to calculate EC₅₀Values (shared as follows, fixed slope), EC₅₀The values are summarized in Table 9. To demonstrate the PD-L1 dependence of the fusion protein on CD137 conjugation, the following isThe same experiment was performed in parallel in the absence of RKO cells. Assays were performed in triplicate.

Fig. 8A-8D depict the results of representative experiments. The data demonstrate that all the tested fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, and SEQ ID NOs: 86 and 93) induced strong CD 137-mediated T cell co-stimulation. FIGS. 8B and 8D show that CD137 activation by the fusion protein is PD-L1 dependent, as no activation of NF-kB-Luc2/CD137 Jurkat cells was detected in the absence of PD-L1 expressing tumor cells. In contrast, the reference anti-CD 137 mAbs (SEQ ID NOs: 28 and 29) showed CD 137-mediated T cell costimulation, regardless of the presence or absence of target cells.

Table 9: assessment of T cell activation using CD137 bioassay

Example 11: evaluation of T cell activation Using human Peripheral Blood Mononuclear Cells (PBMC)

The ability of selected fusion proteins to co-stimulate T cell responses and prevent co-suppression mediated by PD-L1 binding to PD-1 was evaluated using a T cell assay. For this purpose, different concentrations of fusion proteins were added to staphylococcal enterotoxin b (seb) -stimulated human Peripheral Blood Mononuclear Cells (PBMCs) and incubated at 37 ℃ for 4 days. The level of IL-2 secretion in the supernatant was measured.

PBMCs of healthy volunteer donors were isolated from buffy coats by centrifugation through a ficoll density gradient (Biocoll 1.077g/mL, Biochrom) according to the protocol of Biochrom. Purified T cells were resuspended in buffer consisting of 90% FCS and 10% DMSO, immediately frozen and stored in liquid nitrogen until further use. For the assay, PBMCs were thawed and placed in 5% CO at 37 ℃ under humidity₂Atmosphere supplemented with 10% FCS and 1% penicillinStreptomycin (Life Technologies) for 16h in medium (RPMI 1640, Life Technologies).

For each experimental condition, the following procedure was performed in triplicate: 2.5x10⁴Individual PBMCs were incubated in medium in each well of a 384-well flat-bottom tissue culture plate. Fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, and SEQ ID NOs: 90 and 91), the building block PD-L1 antibody (SEQ ID NOs: 86 and 87), the mixture (cocktail) of the reference PD-L1 antibody (SEQ ID NOs: 26 and 27), the reference CD137 antibody (SEQ ID NOs: 28 and 29), and the PD-L1 antibody (SEQ ID NOs: 86 and 87 or SEQ ID NOs: 26 and 27) or a dilution series (typically ranging from 10 to 0.002nM) of the iso type (SEQ ID NOs: 24 and 25) control (SEB) and 0.1ng/ml were added to the corresponding wells. The plates were covered with a gas-permeable sealing film (4 title) and humidified 5% CO at 37 ℃ ₂Incubate in atmosphere for 4 days. Subsequently, the human IL-2DuoSet kit (R) described in the following procedure was used&D Systems) to assess IL-2 levels in the supernatant.

The 384 well plates were coated with 1. mu.g/mL "human IL-2 capture antibody" in PBS for 2h at room temperature. Subsequently, wells were washed 5 times with 80 μ L PBS supplemented with 0.05% tween (PBS-T). After blocking for 1h in PBS-0.05% T containing 1% casein (w/w), assay supernatants and concentration-series IL-2 standards diluted in culture medium were transferred to corresponding wells and incubated overnight at 4 ℃. The following day, a mixture of 100ng/mL goat anti-hIL-2-Bio detection antibody (R & D Systems) and 1. mu.g/mL Sulfotag-labeled streptavidin (Mesoscale Discovery) was added to PBS-T containing 0.5% casein and incubated at room temperature for 1 h. After washing, 25 μ L of reading buffer (Mesoscale Discovery) was added to each well and the resulting Electrochemiluminescence (ECL) signal was detected by a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.

Fig. 9 shows the results of a representative experiment. Bispecific fusion proteins SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, and SEQ ID NOs: 90 and 91 were able to induce T cell activation as evidenced by increased levels of IL-2 secretion compared to isotype control (hIgG4, Sigma). The strongest increase in IL-2 secretion was observed in the tetravalent fusion protein to CD137 (SEQ ID NOs: 94 and 87 and SEQ ID NOs: 90 and 91), followed by a bivalent fusion protein to CD137 (SEQ ID NOs: 90 and 87 and SEQ ID NOs: 86 and 91) in which the lipocalin mutein was fused to the C-terminus of an antibody specific for PD-L1. The lowest increase was observed in the fusion protein bivalent to CD137 (SEQ ID NOs: 92 and 87 and SEQ ID NOs: 86 and 93) in which the lipocalin mutein was fused to the N-terminus of an antibody specific for PD-L1, but still comparable to the mixture of the reference CD137 antibody (SEQ ID NOs: 28 and 29) and the reference PD-L1 antibody (SEQ ID NOs: 26 and 27). All fusion proteins showed higher IL-2 secretion levels compared to the single building block, i.e., CD 137-specific lipocalin mutein-Fc (SEQ ID NO: 88 or SEQ ID NO: 89) or the building block PD-L1 mAb (SEQ ID NOs: 86 and 87).

Example 12: assessment of T cell activation in the Presence of tumor cells expressing different levels of PD-L1

Another T cell assay was used to assess the ability of the fusion protein to co-stimulate T cell activation in a PD-L1 target-dependent manner. Different concentrations of the fusion protein were applied to anti-CD 3 stimulated T cells in the presence of tumor cell lines with different PD-L1 expression levels. Tumor cell lines tested included RKO (PD-L1 high), HCC827(PD-L1 medium) and Hep-G2(PD-L1 negative). The level of IL-2 secretion in the supernatant was measured.

PBMCs from healthy volunteer donors were isolated from buffy coat as described in example 11. T lymphocytes were further purified from PBMCs by magnetic cell sorting using the Pan T cell purification kit (Miltenyi Biotec GmbH) according to the manufacturer's instructions. Purified Pan T cells were resuspended in buffer consisting of 90% FCS and 10% DMSO, immediately frozen and stored in liquid nitrogen until further use.

For the assay, T cells were thawed and placed in humidified 5% CO at 37 ℃₂Culture supplemented with 10% FCS and 1% penicillin-streptomycin (Life Technologies) in atmosphereIn basal (RPMI 1640, Life Technologies) for 16 h.

For each experimental condition, the following procedure was performed in triplicate: flat bottom tissue culture plates were pre-coated with 0.25. mu.g/mL anti-CD 3 antibody for 1h at 37 ℃ and washed twice with PBS. Tumor cell lines RKO, HCC827 or Hep-G2 were treated with 30. mu.g/ml mitomycin C (Sigma Aldrich) for 30min to block proliferation. The mitomycin-treated tumor cells were then washed twice with PBS and at 2.5x 10 per well ⁴Individual cells were plated in culture medium to allow for wetting at 37 ℃ with 5% CO₂Adhere to the wall overnight in an atmosphere. Target cells that had previously been grown under standard conditions were exfoliated using Accutase (PAA laboratories) and then resuspended in culture medium.

The following day, after washing the plates twice with PBS, each well was washed 1.25x10⁴Individual T cells were added to tumor cells. Fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, and SEQ ID NOs: 86 and 93), a dilution series (typically ranging from 0.005 to 10nM) of the reference PD-L1 antibody (SEQ ID NOs: 26 and 27) and the reference CD137 antibody (SEQ ID NOs: 28 and 29) or isotype control (SEQ ID NOs: 24 and 25), used alone or in combination, were added to the corresponding wells. The plates were covered with a gas permeable sealing film and humidified 5% CO at 37 deg.C₂Incubate in atmosphere for 3 days.

After 3 days of co-culture, the IL-2 level in the supernatant was evaluated as described in example 11.

Fig. 10 shows exemplary data. Co-culture of Pan T cells with RKO cells (PD-L1 high) or HCC827(PD-L1 medium) in the presence of fusion proteins (SEQ ID NOs: 90 and 87 and SEQ ID NOs: 86 and 91) in which a lipocalin mutein is fused to the C-terminus of an antibody specific for PD-L1 resulted in a significant increase in IL-2 secretion compared to the hIgG4 isotype control. The increase in IL-2 secretion induced by the fusion proteins in which the lipocalin mutein is fused to the N-terminus of an antibody specific for PD-L1 (SEQ ID NOs: 92 and 87 and SEQ ID NOs: 86 and 93) is weaker, but still higher than the mixture of the reference CD137 antibody (SEQ ID NOs: 28 and 29) and the reference PD-L1 antibody (SEQ ID NOs: 26 and 27). No increase in IL-2 secretion was observed in the mixture using the building blocks, CD137 specific lipocalin mutein (Fc fusion protein, SEQ ID NO: 89) and PD-L1 antibody (SEQ ID NO: 86 and 87). Furthermore, co-culture of Hep-G2 (negative PD-L1) with any fusion protein did not increase IL-2 secretion levels, but co-culture with a mixture of the reference CD137 antibody (SEQ ID NOs: 28 and 29) and the reference PD-L1 antibody (SEQ ID NOs: 26 and 27) increased IL-2 secretion levels.

The data indicate that the functional activity of the fusion protein as measured by its ability to activate T cells or increase IL-2 secretion is PD-L1 dependent. In contrast, the T cell activation or IL-2 secretion induced by the reference CD137 antibodies (SEQ ID NOs: 28 and 29) when used in combination with the reference PD-L1 antibodies (SEQ ID NOs: 26 and 27) was not necessarily PD-L1 dependent and difficult to predict. Furthermore, the data indicate that the bispecific format targeting PD-L1 and CD137 is superior to a mixture of two independent molecules targeting CD137 and PD-L1 in the presence of target cells expressing PD-L1.

Example 13: evaluation of storage stability of fusion protein

To assess storage stability, exemplary fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, SEQ ID NO: 88, and SEQ ID NO: 89) at a concentration of 1mg/mL in PBS were incubated at 37 ℃ for 1 week. Monomeric fusion proteins were then assayed using analytical grade size exclusion by applying 20 μ g of sample to a Superdex 200, 3.2/300 Increatase (GE healthcare) column with a flow rate of 0.15ml/min and PBS as running buffer. All tested fusion proteins were stable after 1 week of incubation in PBS at 37 ℃. Fig. 11A shows an exemplary result.

Further storage stability evaluations were performed on the selected fusion proteins (SEQ ID NOs: 90 and 87). 20mg/ml of the fusion protein was incubated in 25mM histidine, 60mM NaCl, 200mM arginine pH 6 at 40 ℃ for 4 weeks. The content of functional fusion protein was measured in a quantitative ELISA setup using a simultaneous binding assay as described in example 5. Exemplary results are shown in fig. 11B.

Example 14: using CD4⁺Mixed Lymphocyte Reaction (MLR) assessment of T cells

Mixed Lymphocyte Reaction (MLR) assays to assess induction of CD4 by exemplary fusion proteins in the presence of antigen presenting cells⁺The ability of T cells to activate. In monocyte-derived dendritic cells (moDC) and CD4 from mismatched healthy donors⁺Different concentrations of fusion proteins (SEQ ID NOs: 90 and 87) were tested in the presence of T cells. After 6 days of incubation in the presence of the test molecule, secretion of IL-2 and IFN-. gamma.in the supernatant was quantified.

PBMCs were purified from platelet apheresis bags using Lymphoprep solution according to the manufacturer's instructions (StemCell). Purification of total CD4 from PBMC using the Miltenyi kit⁺T lymphocytes, and were frozen in a solution of 90% FBS and 10% DMSO. Using CD14⁺Bead kit (Miltenyi) purified CD14 ⁺Monocytes, and used freshly.

By 2X10 in RPMI1640 supplemented with 10% FBS and Pen/strep (Life Tech) in the presence of 50ng/mL IL-4 and 100ng/mL GMCSF (Miltenyi)⁶Individual cell/mL cultured CD14⁺Monocytes were harvested for 6 days to obtain modcs. On day 3, 10ml of fresh medium containing cytokines was added. Phenotypes were assessed by FACS at day 7 of differentiation (CD14, CD1a, HLADR, PD-L1).

In the presence of test molecules, in the presence of 50000 CD4⁺10000 modcs were cultured in triplicate wells of RPMI for 6 days in complete RPMI medium in 96 wells of U-bottom of T cells. At the end of the culture, the supernatant was immediately frozen and stored for cytokine quantification.

IL-2 levels in the supernatants were measured by employing the Luminex technique and exemplary data are shown in FIG. 12. Figure 12A shows that in several sets of MLR experiments (N ═ 8), fusion proteins of 10 and 0.1 μ g/mL (SEQ ID NOs: 90 and 87) were significantly better in IL-2 induction than the corresponding individual building blocks (SEQ ID NOs: 89 and SEQ ID NOs: 86 and 87) or the reference CD137 or PD-L1 antibodies (SEQ ID NOs: 28 and 29 or SEQ ID NOs: 26 and 27). FIG. 12B shows that the fusion proteins (SEQ ID NOs: 90 and 87) induce dose-dependent IL-2 secretion compared to isotype antibody controls. The fusion proteins (SEQ ID NOs: 90 and 87) induced higher IL-2 levels compared to equimolar concentrations of a mixture of the reference PD-L1 antibody (SEQ ID NOs: 26 and 27) and the reference CD137 antibody (SEQ ID NOs: 28 and 29) in the concentration range of 0.001 to 20. mu.g/mL.

Example 15: using CD8⁺Mixed lymphocyte response assessment of T cells

In view of the human CD8⁺Report of CD137 expression and Activity in T cells, the induction of CD8 by an exemplary fusion protein in the presence of antigen presenting cells was evaluated in an MLR assay⁺The ability of T cells to activate. In mocC and Total CD8 from mismatched healthy donors⁺Fusion proteins were tested in the presence of T cells (SEQ ID NOs: 90 and 87). After 6 days of incubation in the presence of the test molecules, the secretion of IL-2 and CD8 effector molecules (perforin, granzyme B and granzyme A) in the supernatant was quantified.

PBMCs were purified from platelet apheresis bags using Lymphoprep solution according to the manufacturer's instructions (StemCell). Purification of total CD8 from PBMC using the Miltenyi kit⁺T lymphocytes, and used freshly. Using CD14⁺The bead kit (Miltenyi) purified CD14 positive monocytes and was used fresh.

By 2X10 in RPMI1640 supplemented with 10% FBS and Pen/strep (Life Tech) in the presence of 50ng/mL IL-4 and 100ng/mL GMCSF (Miltenyi)⁶Individual cell/mL cultured CD14⁺Monocytes were obtained for 6 days to obtain modcs. On day 3, 10ml of fresh medium containing cytokines was added. Phenotypes were assessed by FACS at day 7 of differentiation (CD14, CD1a, HLADR, PD-L1).

In complete RPMI medium in 96 wells in U-bottom (triplicate wells) with 50000 CD8 in the presence of test molecules⁺T cells were cultured with 10000 modcs for 6 days. At the end of the incubation, the supernatant was immediately frozen and stored for quantitation of secreted factors.

IL-2, perforin, granzyme A and granzyme B in the supernatant were quantified using Luminex technology. Fig. 13 shows exemplary data.

FIG. 13 shows fusion proteins (SEQ ID NOs: 90 and 29) compared to equimolar concentrations (10. mu.g/mL (N-4)) of reference PD-L1 antibody (SEQ ID NOs: 26 and 27), reference CD137 antibody (SEQ ID NOs: 28 and 29) or mixtures of the two87) Showing increased secretion of IL-2, perforin, granzyme B and granzyme A. The data further show that the fusion proteins (SEQ ID NOs: 90 and 87) are cytotoxic to CD8⁺T cells are active.

Example 16: assessment of functional in vivo Activity in human PBMC-implanted xenograft mouse model

To investigate the in vivo activity of the fusion proteins provided, a cell line derived xenograft mouse model was used. Thus, human cancer cell lines were implanted subcutaneously in immunodeficient female NOG mice, which were handed over (deliverer) at 4-6 weeks of age with a quarantine period of at least 1 week. When the tumor reaches about 80-100mm ³After the volume of (c), mice will become substitutes for human PBMCs (substitees). Test compounds were injected at least 3 times and tumor growth and activity were measured continuously. After the study was completed, the mice were sacrificed. Intratumoral infiltration of CD3-, CD 4-and CD 8-positive cells was assessed by immunohistochemistry. IFN-. gamma.RNAscope was performed as a further reading.

Example 17: epitope analysis of fusion proteins

To assess the epitopes recognized by the fusion proteins, and whether these epitopes are clinically relevant, competition between the fusion proteins and the reference CD137 antibody was determined using a competitive ELISA format (format).

Reference CD137 antibodies (SEQ ID NOs: 28 and 29) (4. mu.g/mL) in PBS were coated overnight on microtiter plates at 4 ℃. After each incubation step, plates were washed 5 times with 100 μ L PBS-0.05% T (PBS supplemented with 0.05% (v/v) tween 20). Plates were blocked with 2% BSA (w/v) in PBS-0.1% T (PBS supplemented with 0.1% (v/v) Tween 20) for 1 hour at room temperature, then washed again. The fusion proteins (SEQ ID NOs: 90 and 87 and SEQ ID NOs: 86 and 91), the CD 137-specific lipocalin mutein (SEQ ID NO: 42), the reference CD137 antibody (SEQ ID NOs: 28 and 29) and the control antibody (SEQ ID NOs: 86 and 87) were mixed with 1nM biotinylated human CD137Fc fusion protein (huCD137-Fc-bio) as tracer and incubated for 1 hour at room temperature. The mixture of test molecules and tracer was added to the plate and incubated at room temperature for 20min, followed by 5 wash steps with 100 μ L PBS-0.05% T. Subsequently, a 1: 5000 dilution of Extravidin-HRP (Sigma-Aldrich) in PBS-0.1% T-2% BSA was added to the wells and incubated for 1 h. After additional washing steps, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescent microplate reader.

Competition data for an exemplary experiment is shown in fig. 14, where the x-axis represents the concentration of the test molecule and the y-axis represents the concentration of the tracer molecule measured. Fitting the data to a 1: 1 sigmoidal curve, where IC₅₀The value and maximum signal are free parameters and the slope is fixed to unity. The results demonstrate that the exemplary fusion proteins (SEQ ID NOs: 90 and 87 and SEQ ID NOs: 86 and 91) compete with the CD137 antibody (SEQ ID NOs: 28 and 29) for binding to CD137, indicating that the fusion proteins bind overlapping epitopes to the antibody.

Table 10: competition for fusion proteins

SEQ ID NO	IC₅₀[nM]
		90 and 87	1.10
86 and 91	0.99
		28 and 29	0.20
42	2.60

Example 18: assessment of T cell activation Using PD-1/PD-L1 blocking bioassay

The potential of the selected fusion proteins to block PD-1/PD-L1-mediated inhibition was assessed using PD-1-NFAT-luckat T cells (Jurkat cell line engineered to express PD-1 and an NFAT response element (NFAT-RE) driven luc gene (firefly luciferase gene)) co-cultured with PD-L1 aAPC/CHO-K1 cells (CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate the cognate TCR in an antigen-independent manner). In this bioassay, the PD-1/PD-L1 interaction inhibits TCR signaling and NFAT-RE mediated luminescence when PD-1-NFAT-luc Jurkat T cells are co-cultured with PD-L1 aAPC/CHO-K1 cells. The addition of a PD-1/PD-L1 blocker, such as the fusion protein specific for CD137 and PD-L1 described herein, releases the inhibitory signal and results in TCR activation and NFAT-RE mediated luminescence.

PD-L1 aAPC/CHO-K1 cells were grown in Ham's F12 medium supplemented with 10% FCS at 8.00X 10 per well³Individual cells were plated and allowed to wet 5% CO at 37 deg.C₂Adhere to the wall overnight in an atmosphere. The next day, the medium was discarded. 1.00x 10⁴PD-1-NFAT-luc Jurkat T cells were added to each well, followed by the addition of fusion proteins (SEQ ID NOs: 90 and 87) or PD-L1 antibodies (SEQ ID NOs: 86 and 87 or SEQ ID NOs: 26 and 27) at different concentrations (typically ranging from 0.005nM to 50 nM). The plates were covered with a gas permeable sealing film and humidified 5% CO at 37 ℃₂Incubation in the atmosphere. After 6h, 30. mu.L of Bio-Glo^TMReagents were added to each well and bioluminescent signals were quantified using a luminometer. Using GraphPadFour parameter logistic curve analysis was performed to calculate EC₅₀Value, EC₅₀The values are summarized in Table 11. Assays were performed in triplicate.

The results of representative experiments are depicted in fig. 15. The data demonstrate that the fusion proteins tested with EC of PD-L1 antibodies (SEQ ID NOs: 86 and 87 or SEQ ID NOs: 26 and 27)₅₀Equivalent EC₅₀Values inhibit PD-1/PD-L1 in a dose-dependent manner to block and activate T cells. As a negative control, reference CDNeither the 137 antibody (SEQ ID NOs: 28 and 29) nor the isotype control antibody (SEQ ID NOs: 24 and 25) resulted in an increase in luminescent signal.

Table 11: assessment of T cell activation Using PD-1/PD-L1 blocking bioassay

SEQ ID NO	EC₅₀[nM]
		90 and 87	0.49
86 and 91	0.58
		28 and 29	0.46

Example 19: evaluation of T cell activation using human PBMC

Additional T cell assays were used to assess the ability of selected fusion proteins to co-stimulate T cell responses, where different concentrations of fusion proteins were added to SEB-stimulated human PBMCs and incubated at 37 ℃ for 3 days. The level of IL-2 secretion in the supernatant was measured.

PBMCs from healthy volunteer donors were isolated and stored as described in example 11. For the assay, PBMCs were thawed and placed in 5% CO at 37 ℃ under humidity₂Atmospheric medium (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1% penicillin-streptomycin (Life Technologies) for 24 h.

For each experimental condition, the following procedure was performed in triplicate: 2.5x10⁴Individual PBMCs were incubated in medium in each well of a 384-well flat-bottom tissue culture plate. Selected fusion proteins(SEQ ID NOs: 90 and 87), the building block PD-L1 antibody (SEQ ID NOs: 86 and 87), the reference CD137 antibody (SEQ ID NOs: 28 and 29) used alone or in combination with the reference PD-L1 antibody (SEQ ID NOs: 26 and 27) or a dilution series (typically ranging from 0.0002 to 10nM) of isotype control (SEQ ID NOs: 24 and 25) and SEB at 0.1ng/ml were added to the corresponding wells. The plates were covered with a gas-permeable sealing film (4 title) and humidified 5% CO at 37 ℃ ₂Incubate in atmosphere for 3 days. Subsequently, the supernatant was evaluated for IL-2 as described in example 11.

Fig. 16 depicts the results of a representative experiment. Table 12 summarizes the EC of the test molecules for inducing IL-2 secretion₅₀The value is obtained. Compared to the building block PD-L1 antibody, the reference CD137 antibody, and the mixture of reference PD-L1 and CD137 antibodies, the amino acid sequences of SEQ ID NOs: the bispecific fusion proteins of 90 and 87 induce a dose-dependent increase in strong IL-2 secretion to higher levels and result in potent EC compared to PD-L1 and CD137 antibodies used alone or in combination₅₀The value decreases.

Table 12: evaluation of T cell activation using human PBMC

Example 20: evaluation of PD-L1-dependent T cell activation induced by fusion proteins

The fusion proteins were further analyzed for PD-L1 target-dependent T cell co-stimulation using a T activated cell assay. Different concentrations of the fusion protein were applied to anti-CD 3-stimulated T cells and co-cultured with human PD-L1 transfected or mock transfected Flp-In-CHO cells. The level of IL-2 secretion in the supernatant was measured.

PBMCs from healthy volunteer donors were isolated from buffy coat as described in example 11. T lymphocytes were further purified and stored as described in example 12.

For the assay, T cells were thawed and placed in humidified 5% CO at 37 ℃ ₂Atmospheric medium (RPMI 1640, Life technology) supplemented with 10% FCS and 1% penicillin-streptomycin (Life Technologies)gies) overnight.

For each experimental condition, the following procedure was performed in triplicate: flat bottom tissue culture plates were pre-coated with 0.25. mu.g/mL anti-CD 3 antibody at 37 ℃ for 2h, then washed twice with PBS. CHO cells transfected or mock-transfected with human PD-L1 were treated with 30. mu.g/ml mitomycin C (Sigma Aldrich) for 30min to block proliferation. Cells treated with mitomycin were then washed twice with PBS and at 1.0x 10 per well⁷Individual cells were plated in culture medium to allow for wetting at 37 ℃ with 5% CO₂Adhere to the wall overnight in an atmosphere. CHO cells that had previously been grown under standard conditions were exfoliated using Accutase (PAA laboratories) and then resuspended in culture medium.

The following day, each well will be 8.33x10³Individual T cells were added to CHO cells. Exemplary fusion proteins (SEQ ID NOs: 90 and 87), the building block PD-L1 antibody (SEQ ID NOs: 86 and 87), the reference CD137 antibody (SEQ ID NOs: 28 and 29), and a mixture of the reference PD-L1 antibody (SEQ ID NOs: 26 and 27) and the reference CD137 antibody (SEQ ID NOs: 28 and 29), or a dilution series (typically ranging from 0.003nM to 50nM) of isotype controls (SEQ ID NOs: 24 and 25) were added to the respective wells. The plates were covered with a gas permeable sealing film and humidified 5% CO at 37 deg.C ₂Incubate in atmosphere for 2 days.

After 2 days of co-culture, the IL-2 level in the supernatant was evaluated as described in example 11.

Fig. 17 shows exemplary data. Co-culture of Pan T cells with CHO cells in the presence of fusion proteins (SEQ ID NOs: 90 and 87 and SEQ ID NOs: 86 and 91) resulted in strong dose-dependent IL-2 secretion and was much stronger than the reference antibody, in which case only a slight increase in IL-2 secretion was observed for the reference CD137 antibody or the mixture of the reference CD137 antibody and the reference PD-L1 antibody, compared to the hIgG4 isotype control. When co-cultured with mock-transfected CHO cells (PD-L1 negative), only the reference CD137 antibody and the mixture of the reference CD137 and reference PD-L1 antibodies showed a slight dose-dependent increase in IL-2 secretion. The results demonstrate that activation of T cells by the fusion protein is PD-L1 dependent, in contrast to the reference CD137 antibody (SEQ ID NOs: 28 and 29) which shows CD137 mediated T cell co-stimulation, whether or not the target cells are present.

Example 21: pharmacokinetics of fusion proteins in mice

Pharmacokinetic analyses of representative fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, and SEQ ID NOs: 90 and 91) were performed in mice. Male CD-1 mice (3 mice per time point; Charles River Laboratories, Research Models and Services, Germany GmbH) of about 5 weeks of age were injected with the fusion protein in the tail vein at a dose of 10 mg/kg. The test article was administered as a bolus (bolus) in a volume of 5 mL/kg. Plasma samples from mice were obtained at time points 5 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 hours, 4 days, 8 days, 14 days, 21 days, and 28 days. Sufficient whole blood (collected under isoflurane anesthesia) was collected to obtain at least 100 μ L of Li-Heparin plasma/animal and time. Drug levels were detected using a sandwich ELISA that detects the intact bispecific construct by the targets PD-L1 and CD 137. Data were fitted using a two-chamber model of Prism GraphPad 5 software.

FIG. 18 shows graphs of plasma concentrations over time for the fusion proteins (SEQ ID NOs: 90 and 87, SEQ ID NOs: 86 and 91, SEQ ID NOs: 92 and 87, SEQ ID NOs: 86 and 93, SEQ ID NOs: 94 and 87, and SEQ ID NOs: 90 and 91), together with values obtained for the PD-L1 antibody (SEQ ID NOs: 86 and 87) plotted for reference. In all cases, the pharmacokinetics appeared similar. Starting from a plasma concentration of about 200. mu.g/mL, the plasma levels dropped to a level of about 50. mu.g/mL in about 48 hours, then further dropped at a lower rate until about 10. mu.g/mL at the end of the experiment after 28 days. Non-compartmental (non-compartmental) analysis was performed on these data. The terminal half-lives are summarized in table 13.

The data demonstrate that the fusion protein has a long, antibody-like terminal half-life in mice. Since the assay used to determine the plasma concentration of the fusion protein required retention of activity on PD-L1 and CD137, the results also demonstrated that the bispecific molecule remained intact over a 28 day period.

Table 13: terminal half-life in mice determined by non-compartmental analysis

SEQ ID NO	Terminal half-life [ h]
		90 and 87	295
92 and 87	346
		86 and 93	332
94 and 87	209
		90 and 91	250
86 and 87	390

Example 22: pharmacokinetics of fusion proteins in mice

Pharmacokinetic analyses of representative fusion proteins (SEQ ID NOs: 90 and 87) were performed in mice and compared to two previously described fusion proteins that bind CD 137-and PD-L1 (SEQ ID NO: 147 and SEQ ID NO: 148). Male CD-1 mice (2 mice per time point; Charles River Laboratories, Research Models and Services, Germany GmbH) of about 5 weeks of age were injected caudally with the corresponding molecules at a dose of 2 mg/kg. Plasma samples from mice were obtained at time points 5 minutes, 24 hours, 168 hours, and 336 hours. Sufficient whole blood (collected under isoflurane anesthesia) was collected to obtain at least 30-50 μ L of Li-Heparin plasma/animal and time.

Plasma drug concentrations were then analyzed by ELISA. HuCD137-His (human CD137 with a C-terminal polyhistidine tag) was dissolved in PBS (1 μ g/mL) and coated on microtiter plates overnight at 4 ℃. After each incubation step, plates were washed 5 times with 80 μ L of PBS supplemented with 0.05% (v/v) Tween 20. Plates were blocked with PBS/BSA/Tween (PBS containing 2% BSA (w/v) and 0.1% (v/v) Tween 20) for 1 hour at room temperature, then washed. Plasma samples were diluted to 20% plasma concentration in PBS/BSA/tween, added to the wells and incubated for 1 hour at room temperature. Followed by another washing step. Bound reagents in the study were detected after 1 hour incubation with a mixture of 1. mu.g/mL biotinylated human PD-L1 and streptavidin SULFO-TAG (mesoscale discovery), each diluted in PBS containing 2% BSA (w/v) and 0.1% (v/v) Tween 20. After an additional wash step, 35 μ L of reading buffer was added to each well and the Electrochemiluminescence (ECL) signal of each well was read by a Mesoscale Discovery reader. Data were transferred to Excel for analysis and quantification. Calibration curves were established for standard protein dilutions.

FIG. 19 shows SEQ ID NOs: 90 and 87, SEQ ID NO: 147 and SEQ ID NO: 148 plasma concentration over time. SEQ ID NOs: 90 and 87 exhibit good pharmacokinetic profiles or antibody-like pharmacokinetics, whereas SEQ ID NO: 147 and SEQ ID NO: 148 are not shown. If after 336h, C is_maxWhen the percentage (%) is 10% or more, it is considered that good pharmacokinetic characteristics or antibody-like pharmacokinetics are achieved.

The embodiments exemplarily described herein may be implemented in the absence of any one or more elements, one or more limitations, not specifically disclosed herein, as appropriate. Thus, for example, the terms "comprising," "including," "containing," and the like are to be construed broadly and without limitation. Additionally, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although embodiments of the present invention have been specifically disclosed by preferred embodiments and optional features, modification and variation thereof may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. All patents, patent applications, textbooks, and peer review publications described herein are incorporated by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein, is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Further, where features are described in terms of markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the markush group. Further embodiments will become apparent from the claims below.

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

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