Novel fusion protein specific for cd137 and pd-l1

Patent metadata

Field	Value
Publication number	US-2021363257-A1
Application number	US-201917264080-A
Country	US
Kind code	A1
Filing date	Jul 31, 2019
Priority date	Jul 31, 2018
Publication date	Nov 25, 2021
Grant date	—

Abstract

The disclosure provides fusion proteins specific for both CD 137 and PD-L1, which fusion protein can be used to co-stimulate lymphocyte activation in a PD-L1-target-dependent manner. Such fusion proteins can be used in many pharmaceutical applications, for example, as anti-cancer agents and/or immune modulators for the treatment or prevention of human diseases such as a variety of tumors. The present disclosure also concerns methods of making the fusion proteins described herein as well as compositions comprising such fusion proteins. The present disclosure further relates to nucleic acid molecules encoding such fusion proteins and to methods for generation of such fusion proteins and nucleic acid molecules. In addition, the application discloses therapeutic and/or diagnostic uses of such fusion proteins as well as compositions comprising one or more of such fusion proteins.

First claim

1 . A fusion protein that is capable of binding both 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 an antigen-binding domain thereof and is specific for PD-L1, and wherein a second subunit comprises a lipocalin mutein and is specific for CD137.

2 . The fusion protein of claim 1 , further comprising a third subunit, which third subunit comprises a lipocalin mutein specific for CD137.

3 . The fusion protein of claim 1 or 2 , wherein the fusion protein is capable of binding PD-L1 with a K D value of at most about 2 nM or comparable to or lower than the K D value of the immunoglobulin or an antigen-binding domain thereof that is included in the first subunit alone.

4 . The fusion protein of claim 1 or 2 , wherein the fusion protein is capable of binding CD137 with a K D value of at most about 7 nM or comparable to or lower than the K D value of the lipocalin mutein specific for CD137 that is included in the second subunit alone.

5 . The fusion protein of claim 3 or 4 , wherein the K D value is determined by a surface-plasmon-resonance (SPR) assay.

6 . The fusion protein of claim 1 or 2 , wherein the fusion protein is capable of binding PD-L1 with an EC 50 value of at most about 0.5 nM or comparable to or lower than the EC 50 value of the immunoglobulin or an antigen-binding domain thereof that is included in the first subunit alone.

7 . The fusion protein of claim 1 or 2 , wherein the fusion protein is capable of binding CD137 with an EC 50 value of at most about 0.6 nM or comparable to or lower than the EC 50 value of the lipocalin mutein specific for CD137 that is included in the second subunit alone.

8 . The fusion protein of any one of claims 6 - 7 , wherein the EC 50 value is determined by an 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 is cross-reactive with cynomolgus CD137.

11 . The fusion protein of claim 1 or 2 , wherein the fusion protein is capable of simultaneously binding CD137 and PD-L1 with an EC 50 values of at most about 10 nM, when said fusion protein is measured in an ELISA assay.

12 . The fusion protein of claim 1 or 2 , wherein the fusion protein is capable of binding CD137 expressed on a cell with an EC 50 values of at most about 60 nM.

13 . The fusion protein of claim 1 or 2 , wherein the fusion protein is capable of binding PD-L1 expressed on a cell with an EC 50 values of at most about 10 nM, when said fusion protein is measured in a flow cytometric analysis.

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

15 . The fusion protein of claim 1 or 2 , wherein the fusion protein is capable of binding CD137 in the presence of 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 compete with the antibody shown in SEQ ID NOs: 28 and 29 for binding to CD137.

18 . The fusion protein of claim 1 or 2 , wherein the fusion protein has overlapping CD137-binding epitope with the antibody shown 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 responses.

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-gamma.

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

23 . The fusion protein of any one of claims 1 - 22 , wherein the fusion protein is capable of co-stimulating T-cell responses 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 T-cell responses in a tumor microenvironment.

25 . The fusion protein of any one of claims 1 - 24 , wherein the fusion protein does not co-stimulate T-cell responses 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 the inhibitory signal of PD-1.

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

28 . The fusion protein of any one of claims 1 - 27 , wherein the fusion protein has a more favorable pharmacokinetic profile than SEQ ID NO: 147 or SEQ ID NO: 148.

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 of claim 29 , wherein the amino acid sequence of the lipocalin mutein comprises, 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 hTlc (SEQ ID NO: 1), one or more of the following mutated amino acid residues: 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 of claim 29 or 30 , wherein the amino acid sequence of the lipocalin mutein comprises one of the following sets of mutated amino acid residues in comparison with 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

Assignees

• Pieris Pharmaceuticals Gmbh
• Servier Lab

Inventors

• Pavlidou Marina
• Rothe Christine
• Olwill Shane
• Bel Aiba Rachida
• Hinner Marlon
• Peper Janet
• Pattarini Lucia
• Scholer-Dahirel Alix

Classifications

• C07K14/70596

  Molecules with a "CD"-designation not provided for elsewhere · CPC title

• C07K2317/33

  Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity · CPC title

• C07K14/70578

  NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151) · CPC title

• C07K14/47

  from mammals · CPC title

• C07K2317/76

  Antagonist effect on antigen, e.g. neutralization or inhibition of binding · CPC title