Protease-processed molecules

Abstract

The present invention relates to protein molecules comprising at least one protease-cleavable linker, and to protein molecules obtainable by protease cleavage of such protein molecules and their use in therapy.

Description

[Technical Field]

[0001] The present invention relates to protein molecules comprising at least one protease-cleavable linker, and to protein molecules obtainable by protease processing of such protein molecules and their use in therapy. [Background technology]

[0002] Natural IgG antibodies are bivalent and monospecific. Multispecific, e.g., bispecific, antibodies with binding specificities for multiple different antigens can be produced using recombinant technology and are expected to have a wide range of clinical applications. It is well known that a complete IgG antibody molecule is a Y-shaped molecule containing four polypeptide chains: two heavy chains and two light chains. Each light chain consists of two domains: the N-terminal domain known as the variable or VL domain (or region), and the C-terminal domain known as the constant or CL domain / region (constant kappa (Cκ) or constant lambda (Cλ) domain). Each heavy chain consists of four or five domains, depending on the antibody class. The N-terminal domain is known as the variable (or VH) domain (or region), followed by a first constant (or CH1) domain, a hinge region, and then the second and third constant (or CH2 and CH3) domains. In an assembled antibody, the VL and VH domains associate together to form the antigen-binding site. The CL and CH1 domains also associate together, keeping one heavy chain associated with one light chain. Two heavy-light chain heterodimers associate together through interactions between the CH2 and CH3 domains and the hinge regions of the two heavy chains.

[0003] One of the major problems associated with generating multispecific antibodies is light chain mispairing. While pairing of two different heavy chains can be addressed by techniques such as knobs-into-hole, enforcing correct light chain association is also essential. This is perhaps more challenging from a structural standpoint, since modifications must be applied within the Fab fragment interface, which requires further engineering. In any case, heterodimerization of heavy chains and their association with cognate light chains requires a fairly intensive number of design variations to achieve proper chain association while avoiding the formation of unwanted species. Furthermore, expression of multispecific molecules from different vectors / plasmids encoding the light and heavy chains requires comparable translation of all plasmids to provide the optimal ratio for accurate chain pairing. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention addresses the light chain pairing problem by changing the paradigm from a design-driven multispecific format to a processing-driven multispecific format, where no modification of the antibody sequence is required. [Means for solving the problem]

[0005] In one aspect, the present invention provides a protein having the formula PP1-PCL-PP2 {In the formula, PP1 is the first polypeptide, PP2 is a second polypeptide, PCL is a protease-cleavable linker comprising at least one protease cleavage site comprising the amino acid sequence HRRX1X2RSVDE (SEQ ID NO: 42), wherein X1 and X2 are independently selected from the group consisting of amino acids, preferably natural amino acids. The present invention relates to a protein comprising at least one polypeptide chain having the following structure:

[0006] In one embodiment, the protease is a furin and / or a furin-like protease.

[0007] In one embodiment, at least one protease cleavage site comprises the amino acid sequence HRRRKRSVDE (SEQ ID NO: 43) or the amino acid sequence HRRQQRSVDE (SEQ ID NO: 44).

[0008] In one embodiment, protease cleavage of the linker results in a change in the activity of the protein.

[0009] In one embodiment, the altered activity is binding or increased binding to at least one antigen.

[0010] In one embodiment, the protease cleavage occurs intracellularly.

[0011] In one embodiment, the protease-cleavable linker comprises two protease cleavage sites, wherein the two protease cleavage sites are located at the N-terminus and C-terminus of the protease-cleavable linker, respectively, and the two protease cleavage sites may be the same or different.

[0012] In one embodiment, the protease-cleavable linker has a length of 10 to 80 amino acid residues.

[0013] In one embodiment, at least one protease cleavage site is preceded and / or followed by at least 2, at least 3, at least 4, at least 5, or at least 10 consecutive amino acid residues independently selected from the group consisting of glycine and serine.

[0014] In one embodiment, the protease cleavable linker is (GS) n , (G4S) n (SEQ ID NO: 51), (HH) n , (EAAAK) n (SEQ ID NO: 52), (AP) n A (SEQ ID NO: 53), (KQGKQ)n (SEQ ID NO: 54), and combinations thereof, wherein n is an integer selected from 1-10.

[0015] In one embodiment, the protease-cleavable linker further comprises an amino acid sequence selected from the group consisting of GSHHHHHHHHGGGGS (SEQ ID NO: 45), GGGGSEAAAKEAAAKGGGGS (SEQ ID NO: 46), APAPAPAPAPAPAPAPA (SEQ ID NO: 47), GSKQGKQKQGKQGS (SEQ ID NO: 48), and combinations thereof.

[0016] In one embodiment, PP1 comprises at least one immunoglobulin constant region and / or at least one immunoglobulin variable region, and / or PP2 comprises at least one immunoglobulin constant region and / or at least one immunoglobulin variable region.

[0017] In one embodiment, PP1 comprises an immunoglobulin constant region and PP2 comprises an immunoglobulin variable region, or PP1 comprises an immunoglobulin variable region and PP2 comprises an immunoglobulin constant region, or PP1 comprises an immunoglobulin constant region and PP2 comprises an immunoglobulin constant region, or PP1 contains an immunoglobulin variable region, and PP2 contains an immunoglobulin variable region.

[0018] In one embodiment, the protein is an antibody or antibody derivative.

[0019] In one embodiment, the protein is a single chain antibody, preferably a multispecific single chain antibody.

[0020] In one embodiment, the protein is (a) VL-CL-PCL-VH-CH1-hinge-CH2-CH3 {In the formula, VL is immunoglobulin light chain variable region; CL is the immunoglobulin light chain constant region; VH is an immunoglobulin heavy chain variable region; CH1 is the immunoglobulin CH1 heavy chain constant region; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; PCL is a protease-cleavable linker}; (b) VL a -CL a -PCL-VL b -CL b {In the formula, VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; CL a is a first immunoglobulin light chain constant region; CL b is a second immunoglobulin light chain constant region; PCL is a protease-cleavable linker}; and (c)VL a -CL a -PCL-VL b -CL b -L-hinge-CH2-CH3 {In the formula, VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; CL a is a first immunoglobulin light chain constant region; CL b is a second immunoglobulin light chain constant region; L is an optional linker; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; PCL is a protease-cleavable linker. The polypeptide chain has a formula selected from the group consisting of:

[0021] In one embodiment, the protein comprises a polypeptide chain having the formula VL-CL-PCL-VH-CH1-hinge-CH2-CH3, where the VL pairs with the VH to form the antigen-binding site.

[0022] In one embodiment, the antigen binding site becomes active or exhibits increased activity upon protease cleavage of the protease-cleavable linker.

[0023] In one embodiment, the protein comprises two copies of a polypeptide chain having the formula VL-CL-PCL-VH-CH1-hinge-CH2-CH3, wherein the two copies are associated with each other via at least two disulfide bonds.

[0024] In one embodiment, the protein has the formula VL a -CL a -PCL-VH a -CH1 a a first polypeptide chain having a hinge-CH2-CH3, and a polypeptide chain of formula VL b -CL b -PCL-VH b -CH1 b a second polypeptide chain having a hinge -CH2-CH3, where: VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; CL a is a first immunoglobulin light chain constant region; CL b is a second immunoglobulin light chain constant region; VH a is a first immunoglobulin heavy chain variable region; VH b is a second immunoglobulin heavy chain variable region; CH1 a is a first immunoglobulin CH1 heavy chain constant region; CH1 b is a second immunoglobulin CH1 heavy chain constant region; VL a is VH a and form an antigen-binding site that binds to antigen A, and VL b is VH b and forms an antigen-binding site that binds to antigen B; The first polypeptide chain is associated with the second polypeptide chain through at least two disulfide bonds and, optionally, through at least one additional Fc interaction.

[0025] In one embodiment, antigen-binding site A and / or antigen-binding site B become active or exhibit increased activity upon protease cleavage of the protease-cleavable linker.

[0026] In one embodiment, the at least one additional Fc interaction is at least one knob-into-hole interaction.

[0027] In one embodiment, the protein has the formula VL b -L1-VL a - a first polypeptide chain having the formula VH a -L3-VH b -L4-CH1-hinge-CH2-CH3, and a second polypeptide chain having the formula VL c -CL c -PCL-VH c -CH1 c a third polypeptide chain having a hinge -CH2-CH3, where: VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; VL c is a third immunoglobulin light chain variable region; CL is the immunoglobulin light chain constant region; CL c is an immunoglobulin light chain constant region; VH a is a first immunoglobulin heavy chain variable region; VH b is a second immunoglobulin heavy chain variable region; VH c is a third immunoglobulin heavy chain variable region; CH1 is the immunoglobulin CH1 heavy chain constant region; CH1 c is an immunoglobulin CH1 heavy chain constant region; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; L1, L2, L3 and L4 are linkers; VL a is VH a and form an antigen-binding site that binds to antigen A, and VL b is VH b and VL to form an antigen-binding site that binds to antigen B. c is VH c and forms an antigen-binding site that binds to antigen C; The second polypeptide chain is associated with a third polypeptide chain via at least two disulfide bonds and optionally via at least one additional Fc interaction.

[0028] In one embodiment, antigen binding site C becomes active or exhibits increased activity upon protease cleavage of the protease-cleavable linker.

[0029] In one embodiment, the at least one additional Fc interaction is at least one knob-into-hole interaction.

[0030] In one embodiment, the protein has the formula VL a -CL a -PCL-VL b -CL b and a first polypeptide chain having the formula VH a -CH1 a -L-VH b -CH1 b a second polypeptide chain having a hinge -CH2-CH3, where: VH a is a first immunoglobulin heavy chain variable region; VH b is a second immunoglobulin heavy chain variable region; CH1 a is a first immunoglobulin CH1 heavy chain constant region; CH1 b is a second immunoglobulin CH1 heavy chain constant region; L is an optional linker; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; VL a is VH a and form an antigen-binding site that binds to antigen A, and VL b is VH b It pairs with the nucleotide sequence to form an antigen-binding site that binds to antigen B.

[0031] In one embodiment, antigen binding site B becomes active or exhibits increased activity upon protease cleavage of the protease-cleavable linker.

[0032] In one embodiment, the protein comprises two copies of a first polypeptide chain and two copies of a second polypeptide chain, wherein the two copies of the second polypeptide chain are associated with each other via at least two disulfide bonds.

[0033] In one embodiment, the protein has the formula VL b -CL b -PCL-VL a -CL a a first polypeptide chain having the formula VL c -CL c -PCL-VL d -CL d a second polypeptide chain having the formula VH a -CH1 a -L-VH b -CH1 b a third polypeptide chain having a hinge-CH2-CH3 and a polypeptide of the formula VH c -CH1 c -L-VH d -CH1 d a fourth polypeptide chain having a hinge -CH2-CH3, where: VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; VL c is a third immunoglobulin light chain variable region; VL d is a fourth immunoglobulin light chain variable region; CL a is a first immunoglobulin light chain constant region; CL b is a second immunoglobulin light chain constant region; CL c is a third immunoglobulin light chain constant region; CL d is a fourth immunoglobulin light chain constant region; VH a is a first immunoglobulin heavy chain variable region; VH b is a second immunoglobulin heavy chain variable region; VH c is a third immunoglobulin heavy chain variable region; VH d is a fourth immunoglobulin heavy chain variable region; CH1 a is a first immunoglobulin CH1 heavy chain constant region; CH1 b is a second immunoglobulin CH1 heavy chain constant region; CH1 c is a third immunoglobulin CH1 heavy chain constant region; CH1 d is a fourth immunoglobulin CH1 heavy chain constant region; L is an optional linker; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; VL a is VH a and form an antigen-binding site that binds to antigen A, and VL b is VH b and VL to form an antigen-binding site that binds to antigen B. c is VH c and forms an antigen-binding site that binds to antigen C, and VL d is VH d to form an antigen-binding site that binds to antigen D; The third polypeptide chain is associated with a fourth polypeptide chain via at least two disulfide bonds and optionally via at least one additional Fc interaction.

[0034] In one embodiment, antigen-binding site B and / or antigen-binding site D become active or exhibit increased activity upon protease cleavage of the protease-cleavable linker.

[0035] In one embodiment, the at least one additional Fc interaction is at least one knob-into-hole interaction.

[0036] In one embodiment, the protein has the formula VL a -CL a -PCL-VL b -CL b - a first polypeptide chain having a L-hinge-CH2-CH3, and a polypeptide chain of the formula VH a -CH1 a -L-VH b -CH1 b a second polypeptide chain having a hinge -CH2-CH3, where: VH a is a first immunoglobulin heavy chain variable region; VH b is a second immunoglobulin heavy chain variable region; CH1 a is a first immunoglobulin CH1 heavy chain constant region; CH1 b is a second immunoglobulin CH1 heavy chain constant region; L is an optional linker; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; VL a is VH a and form an antigen-binding site that binds to antigen A, and VL b is VH b and forms an antigen-binding site that binds to antigen B; The first polypeptide chain is associated with the second polypeptide chain through at least two disulfide bonds and, optionally, through at least one additional Fc interaction.

[0037] In one embodiment, antigen binding site B becomes active or exhibits increased activity upon protease cleavage of the protease-cleavable linker.

[0038] In one embodiment, the at least one additional Fc interaction is at least one knob-into-hole interaction.

[0039] In another aspect, the present invention relates to a nucleic acid or set of nucleic acids that encodes a protein as defined above.

[0040] In another aspect, the present invention relates to a vector or set of vectors comprising a nucleic acid or set of nucleic acids as defined above.

[0041] In another aspect, the present invention relates to a host cell comprising a protein as defined above, a nucleic acid or set of nucleic acids as defined above, or a vector or set of vectors as defined above.

[0042] In one embodiment, the host cell expresses endogenous or exogenous furin and / or furin-like proteases.

[0043] In one embodiment, the host cell is a mammalian cell.

[0044] In one aspect, the present invention provides a method for producing a protein, comprising: i) culturing a host cell as defined above; ii) isolating the protein from the host cell; The present invention relates to a method comprising: In another aspect, the present invention relates to a protein obtainable by the method defined above.

[0045] In yet another aspect, the present invention relates to proteins obtainable by furin and / or furin-like protease cleavage of the proteins defined above.

[0046] In one embodiment, the protein as defined above is a therapeutically active protein.

[0047] In one aspect, the present invention relates to a protein as defined above for use in therapy.

[0048] In another aspect, the present invention relates to the use of a protein as defined above in the manufacture of a medicament.

[0049] In yet another aspect, the present invention relates to a method for treating or preventing a disease or disorder, comprising administering to a subject in need thereof an effective amount of a protein as defined above.

[0050] In yet another aspect, the present invention relates to a pharmaceutical composition or kit comprising a protein as defined above.

[0051] In another aspect, the present invention relates to a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 42 to 44, a protein comprising said peptide, a nucleic acid encoding said peptide or protein, a vector comprising said nucleic acid, or a host cell comprising said peptide, protein, nucleic acid, or vector. [Brief explanation of the drawings]

[0052] [Figure 1] Protease-cleavable homodimeric antibody formats. (A) Single-chain (sC) monoclonal IgG. The CL (e.g., CK) domain is extended at its C-terminus by the N-terminus of the VH domain of the heavy chain via a protease-cleavable linker (PCL) sequence. (B) Single-light-chain (sLC) bivalent, bispecific tandem-IgG. The VH-CH1 (VHb) domain of the heavy chain is extended at its N-terminus by a second VH-CH1 domain (VHa) via, for example, a (G4S)3 linker (SEQ ID NO: 55). The VL-CL domain of the light chain (VLb) is extended at its N-terminus by a second VL-CL domain (VLa) using a PCL sequence. [Figure 2-1]Protease-cleavable homodimeric antibody formats. (A) Single light chain (sLC) multispecific tandem-IgG. The VH-CH1 (VHb and VHd) domains of the heavy chain are extended at the N-terminus by a second VH-CH1 domain (VHa and VHc), e.g., via a (G4S)3 linker (SEQ ID NO: 55). The VL-CL domains of the light chain (VLb and VLd) are extended at the N-terminus by a second VL-CL domain (VLa and VLc) using a PCL sequence. (B) Single chain (sC) monovalent bispecific tandem-IgG. The VH-CH1 domain of the first heavy chain (VHb) is extended at its N-terminus by the second VH-CH1 domain (VHa) via, for example, a (G4S)3 linker (SEQ ID NO: 55); the hinge region of the second heavy chain is extended by two VL-CL domains, where VLb and Fv a are connected via a PCL sequence. (C) Single-chain (sC) Fab containing trispecific CODV-IgG. One arm of the antibody contains CODV-LC (VLb-linker-VLa-linker-CL) associated with CODV-HC (VHa-linker-VHb-linker-CH1-hinge-CH2-CH3), and the other arm contains a single-chain (sC) Fab arm, where the VHc domain is extended at its N-terminus by the VLc-CL domain using a PCL sequence. (D) Single-chain (sC) bispecific IgG. The VHa and VHb domains of each HC are extended at their N-termini by VLa-CL and VLb-CL domains, respectively, using a PCL sequence. The heavy chains of all heterodimeric molecules preferably contain one or more knob-into-hole (KIH) mutations to form asymmetric antibodies. [Figure 2-2] Continued from Figure 2-1. [Figure 3-1]Analysis of tandem-anti-IL4 x anti-IL13-human IgG1 constructs from HEK293-FS cells. Panel (A) shows the purity of the tandem-IgG products (tandem-IgG control; sLC-PCL1-tandem-IgG and sLC-PCL2-tandem-IgG) after affinity and preparative SEC using analytical size-exclusion chromatography. Panel (B) shows the homogeneity of the antibody products using analytical hydrophobic interaction chromatography. Panel (C) shows the reduced (one heavy chain and two light chains / one single LC, respectively) and oxidized (intact tandem-IgG) forms of the antibody using 4-12% Bis / Tris-MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). [Figure 3-2] Continued from Figure 3-1. [Figure 4-1] Analysis of tandem-anti-IL4 x anti-IL13-human IgG1 constructs from HEK293-FS cells. Panel (A) shows the purity of the tandem-IgG products (sLC-PCL1-tandem-IgG, sLC-delta-PCL tandem-IgG, and sLC-PCL2-tandem-IgG) after affinity and preparative SEC using analytical size-exclusion chromatography. Panel (B) shows the homogeneity of the antibody products using analytical hydrophobic interaction chromatography. Panel (C) shows the reduced (one heavy chain and two light chains / one single LC, respectively) and oxidized (intact tandem-IgG) forms of the antibody using 4-12% Bis / Tris-MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). [Figure 4-2] Continued from Figure 4-1. [Figure 5-1]Analysis of sLC-tandem-IgG coexpressed with proteases in HEK293-FS cells. Panel (A) shows the purity of the tandem-IgG products (sLC-PCL1-tandem-IgG, sLC-delta-PCL-tandem-IgG) after affinity and preparative SEC using analytical size-exclusion chromatography. Panel (B) shows the homogeneity of the antibody products using analytical hydrophobic interaction chromatography. Panel (C) shows the reduced (one heavy chain and two light chains / one single LC, respectively) and oxidized (intact tandem-IgG) forms of the antibody using 4-12% Bis / Tris-MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Panel (D) shows LC-MS analysis of sLC-tandem-IgG-PCL1 after coexpression with furin and furin-KDEL under reducing conditions after deglycosylation. [Figure 5-2] Continued from Figure 5-1. [Figure 6] Analysis of sLC tandem-IgG with and without protease co-expression in stable CHO 9E4 cell pools. The oxidized (intact tandem-IgG) and reduced forms (one heavy and two light chains / one single LC, respectively) of purified antibody expressed with (left panel) and without (right panel) protease co-expression in stable cell pools are shown using 4-12% Bis / Tris MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). [Figure 7] SDS-PAGE analysis of sC-IgG and tandem-like IgG expressed in HEK293-FS and ExpiCHO cells. Panel (A) shows the reduced (one heavy and one light chain / one sC (left), one heavy chain and unprocessed / processed sC (right)) and oxidized (intact tandem-IgG) forms of two antibody formats expressed in HEK293-FS cells, and panel (B) shows the corresponding constructs expressed in ExpiCHO cells after Protein A and SEC purification using 4-12% Bis / Tris-MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). [Figure 8] Analysis of sC-IgG and tandem-like IgG coexpressed with proteases in HEK293-FS cells. Panel (A) shows the reduced (one heavy chain and one light chain / one sC) and oxidized forms of sC-IgG after coexpression with PCSK family members in HEK293-FS cells. Panel (B) shows the reduced (one heavy chain and unprocessed / processed sLC) and oxidized (intact tandem-like IgG) forms of tandem-like IgG after coexpression with PCSK family members in HEK293-FS cells after Protein A and SEC purification using 4-12% Bis / Tris-MOPS sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). [Figure 9] Analysis of trispecific CODV-sCFab-IgG expressed in HEK293FS cells. Panel (A) shows the preparative SEC chromatogram, and panel (B) shows the oxidized (intact trispecific CODV-Fab-IgG) and reduced (CODV heavy and light chains and single / processed Fab heavy and light chains) forms after preparative SEC using a LabCHip electrophoresis device. DETAILED DESCRIPTION OF THE INVENTION

[0053] Before describing the present invention in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," edited by Leuenberger, H.G.W., Nagel, B., and Kolbl, H. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0054] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0055] The elements of the present invention are described below. These elements are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred / specific embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all described elements in this application should be considered to be disclosed by the description of this application unless the context indicates otherwise.

[0056] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" should be understood to imply the inclusion of a specified integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0057] In one aspect, the present invention provides a protein having the formula PP1-PCL-PP2 {In the formula, PP1 is the first polypeptide, PP2 is a second polypeptide, PCL is a protease-cleavable linker comprising at least one protease cleavage site comprising the amino acid sequence HRRX1X2RSVDE (SEQ ID NO: 42), wherein X1 and X2 are independently selected from the group consisting of amino acids, preferably natural amino acids. The present invention relates to a protein comprising at least one polypeptide chain having the following structure:

[0058] When a protein comprises two or more polypeptide chains as defined above, these polypeptide chains may be the same or different.

[0059] The term "peptide" according to the present invention refers to a substance comprising two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably nine or more, preferably ten or more, preferably thirteen or more, preferably sixteen or more, preferably twenty one or more, and up to preferably 8, 10, 20, 30, 40 or 50, especially 100 amino acids covalently linked by peptide bonds. The term "protein" or "polypeptide" refers to large peptides, preferably peptides having more than 100 amino acid residues, although in general the terms "peptide", "polypeptide" and "protein" are synonymous and are used interchangeably herein.

[0060] In one embodiment, the protein is a recombinant protein. In one embodiment, the protein is a fusion protein.

[0061] The term "recombinant" in the context of the present invention means "produced through genetic engineering." Preferably, "recombinant" is not naturally occurring.

[0062] The term "naturally occurring," as used herein, when applied to an object, refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and is present in an organism (including a virus) that has not been intentionally modified by man in the laboratory is naturally occurring.

[0063] The term "fusion protein" (or "chimeric protein") generally refers to a protein created by joining, especially covalently linking, two or more separate proteins and / or peptides to result in a single protein possessing functional properties derived from each of the original proteins and / or peptides.

[0064] The term "linker" as used herein preferably refers to a peptide linker, i.e., a linker consisting of amino acids connected via peptide bonds. However, linkers according to the present invention may also include non-peptide components, such as non-peptide polymers (e.g., PEG).

[0065] The peptide linker according to the present invention can have any length, i.e., contain any number of amino acid residues. However, it is preferably long enough to provide a sufficient degree of flexibility so that the connected / linked moieties can, for example, interact / pair with each other or other moieties, and to allow proper protein folding; yet it is preferably short enough to provide stability (e.g., proteolytic stability) in cells. Suitable peptide linkers are described, for example, in Chen et al., Adv Drug Deliv Rev. 2013, 65(10):1357-69, the entire contents of which are incorporated herein. Preferably, the peptide linker according to the present invention has a length of 1 to 100 amino acids.

[0066] The flexibility of a peptide linker is generally increased when its amino acids are small and do not have bulky side chains that hinder the rotation or bending of the amino acid chain. Therefore, the peptide linkers of the present invention preferably have a high content of small amino acids, particularly glycine, alanine, serine, threonine, leucine, and isoleucine. Preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the amino acids of the peptide linker are such small amino acids. In certain embodiments, the peptide linker according to the present invention is a glycine-serine-rich linker in which at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, and even more preferably at least 85% of the amino acids are glycine or serine residues, respectively. Alternatively, the peptide linker according to the present invention may consist exclusively of glycine and / or serine residues (referred to as a glycine linker, a serine linker, or a glycine-serine linker, respectively). Exemplary peptide linkers include those having the amino acid formula (G l S m ) n wherein l is an integer of 1 to 4, m is 1 or 2, and n is an integer of 1 to 12, preferably 1 to 10, for example, an integer of 2 to 10 or 4 to 10. The peptide linker according to the present invention may also comprise or consist of other sequence elements, for example, a His tag.

[0067] In some embodiments, the peptide linker according to the present invention may also be a rigid peptide linker. Such rigid peptide linkers are known to those skilled in the art and may be, for example, represented by the general formula (XP): n where X represents any amino acid, preferably alanine, lysine or glutamic acid, and n is an integer, preferably an integer between 1 and 12. A particular example of this motif is (AP) n A (SEQ ID NO: 53). Another example of a rigid peptide linker sequence motif is (EAAAK) n(SEQ ID NO: 52), where n is an integer, preferably an integer of 1 to 12.

[0068] The term "protease-cleavable linker" ("PCL"), as used herein, refers to a linker that includes at least one protease cleavage site.

[0069] A "protease cleavage site" ("PCS"; also referred to herein as a protease recognition site) according to the present invention is a type of enzyme cleavage site in a protein that is a target for enzymes (proteases) that function after protein translation. In one embodiment, such enzymes function during transport from the Golgi lumen to the trans-Golgi compartment. Intracellular processing enzymes (proteases) cleave the polypeptide prior to secretion of the protein from the cell.

[0070] In one embodiment, the protease is a furin and / or a furin-like protease. The terms "furin" and "furin-like protease" refer to enzymes corresponding to EC number 3.4.21.75. Furin is a subtilisin-like proprotein convertase, also known as PACE (Paired basic Amino acid Cleaving Enzyme). Furin removes segments of inactive precursor proteins, converting them into biologically active proteins. Examples of furin and / or furin-like proteases (sometimes referred to as members of the furin family of proteases) include, but are not limited to, PCSK1 (also known as PC1 / Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furin or PACE), PCSK4 (also known as PC4), PCSK5 (also known as PCS or PC6), PCSK6 (also known as PACE4), and PCSK7 (also known as PC7 / LPC, PC8, or SPC7).

[0071] The term "amino acid" or "amino acid residue," as used herein, refers to all natural amino acids, unnatural amino acids, amino acid analogs and amino acid mimetics that function in a manner similar to the natural amino acids, and their D and L stereoisomers, where their structure allows for such forms. Amino acids are referred to herein by their name, their commonly known three-letter symbols, or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0072] The term "naturally occurring" when used in connection with amino acids refers to the 20 conventional amino acids (i.e., alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y)), as well as selenocysteine, pyrrolysine, and pyrrolysine.

[0073] The term "unnatural amino acid," as used herein, is meant to refer to amino acids that are not naturally encoded or found in the genetic code of any organism. They may, for example, be purely synthetic compounds. Examples of unnatural amino acids include hydroxyproline, gamma-carboxyglutamic acid, O-phosphoserine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-methyl ... Examples of amino acids that may be used include, but are not limited to, glycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine, D-ornithine, D-arginine, p-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline.

[0074] The term "amino acid analog" as used herein refers to a compound having the same basic chemical structure as a natural amino acid. Amino acid analogs include natural and unnatural amino acids that have been chemically blocked, reversibly or irreversibly, or their C-terminal carboxy group, their N-terminal amino group, and / or their side chain functional groups have been chemically modified. Such analogs include, but are not limited to, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine ​​sulfoxide, S-(carboxymethyl)-cysteine ​​sulfone, aspartic acid-(betamethyl ester), N-ethylglycine, alanine carboxamide, homoserine, norleucine, and methionine methylsulfonium.

[0075] The term "amino acid mimetic," as used herein, refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0076] In one embodiment, X1 and X2 are independently selected from the group consisting of lysine (K), glutamine (Q), and arginine (R). In one embodiment, X1X2 is selected from the group consisting of KK, KQ, KR, QK, QQ, QR, RK, RQ, and RR.

[0077] In one embodiment, at least one protease cleavage site comprises the amino acid sequence HRRRKRSVDE (SEQ ID NO: 43) or the amino acid sequence HRRQQRSVDE (SEQ ID NO: 44).

[0078] In one embodiment, protease cleavage of the linker results in a change in the activity of the protein.

[0079] In one embodiment, the altered activity is binding or increased binding to at least one antigen.

[0080] The term "binding" according to the present invention preferably relates to specific binding. A binding agent, such as an antibody or antibody derivative, is specific for a given target if it is able to bind to said target, while it is (substantially) unable to bind to other targets.

[0081] According to the present invention, a binding agent, such as an antibody or antibody derivative, is capable of binding to a predetermined target if it has significant affinity for and binds to the predetermined target in a standard assay. "Affinity" or "binding affinity" is often defined as the equilibrium dissociation constant (K D ) Preferably, the term "significant affinity" refers to a -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or a lower dissociation constant (K D ) to a predetermined target.

[0082] An agent is incapable (substantially incapable) of binding to a target if it has no significant affinity for the target in a standard assay, does not bind significantly to the target, and in particular does not bind detectably to the target. Preferably, the agent does not detectably bind to the target when present at a concentration of at most 2, preferably 10, more preferably 20, and in particular 50 or 100 μg / ml or more. Preferably, the agent is such that it has a K D At least 10 times, 100 times, or 10 times 3 double, 10 4 double, 10 5 double or ten 6 Twice as high as K D For example, if a drug binds to a target by a K D is 10 -7 M, the K for binding to a target for which the drug has no significant affinity D is at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 It can be M.

[0083] The binding of an agent to a target can be experimentally determined using any suitable method; see, for example, Berzofsky et al., "Antibody-Antigen Interactions," Fundamental Immunology, Paul, WE ed., Raven Press, New York, NY (1984); Kuby, Janis, Immunology, WH Freeman and Company, New York, NY (1992), and the methods described therein. Affinity can be readily determined using conventional techniques, such as by equilibrium dialysis; by using a BIAcore 2000 instrument using the general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by another method known to those skilled in the art. Affinity data may be analyzed, for example, by the method of Scatchard et al., Ann NY Acad. ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction may vary when measured under different conditions, such as salt concentration and pH. Thus, affinity and other antigen binding parameters, such as K D ,I C 50 The measurement is preferably carried out with standardized solutions of antibody and antigen, and standardized buffers.

[0084] The term "increased binding" can refer to an increase in binding of at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% compared to binding before protease cleavage.

[0085] In one embodiment, protease cleavage occurs intracellularly, e.g., within a host cell as defined herein. Thus, the protease cleavage site contained in the protease-cleavable linker may also be referred to as an intracellular processing site. A protease-cleavable linker according to the present invention may also be referred to as an in vivo cleavable linker.

[0086] Preferably, the protease-cleavable linker according to the present invention comprising at least one protease cleavage site according to the present invention is cleaved / processed intracellularly / in vivo without co-expression of a protease (e.g., a furin and / or a furin-like protease), in particular an exogenous protease. The term "co-expression", as used herein, is meant to refer to artificial co-expression, for example by genetically engineering a host cell to express a protease, for example via transfection of the host cell with a nucleic acid encoding the protease.

[0087] In one embodiment, the protease-cleavable linker comprises two protease cleavage sites, wherein the two protease cleavage sites are located at the N-terminus and C-terminus of the protease-cleavable linker, respectively, and the two protease cleavage sites may be the same or different.

[0088] In one embodiment, the protease-cleavable linker has a length of 10 to 80 amino acid residues, preferably 20 to 80 amino acids, more preferably 20 to 70 amino acids, more preferably 30 to 70 amino acids, and even more preferably 30 to 60 amino acids.

[0089] In one embodiment, at least one protease cleavage site is preceded and / or followed by at least 2, at least 3, at least 4, at least 5, or at least 10 consecutive amino acid residues independently selected from the group consisting of glycine and serine.

[0090] In one embodiment, the protease cleavable linker is (GS) n , (G4S) n (SEQ ID NO: 51), (HH) n , (EAAAK) n (SEQ ID NO: 52), (AP) n A (SEQ ID NO: 53), (KQGKQ) n(SEQ ID NO: 54), and combinations thereof, wherein n is an integer selected from 1 to 12, preferably 1 to 10. In one embodiment, the protease-cleavable linker further comprises the amino acid sequence (G4S) n (SEQ ID NO: 51), where n is an integer selected from 2 to 10, preferably 4 to 10.

[0091] In one embodiment, the protease-cleavable linker further comprises an amino acid sequence selected from the group consisting of GSHHHHHHHHGGGGS (SEQ ID NO: 45), GGGGSEAAAKEAAAKGGGGS (SEQ ID NO: 46), APAPAPAPAPAPAPAPA (SEQ ID NO: 47), GSKQGKQKQGKQGS (SEQ ID NO: 48), and combinations thereof.

[0092] In one embodiment, the protease cleavable linker (PCL) has the structure PCS A -L-PCS B where PCS A and PCS B are protease cleavage sites as defined herein, which may be the same or different, and L is SEQ ID NOs: 45-48 and 51-54, (GS) n , (HH) n and combinations thereof.

[0093] In one embodiment, PP1 comprises at least one immunoglobulin constant region and / or at least one immunoglobulin variable region, and / or PP2 comprises at least one immunoglobulin constant region and / or at least one immunoglobulin variable region.

[0094] In one embodiment, the protein is an antibody or antibody derivative.

[0095] In one embodiment, the protein is a single-chain antibody, preferably a multispecific (e.g., bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, etc.) single-chain antibody. The term "single-chain antibody," as used herein, also refers to an antibody or antibody derivative comprising two or more (e.g., two) single chains, preferably associated with each other via at least one covalent or non-covalent bond (e.g., two disulfide bonds).

[0096] The term "antibody" (or "immunoglobulin") refers to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination of the above. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable domains and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are referred to as HCDR1, HCDR2, and HCDR3, and the CDRs of VL are referred to as LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), where the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from the amino terminus to the carboxy terminus: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. In one embodiment, the CL region / domain referred to herein is a Cκ region / domain.

[0097] Antibodies may be derived from different species, including but not limited to mouse, rat, rabbit, guinea pig and human.

[0098] Antibodies described herein include IgA, such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, more particularly an IgG1 kappa or IgG1 lambda isotype (i.e., IgG1, κ, λ), an IgG2a antibody (e.g., IgG2a, κ, λ), an IgG2b antibody (e.g., IgG2b, κ, λ), an IgG3 antibody (e.g., IgG3, κ, λ), or an IgG4 antibody (e.g., IgG4, κ, λ). In one embodiment, the antibody or antibody derivative is an IgG1 antibody or an IgG1 antibody derivative, respectively.

[0099] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by heavy chain constant region genes. "Isotype switching" refers to the phenomenon in which the class or isotype of an antibody changes from one Ig class to one of the other Ig classes.

[0100] The term "monoclonal antibody," as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody exhibits a single binding specificity and affinity. In one embodiment, a monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a non-human animal, e.g., a mouse, fused to an immortalized cell.

[0101] The term "hinge" or "hinge region," as used herein, refers to a flexible stretch of amino acids in the central part of the heavy chain of IgG and IgA, particularly of the IgG (i.e., IgG1, IgG2, IgG3 or IgG4, especially IgG1) immunoglobulin class, that links these two chains by a disulfide bond.

[0102] The term "antibody derivative," as used herein, refers to a molecule that contains at least the domains identified as containing the molecule, but does not possess the entire structure of an antibody such as IgA, IgD, IgE, IgG, IgM, IgY, or IgW, and yet is still capable of binding to a target molecule. The derivative may be, but is not limited to, a functional (i.e., target-binding, particularly specific target-binding) antibody fragment thereof, such as Fab2, or a combination of such derivatives, e.g., a bivalent Fab. It also relates to an antibody to which additional antibody domains, such as additional variable domains, have been added. In one embodiment, the term "antibody derivative" refers to a side chain antibody as described herein. Accordingly, the term "antibody derivative" also includes multispecific (e.g., bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, etc.) and multivalent (e.g., bivalent, trivalent, tetravalent, etc.) antibodies.

[0103] In one embodiment, PP1 comprises an immunoglobulin constant region and PP2 comprises an immunoglobulin variable region, or PP1 comprises an immunoglobulin variable region and PP2 comprises an immunoglobulin constant region, or PP1 comprises an immunoglobulin constant region and PP2 comprises an immunoglobulin constant region, or PP1 contains an immunoglobulin variable region, and PP2 contains an immunoglobulin variable region.

[0104] In one embodiment, the protein is a single-chain (sC) monoclonal IgG (e.g., as described in Spies et al., J Mol Imm. 2015, 67:95-106), a single-light-chain (sLC) bivalent bispecific tandem-IgG (e.g., as described in WO 2009 / 052081 A2), a single-light-chain (sLC) multispecific tandem-IgG (e.g., as described in Wu et al., Nat. Biotechnol. 2007, 25:1290-1297), a single-chain (sC) monovalent bispecific tandem-like IgG (e.g., as described in Brinkmann and Kontermann, MAbs 2017, 9:182-212), a trispecific CODV-sC-Fab-IgG (e.g., as described in WO 2017 / 180913 A2 and / or Xu et al., Science 2017, 358(6359):85-90) and single-chain (sC) bispecific IgG (e.g., as described in Fitzgerald et al., Mol Cancer Ther. 2013, 13:410-25).

[0105] In one embodiment, the protein is: (a) VL-CL-PCL-VH-CH1-hinge-CH2-CH3 {In the formula, VL is immunoglobulin light chain variable region; CL is the immunoglobulin light chain constant region; VH is an immunoglobulin heavy chain variable region; CH1 is the immunoglobulin CH1 heavy chain constant region; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; PCL is a protease-cleavable linker}; (b) VL a -CL a -PCL-VL b -CL b {In the formula, VLa is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; CL a is a first immunoglobulin light chain constant region; CL b is a second immunoglobulin light chain constant region; PCL is a protease-cleavable linker}; and (c)VL a -CL a -PCL-VL b -CL b -L-hinge-CH2-CH3 {In the formula, VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; CL a is a first immunoglobulin light chain constant region; CL b is a second immunoglobulin light chain constant region; L is an optional linker; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; PCL is a protease-cleavable linker. The polypeptide chain has a formula selected from the group consisting of:

[0106] The symbol (-), when used in reference to the formulae disclosed herein, is meant to refer to a covalent bond, particularly a peptide bond, or one or more amino acid residues, such as a peptide linker as described herein.

[0107] In one embodiment, L is a peptide linker as defined herein, preferably having a length of 5 to 25 amino acid residues, preferably 10 to 20 amino acid residues, preferably 12 to 18 amino acid residues, more preferably 13 to 17 amino acid residues, even more preferably 14 to 16 amino acid residues, for example 15 amino acid residues. In one embodiment, L is a glycine-serine rich linker, wherein at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 85% of the amino acids are glycine or serine residues, respectively. In one embodiment, L is a glycine linker, a serine linker, or a glycine-serine linker. In one embodiment, L is a linker having the sequence (G4S) n (SEQ ID NO: 51), where n is an integer selected from 1 to 5, for example 1 to 3 or 2 to 4. In one embodiment, L has the sequence (G4S)3 (SEQ ID NO: 55).

[0108] In one embodiment, the protein comprises a polypeptide chain having the formula VL-CL-PCL-VH-CH1-hinge-CH2-CH3, where the VL pairs with the VH to form the antigen-binding site.

[0109] In one embodiment, the antigen binding site becomes active or exhibits increased activity upon protease cleavage of the protease-cleavable linker.

[0110] Preferably, the term "pairing with," as used herein, refers to the dimerization of immunoglobulin constant regions or immunoglobulin variable regions, which occurs in authentic, e.g., native, immunoglobulins, particularly IgG.

[0111] The term "antigen-binding site" (also called "paratope"), as used herein, refers to the portion of an antibody that recognizes and binds to an antigen.

[0112] In one embodiment, the protein comprises two copies of a polypeptide chain having the formula VL-CL-PCL-VH-CH1-hinge-CH2-CH3, wherein the two copies are associated with each other via at least two disulfide bonds.

[0113] In one embodiment, the protein has the formula VL a -CL a -PCL-VH a -CH1 a a first polypeptide chain having a hinge-CH2-CH3, and a polypeptide chain of formula VL b -CL b -PCL-VH b -CH1 b a second polypeptide chain having a hinge -CH2-CH3, where: VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; CL a is a first immunoglobulin light chain constant region; CL b is a second immunoglobulin light chain constant region; VH a is a first immunoglobulin heavy chain variable region; VH b is a second immunoglobulin heavy chain variable region; CH1 a is a first immunoglobulin CH1 heavy chain constant region; CH1 b is a second immunoglobulin CH1 heavy chain constant region; VL a is VH a and form an antigen-binding site that binds to antigen A, and VL b is VH b and forms an antigen-binding site that binds to antigen B; The first polypeptide chain is associated with the second polypeptide chain through at least two disulfide bonds and, optionally, through at least one additional Fc interaction.

[0114] In one embodiment, antigen-binding site A and / or antigen-binding site B become active or exhibit increased activity upon protease cleavage of the protease-cleavable linker.

[0115] The term "additional Fc interactions," as used herein, refers to interactions between two Fc molecules (e.g., two heterologous Fc molecules), in addition to the at least two disulfide bonds referred to herein, particularly between the CH3 domains of two Fc molecules. Examples of additional Fc interactions include knob-into-hole interactions (see below for details), hydrophobic interactions (e.g., by introducing specific mutations; see, e.g., Von Kreudenstein et al., MAbs 2013, 5(5):646-654), electrostatic interactions (e.g., by electrostatic steering; see, e.g., Gunasekaran et al., J Biol Chem 2010, 285(25):19637-19646), interactions with alternating CH3 segments of IgG and IgA (SEED technology; see, e.g., Davis et al., Protein Eng Des Sel 2010, 23(4):195-202), interactions through the fusion of heterodimeric modules, such as cleavable leucine zippers, at the C-terminus of the CH3 domain (LUZ-Y technology; see, e.g., Wranik et al., J Biol Chem 2012, 287(52):43331-43339).

[0116] In one embodiment, the at least one additional Fc interaction is at least one knobs-into-hole interaction (eg, two knobs-into-hole interactions).

[0117] The term "knobs-into-hole interactions," as used herein, refers to interactions based on knobs-into-hole amino acid changes. Such changes represent well-known rational design methods in antibody engineering used for heterodimerization of heavy (H) immunoglobulin chains, e.g., in the production of bispecific antibodies, as described, for example, in Merchant et al., Nat. Biotechnol. 1998, 16:677-681. The amino acid changes are engineered to create a knob (e.g., in CH3) in the constant region of a first immunoglobulin chain or antibody and a hole (e.g., in CH3) in the constant region of a second immunoglobulin chain or antibody. Knobs are represented by amino acids belonging to the "very large" IMGT volume class of amino acids (e.g., tyrosine, Y), while holes are represented by amino acids belonging to the "small" IMGT volume class (e.g., threonine; T) - for the 20 conventional amino acid IMGT classes, see Pommie et al., J. Mol. Recognit. 2004, 17:17-32. Particularly preferred knob-into-hole amino acid changes according to the present invention include, but are not limited to, those described in Spies et al., J. Mol. Imm. 2015, 67:95-106.

[0118] In one embodiment, the protein has the formula VL b -L1-VL a - a first polypeptide chain having the formula VH a -L3-VH b -L4-CH1-hinge-CH2-CH3, and a second polypeptide chain having the formula VL c -CL c -PCL-VH c -CH1 c a third polypeptide chain having a hinge -CH2-CH3, where: VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; VLc is a third immunoglobulin light chain variable region; CL is the immunoglobulin light chain constant region; CL c is an immunoglobulin light chain constant region; VH a is a first immunoglobulin heavy chain variable region; VH b is a second immunoglobulin heavy chain variable region; VH c is a third immunoglobulin heavy chain variable region; CH1 is the immunoglobulin CH1 heavy chain constant region; CH1 c is an immunoglobulin CH1 heavy chain constant region; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; L1, L2, L3 and L4 are linkers; VL a is VH a and form an antigen-binding site that binds to antigen A, and VL b is VH b and VL to form an antigen-binding site that binds to antigen B. c is VH c and forms an antigen-binding site that binds to antigen C; The second polypeptide chain is associated with a third polypeptide chain via at least two disulfide bonds and optionally via at least one additional Fc interaction.

[0119] In one embodiment, antigen binding site C becomes active or exhibits increased activity upon protease cleavage of the protease-cleavable linker.

[0120] In one embodiment, the at least one additional Fc interaction is at least one knobs-into-hole interaction (eg, two knobs-into-hole interactions).

[0121] In one embodiment, L1, L2, L3, and L4 are independently selected from peptide linkers as defined herein, preferably having a length of 1 to 25, preferably 2 to 25, more preferably 5 to 20, and more preferably 10 to 20 amino acid residues. In one embodiment, L1, L2, L3, and L4 are glycine-serine rich linkers, wherein at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, and even more preferably at least 85% of the amino acids are glycine or serine residues, respectively. In one embodiment, L1, L2, L3, and L4 are glycine linkers, serine linkers, or glycine-serine linkers, respectively. In one embodiment, L1, L2, L3, and L4 are the same or similar to the sequence (G4S) n (SEQ ID NO: 51), where n is independently an integer selected from 1 to 5, for example 1 to 3 or 2 to 4.

[0122] In one embodiment, the protein has the formula VL a -CL a -PCL-VL b -CL b and a first polypeptide chain having the formula VH a -CH1 a -L-VH b -CH1 b a second polypeptide chain having a hinge -CH2-CH3, where: VH a is a first immunoglobulin heavy chain variable region; VH b is a second immunoglobulin heavy chain variable region; CH1 a is a first immunoglobulin CH1 heavy chain constant region; CH1 b is a second immunoglobulin CH1 heavy chain constant region; L is an optional linker; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; VL a is VH a and form an antigen-binding site that binds to antigen A, and VL b is VH b It pairs with the nucleotide sequence to form an antigen-binding site that binds to antigen B.

[0123] In one embodiment, antigen binding site B becomes active or exhibits increased activity upon protease cleavage of the protease-cleavable linker.

[0124] In one embodiment, L is a peptide linker as defined herein. In one embodiment, L has a length of 5 to 25 amino acid residues, preferably 10 to 20 amino acid residues.

[0125] In one embodiment, the protein comprises two copies of a first polypeptide chain and two copies of a second polypeptide chain, wherein the two copies of the second polypeptide chain are associated with each other via at least two disulfide bonds.

[0126] In one embodiment, the protein has the formula VL a -CL a -PCL-VL b -CL b a first polypeptide chain having the formula VL c -CL c -PCL-VL d -CL d a second polypeptide chain having the formula VH a -CH1 a -L-VH b -CH1 b a third polypeptide chain having a hinge-CH2-CH3, and a polypeptide chain of the formula VH c -CH1 c -L-VH d -CH1 da fourth polypeptide chain having a hinge -CH2-CH3, where: VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; VL c is a third immunoglobulin light chain variable region; VL d is a fourth immunoglobulin light chain variable region; CL a is a first immunoglobulin light chain constant region; CL b is a second immunoglobulin light chain constant region; CL c is a third immunoglobulin light chain constant region; CL d is a fourth immunoglobulin light chain constant region; VH a is a first immunoglobulin heavy chain variable region; VH b is a second immunoglobulin heavy chain variable region; VH c is a third immunoglobulin heavy chain variable region; VH d is a fourth immunoglobulin heavy chain variable region; CH1 a is a first immunoglobulin CH1 heavy chain constant region; CH1 b is a second immunoglobulin CH1 heavy chain constant region; CH1 c is a third immunoglobulin CH1 heavy chain constant region; CH1 d is a fourth immunoglobulin CH1 heavy chain constant region; L is an optional linker; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; VL a is VH a and form an antigen-binding site that binds to antigen A, and VL b is VH b and VL to form an antigen-binding site that binds to antigen B. c is VH c and forms an antigen-binding site that binds to antigen C, and VL d is VH d to form an antigen-binding site that binds to antigen D; The third polypeptide chain is associated with a fourth polypeptide chain via at least two disulfide bonds and optionally via at least one additional Fc interaction.

[0127] In one embodiment, antigen-binding site B and / or antigen-binding site D become active or exhibit increased activity upon protease cleavage of the protease-cleavable linker.

[0128] In one embodiment, the at least one additional Fc interaction is at least one knobs-into-hole interaction (eg, two knobs-into-hole interactions).

[0129] In one embodiment, L is a peptide linker as defined herein. In one embodiment, L has a length of 5 to 25 amino acid residues, preferably 10 to 20 amino acid residues.

[0130] In one embodiment, the protein has the formula VL a -CL a -PCL-VL b -CL b - a first polypeptide chain having a L-hinge-CH2-CH3, and a polypeptide chain of the formula VH a -CH1 a -L-VH b -CH1 b a second polypeptide chain having a hinge -CH2-CH3, where: VH ais a first immunoglobulin heavy chain variable region; VH b is a second immunoglobulin heavy chain variable region; CH1 a is a first immunoglobulin CH1 heavy chain constant region; CH1 b is a second immunoglobulin CH1 heavy chain constant region; L is an optional linker; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; VL a is VH a and form an antigen-binding site that binds to antigen A, and VL b is VH b and forms an antigen-binding site that binds to antigen B; The first polypeptide chain is associated with the second polypeptide chain through at least two disulfide bonds and, optionally, through at least one additional Fc interaction.

[0131] In one embodiment, antigen binding site B becomes active or exhibits increased activity upon protease cleavage of the protease-cleavable linker.

[0132] In one embodiment, the at least one additional Fc interaction is at least one knobs-into-hole interaction (eg, two knobs-into-hole interactions).

[0133] In one embodiment, L is a peptide linker as defined herein. In one embodiment, L has a length of 5 to 25 amino acid residues, preferably 10 to 20 amino acid residues.

[0134] In another aspect, the present invention relates to a nucleic acid or set of nucleic acids that encodes a protein as defined herein.

[0135] A "nucleic acid" (or "nucleic acid molecule"), according to the present invention, is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A nucleic acid molecule, according to the present invention, can be single-stranded or double-stranded and can exist in the form of a molecule that is linear or covalently closed to form a circle.

[0136] The term "DNA" refers to a molecule that contains, and preferably consists entirely or substantially of, deoxyribonucleotide residues. "Deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the beta-D-ribofuranosyl group. The term "DNA" includes isolated DNA, such as partially or completely purified DNA, essentially pure DNA, synthetic DNA, and recombinantly produced DNA, and includes modified DNA that differs from natural DNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may include the addition of non-nucleotide material, for example, at one or more nucleotides of the DNA, at the end(s) of the DNA, or within the DNA. Nucleotides in DNA molecules may also include non-standard nucleotides, such as unnatural or chemically synthesized nucleotides. These modified DNAs may be referred to as analogs or analogs of natural DNA. When used with respect to nucleotides, the term "natural" refers to the bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).

[0137] The term "RNA" refers to a molecule that contains, and preferably consists entirely or substantially of, ribonucleotide residues. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a beta-D-ribofuranosyl group. The term "RNA" includes isolated RNA, such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly produced RNA, and includes modified RNA that differs from natural RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may include the addition of non-nucleotide material, for example, at one or more nucleotides of the RNA, at the end(s) of the RNA, or internally. Nucleotides in RNA molecules may also include non-natural nucleotides or non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be referred to as analogs or analogs of natural RNA. According to the present invention, "RNA" refers to single-stranded or double-stranded RNA. In one embodiment, the RNA is mRNA, such as in vitro transcribed RNA (IVT RNA), or synthetic RNA. The RNA may also be modified, e.g., with one or more modifications that increase the stability (e.g., half-life) of the RNA. Such modifications are known to those of skill in the art and include, e.g., a 5'-cap or 5' cap analog.

[0138] In another aspect, the present invention relates to a vector or set of vectors comprising a nucleic acid or set of nucleic acids as defined herein.

[0139] The term "vector" as used herein includes all vectors known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). These vectors include expression vectors and cloning vectors. Expression vectors, including plasmids and viral vectors, generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a specific desired DNA fragment and may lack functional sequences required for expression of the desired DNA fragment.

[0140] Alternatively, the nucleic acid molecule according to the invention may be integrated into a genome, for example the genome of a host cell. Means and methods for integrating particular nucleic acid molecules into a genome are known to those skilled in the art.

[0141] In another aspect, the present invention relates to a host cell comprising a protein as defined herein, a nucleic acid or set of nucleic acids as defined herein, or a vector or set of vectors as defined herein.

[0142] The term "cell" or "host cell" preferably relates to an intact cell, i.e., a cell with an intact membrane that has not released its normal intracellular components, such as enzymes, organelles, or genetic material. An intact cell is preferably a viable cell, i.e., a living cell that is capable of carrying out its normal metabolic functions. Preferably, the term relates, according to the present invention, to any cell that can be transfected with an exogenous nucleic acid. Preferably, the cell is capable of transfecting an exogenous nucleic acid and expressing the nucleic acid in the recipient when transferred to the recipient. The term "cell" includes bacterial cells; other useful cells are yeast cells, fungal cells, or mammalian cells. Suitable bacterial cells include cells from Gram-negative bacterial strains, such as strains of Escherichia coli, Proteus, and Pseudomonas, and Gram-positive bacterial strains, such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cells include cells from species of the genera Trichoderma, Neurospora, and Aspergillus. Suitable yeast cells include cells from species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, HEK293 (e.g., HEK293-FS), and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for the expression of heterologous proteins can also be used. A "cell" or "host cell" can be an isolated tissue or organism, particularly a "non-human organism," or part of a tissue or organism, particularly a "non-human organism." The term "non-human organism," as used herein, is meant to include non-human primates or other animals, particularly mammals such as cows, horses, pigs, sheep, goats, dogs, cats, rabbits, or rodents, such as mice, rats, guinea pigs, and hamsters.

[0143] In one embodiment, the host cell expresses endogenous or exogenous furin and / or furin-like proteases.

[0144] In one embodiment, the host cell is a mammalian cell, hi one embodiment, the mammalian cell is a CHO cell, preferably selected from the group consisting of CHO 9E4, ExpiCHO, CHO DG44 and CHO K1.

[0145] In one aspect, the present invention provides a method for producing a protein, such as a protein defined herein, comprising: i) culturing a host cell as defined herein; ii) isolating the protein from the host cell; The present invention relates to a method comprising:

[0146] In one embodiment, the host cells are cultured under conditions that allow expression of the protein.

[0147] In another aspect, the present invention relates to a protein obtainable (or obtained) by a method as defined herein.

[0148] In yet another aspect, the present invention relates to proteins obtainable (or obtained) by furin and / or furin-like protease cleavage of the proteins defined herein.

[0149] In one embodiment, a protein as defined herein is a therapeutically active protein.

[0150] The term "therapeutically active protein," as used herein, refers to a protein that is suitable for therapy, i.e., that can be used to treat a disease or disorder. In one embodiment, the therapeutically active protein is an antibody or antibody derivative that binds to a therapeutically relevant antigen.

[0151] For all antibodies and antibody derivatives described herein, the therapeutically relevant antigens are, for example, independently selected for each specificity from the group consisting of IL-4, IL-13, PD-1, 4.1BB, OX40, and GITR. For all bispecific antibodies and antibody derivatives described herein, the antigen pair is, for example, selected from the group consisting of the antigen pairs IL-4 and IL-13, PD-1 and OX40, PD-1 and GITR, and PD-1 and 4.1BB. For all trispecific antibodies and antibody derivatives described herein, the two antigens are, for example, selected from the group consisting of the antigen pairs IL-4 and IL-13, PD-1 and OX40, PD-1 and GITR, and PD-1 and 4.1BB. The third and fourth antigens may be additional antigens selected from the above list. In one embodiment of the trispecific antibody or antibody derivative described herein, the antigens are PD-1, GITR, and OX40.

[0152] In one aspect, the present invention relates to a protein as defined herein for use in therapy.

[0153] In another aspect, the present invention relates to the use of a protein as defined herein in the manufacture of a medicament.

[0154] In yet another aspect, the present invention relates to a method of treating a disease or disorder, comprising administering to a subject in need thereof an effective amount of a protein as defined herein.

[0155] The term "medicament" as used herein refers to a substance / composition used in therapy, i.e., in the treatment of a disease or disorder.

[0156] By "treating" is meant administering a compound or composition, or a combination of compounds or compositions, to a subject to prevent or eliminate a disease or disorder; arrest or slow the onset of a disease or disorder in a subject; inhibit or slow the onset of a new disease or disorder in a subject; reduce the frequency or severity of symptoms and / or recurrence in a subject who currently has or previously had a disease or disorder; and / or prolong, i.e., increase, the longevity of the subject.

[0157] In particular, the term "treating a disease or disorder / treatment of a disease or disorder" includes curing, shortening the duration of, ameliorating, preventing, slowing or inhibiting the progression or worsening of, or preventing or delaying the onset of, a disease or disorder or a symptom thereof.

[0158] According to the present invention, the term "disease" refers to any pathological condition, in particular cancer, infectious diseases, inflammatory diseases, autoimmune disorders and transplant rejection.

[0159] As used herein, the term "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. By "cancer cells" is meant abnormal cells that grow by rapid, uncontrolled cell proliferation and continue to grow after stimuli to initiate new growth have ceased. The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, bowel cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose, and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer, as well as metastases thereof. Examples of these are lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer, or metastases of the cancer types or tumors described above.

[0160] The term "cancer" according to the present invention also includes cancer metastasis. By "metastasis" is meant the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process, dependent on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and vessels, and subsequent infiltration of the target organ after transport by the blood. Finally, the growth of new tumors, i.e., secondary or metastatic tumors, at the target site depends on angiogenesis. Even after removal of the primary tumor, tumor metastasis often occurs because tumor cells or components remain and can develop metastatic potential. In one embodiment, the term "metastasis" according to the present invention relates to "distant metastasis," which refers to metastasis that is distant from the primary tumor and the regional lymph node system.

[0161] The term "infectious disease" refers to any disease that can be transmitted from individual to individual or organism to organism and is caused by a microbial pathogen (e.g., the common cold). Examples of infectious diseases include AIDS (HIV), hepatitis A, B, or C, herpes, shingles (chickenpox), rubella (rubella virus), yellow fever, dengue, etc., flaviviruses, influenza viruses, hemorrhagic infectious diseases (Marburg virus or Ebola virus), and viral infectious diseases such as severe acute respiratory syndrome (SARS), Legionnaires' disease (Legionella), sexually transmitted diseases (e.g., chlamydia or gonorrhea), stomach ulcers (Helicobacter), cholera (Vibrio), tuberculosis, diphtheria, E. coli, Staphylococcus aureus, and the like. bacterial infectious diseases such as infections with bacteria, salmonella or streptococcus (tetanus); infections with pathogenic protozoa such as malaria, sleeping sickness and leishmaniasis; toxoplasmosis, i.e. infections with malaria parasites, trypanosoma, leishmania and toxoplasma; or fungal infections caused by, for example, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

[0162] The term "inflammatory disease" refers to any disease characterized by or associated with high levels of inflammation in tissues, especially connective tissues, or degeneration of these tissues. Chronic inflammatory diseases are medical conditions characterized by persistent inflammation. Examples of (chronic) inflammatory diseases include celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel syndrome, atherosclerosis, arthritis, ankylosing spondylitis, Crohn's disease, colitis, chronic active hepatitis, dermatitis, and psoriasis.

[0163] The term "autoimmune disorder" refers to any disease or disorder in which the body produces an immunogenic (i.e., immune system) response to some components of its own tissues. In other words, the immune system loses the ability to recognize some tissues or systems within the body as self and targets and attacks them as if they were foreign. Autoimmune diseases can be classified into those that primarily affect one organ (e.g., hemolytic anemia and antiimmune thyroiditis) and those in which the autoimmune disease process spreads through many tissues (e.g., systemic lupus erythematosus). For example, multiple sclerosis is thought to be caused by T cells attacking the sheaths surrounding nerve fibers in the brain and spinal cord. This results in loss of coordination, weakness, and blurred vision. Autoimmune diseases are known in the art and include, for example, Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.

[0164] The term "transplant rejection" refers to the rejection of a transplanted tissue or organ by the recipient's immune system, which can ultimately destroy the transplanted tissue or organ.

[0165] The term "effective amount," as used herein, particularly refers to a "therapeutically effective amount," which is an amount that, alone or together with further doses, achieves the desired therapeutic response or desired therapeutic effect and preferably does not cause unacceptable side effects. In the case of the treatment of a specific disease or a specific condition, the desired response preferably relates to the inhibition of the disease process. This includes slowing the progression of the disease, and particularly halting or reversing the progression of the disease. The desired response in the treatment of a disease or condition may also be delaying or preventing the onset of the disease or condition. The effective amount of the protein described herein depends on the condition being treated, the severity of the disease, individual parameters of the subject, including age, physiological condition, size, and weight, the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Thus, the administered dose of the agent described herein may depend on several of such parameters. If the response in the subject is insufficient with the initial dose, a higher dose (or a more effectively higher dose achieved by a different, more localized route of administration) may be used.

[0166] The term "subject," according to the present invention, means a subject for treatment, particularly a diseased subject (also referred to as a "patient"), including mammals such as humans, non-human primates, or other animals, particularly cows, horses, pigs, sheep, goats, dogs, cats, rabbits, or rodents, such as mice, rats, guinea pigs, and hamsters. In one embodiment, the subject / patient is a human.

[0167] In yet another aspect, the present invention relates to a pharmaceutical composition or kit comprising the protein as defined above. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0168] The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

[0169] The terms "pharmaceutically acceptable carrier" or "physiologically acceptable carrier," as used herein, refer to one or more formulation materials suitable for achieving or enhancing delivery of an antibody-like binding protein. The primary carrier of a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a vehicle or carrier suitable for injection can be water, saline, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which may further contain sorbitol or a suitable substitute. In one embodiment of the present invention, antibody-like binding protein compositions can be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing selected compositions having the desired degree of purity with optional formulating agents. Additionally, antibody-like binding proteins may be formulated as lyophilizates using appropriate excipients, such as sucrose.

[0170] Pharmaceutical compositions may contain formulatory materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, separation or release rate, adsorption, or permeation of the composition. Suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl beta-cyclodextrin), bulking agents, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin), proteins (such as serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), and the like. etc.), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide), solvents (such as glycerin, propylene glycol or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as Pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapol), stability enhancers (such as sucrose or sorbitol), tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, or mannitol sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants (e.g., REMINGTON'S PHARMACEUTICAL SCIENCES (18th ed., AR Gennaro, ed., Mack Publishing Company 1990) and subsequent editions).

[0171] As used herein, the term "kit of parts" (or "kit") refers to an article of manufacture containing one or more containers and, optionally, a data carrier. The one or more containers may be filled with one or more of the agents (reagents) listed above. For example, additional containers containing diluents, buffers, and additional reagents may be included in the kit. The data carrier may be a non-electronic data carrier, such as a graphic data carrier such as an information leaflet, information sheet, barcode, or access code, or an electronic data carrier such as a compact disc (CD), digital versatile disc (DVD), microchip, or another semiconductor-based electronic data carrier. The access code may enable access to a database, such as an Internet database, a centralized database, or a distributed database. The data carrier may contain instructions for use of the agents of the present invention, such as proteins and pharmaceutical compositions, and related agents, such as nucleic acid molecules and host cells, described herein.

[0172] In another aspect, the present invention provides a compound of formula VL b -L1-VL a - a first polypeptide chain having the formula VH a -L3-VH b -L4-CH1-hinge-CH2-CH3, and a second polypeptide chain having the formula VL c -CL c -PCL-VH c -CH1 c - for a protein comprising a third polypeptide chain having a hinge -CH2-CH3, where: VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; VL c is a third immunoglobulin light chain variable region; CL is the immunoglobulin light chain constant region; CL c is an immunoglobulin light chain constant region; VH a is a first immunoglobulin heavy chain variable region; VH b is a second immunoglobulin heavy chain variable region; VH c is a third immunoglobulin heavy chain variable region; CH1 is the immunoglobulin CH1 heavy chain constant region; CH1 c is an immunoglobulin CH1 heavy chain constant region; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; PCL is a protease-cleavable linker; L1, L2, L3 and L4 are linkers; VL a is VH a and form an antigen-binding site that binds to antigen A, and VL b is VH b and VL to form an antigen-binding site that binds to antigen B. c is VH c and forms an antigen-binding site that binds to antigen C; The second polypeptide chain is associated with a third polypeptide chain via at least two disulfide bonds and optionally via at least one additional Fc interaction.

[0173] In one embodiment, antigen binding site C becomes active or exhibits increased activity upon protease cleavage of the protease-cleavable linker.

[0174] In one embodiment, the protease-cleavable linker is a protease-cleavable linker as defined herein.

[0175] In one embodiment, the at least one additional Fc interaction is at least one knobs-into-hole interaction (eg, two knobs-into-hole interactions).

[0176] In one embodiment, L1, L2, L3, and L4 are independently selected from peptide linkers as defined herein, preferably having a length of 1 to 25, preferably 2 to 25, more preferably 5 to 20, and more preferably 10 to 20 amino acid residues. In one embodiment, L1, L2, L3, and L4 are glycine-serine rich linkers, wherein at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, and even more preferably at least 85% of the amino acids are glycine or serine residues, respectively. In one embodiment, L1, L2, L3, and L4 are glycine linkers, serine linkers, or glycine-serine linkers, respectively. In one embodiment, L1, L2, L3, and L4 are the same or similar to the sequence (G4S) n (SEQ ID NO: 51), where n is an integer independently selected from 1 to 5, for example 1 to 3 or 2 to 4.

[0177] The present invention also includes (the use of) functional (i.e., protease-cleavable) variants of the amino acid sequence of any one of SEQ ID NOs: 42-44 in any aspect of the invention described herein. In one embodiment, the variant comprises up to three, two, or one amino acid substitution(s), preferably conservative amino acid substitution(s), deletion of up to three, two, or one amino acid residue (e.g., at the N-terminus and / or C-terminus) and / or addition of up to three, two, or one amino acid residue (e.g., to the N-terminus and / or C-terminus), in / from / to the amino acid sequence of any one of SEQ ID NOs: 42-44. Conservative amino acid substitutions include substitution of an amino acid with another amino acid from the same family of amino acids, i.e., amino acids that are related in their side chains (e.g., in terms of charge and / or size). Natural amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. Conservative substitutions are well known in the art (see, e.g., Creighton (1984) Proteins. W.H. Freeman and Company). In one embodiment, the variant comprises one or more (e.g., up to three, two, or one) of the following amino acid substitutions: R→K, S→T / A (i.e., S→T or S→A), V→A / I / L (i.e., V→A or V→I or V→L), D→E, E→D. In one embodiment, the variant (e.g., a variant of SEQ ID NO: 42) comprises one or more (e.g., up to three, two, or one) of the following amino acid substitutions: S→T / A, V→A / I / L, D→E, E→D.

[0178] The present invention will now be further described with reference to the following examples, which are intended to illustrate the invention and not to limit the scope of the invention. [Example]

[0179] material and method Expression of protease-cleavable monospecific and bispecific molecules in HEK 293-FS, ExpiCHO cells and stable CHO 9E4 cell pools For monoclonal antibodies, the expression plasmids encoded a single chain containing a heavy chain and a light chain (anti-IL4-hIgG1) linked via a protease-cleavable linker; for bispecific tandem-IgG antibodies, the expression plasmids encoded either a heavy chain or a single light chain (two Fab fragments linked by a protease-cleavable linker); for bispecific tandem-like antibodies, the expression plasmids encoded either a heavy chain or a heavy-light chain-linked construct. For proteases, the expression plasmids encoded the human propeptide sequence of either furin, PCSK5, PCSK6, PCSK7, or furin with an additional KDEL-signal peptide. All expression plasmids were propagated in E. coli DH5a. Plasmids used for transfection were prepared from E. coli using the Qiagen EndoFree Plasmid Mega Kit.

[0180] HEK 293-FS cells growing in F17 serum-free suspension culture (Invitrogen) were transfected with the indicated single chain or LC and HC plasmids using polyethylenimine transfection reagent. ExpiCHO cells growing in F17 serum-free suspension culture (Invitrogen) were transfected with the indicated plasmids using the appropriate transfection reagent.

[0181] After 7 days of incubation at 37°C, the cells were removed by centrifugation and the supernatant was passed through a 0.22 μm filter to remove particles.

[0182] For co-transfection of HEK293-FS or ExpiCHO cells, the plasmids for the antibody constructs were mixed in a 1:1 molar ratio with the respective expression plasmids encoding the propeptide sequences of the proteases.

[0183] To generate stable CHO 9E4 cell pools, CHO 9E4 cells were cultured in CD CHO serum-free medium (Invitrogen) and electroporated with transposon-based plasmids encoding single light and heavy chains, respectively, using a MaxCyte STX instrument and buffer reagents (MaxCyte Inc.). For protease coexpression, 30% of the plasmid content was a transposon-based protease-encoding plasmid. Stable cell pools were selected for 6 days in CD CHO medium containing the MSX selection marker. For antibody expression, cells were cultured under selective pressure in OPTi CHO medium (Invitrogen) containing FeedB (Invitrogen) at 37°C for 13 days, after which the supernatant was separated by centrifugation and passed through a 0.22 μm filter to remove particles.

[0184] For purification, the antibody was captured on a MabSelect SuRe column (catalog number 11-0034-93, GE Healthcare) and eluted with 0.1 M citrate buffer pH 3.0. The eluted fraction was directly neutralized by adding 1:6 (v / v) Trizma pH 8.0 (Sigma Aldrich). After protein purification by size exclusion chromatography (SEC) using Superdex200 16 / 60 (GE) and a final ultrafiltration concentration step, the protein was used for further characterization.

[0185] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis For SDS-PAGE, 2 μg of protein sample was mixed with NuPage LDS Sample Buffer (ThermoFisher Scientific); for reducing conditions, 2 μg of protein was mixed with NuPage LDS Sample Buffer containing DTT (dithiothreitol) and heated to 95°C for 5 minutes. The sample and Color Protein Standard Broad Range Ladder (New England Biolabs) were loaded onto a 4-12% Bis / Tris gel (Invitrogen) and run at 200V for 45 minutes in NuPage MOPS SDS Running Buffer (ThermoFisher Scientific). The gel was stained with Instant Blue Coomassie protein stain (Expedeon). For capillary electrophoresis, protein samples were prepared under reducing and oxidizing conditions using the Protein Clear HR Assay Kit and a LabChip Touch SP2 instrument (Perkin Elmer) according to the manufacturer's protocol.

[0186] Analytical Size Exclusion Chromatography (SEC) Analytical SEC was performed at 25°C using a BioSECcurity instrument (PSS Polymer) with an AdvanceBio 300 column (4.6 mm × 300 mm) and an AdvanceBio 300 guard column (Agilent Technologies). Analysis was performed at a flow rate of 0.5 ml / min using 2x concentrated D-PBS buffer (Thermo Fisher Scientific) with detection at 280 nm. 10 μl of protein sample (1 mg / ml) was applied to the column. Data evaluation was performed using WinGPC software v8.1 (PSS Polymer). For molecular weight estimation, the SEC column was calibrated with a protein calibration standard mixture (Agilent Technologies).

[0187] Analytical Hydrophobic Interaction Chromatography (HIC) Analytical HIC was performed at 25°C using an LC10 HPLC instrument (Shimadzu) or a Vanquish HPLC instrument (Thermo Fisher Scientific) equipped with a TSKgel Butyl-NPR column (2.5 μm, 4.6 × 35 mm) (Tosoh Bioscience). Analysis was performed at a flow rate of 1 ml / min with detection at 280 nm. 5 μg of undiluted protein sample was applied to the column. Gradient elution was performed from 15% B to 85% B for 7 min, followed by 100% B for 1 min, then 15% B for 1 min, and then equilibration at 15% B for 3 min. Buffer A consisted of 1.5 M ammonium sulfate, 25 mM sodium phosphate pH 7.0. Buffer B consisted of 25 mM sodium phosphate pH 7.0. Data evaluation was performed using either LabSolutions software v5.85 (Shimadzu) or Chromeleon 7 software (Thermo Fisher Scientific).

[0188] Mass spectrometry (MS) The protein integrity and potential mispairing of the heterodimer constructs were analyzed by LC-mass spectrometry (LC-MS). 12.5 μg of protein was diluted to 0.17 mg / ml in LC-MS-grade water (Thermo Scientific) treated with 0.5 μl of PNGaseF (glycerol-free, New England Biolabs), and the protein sample was deglycosylated for 16 hours at 37°C. LC-MS analysis was performed using a Thermo Fisher Orbitrap Lumos LC / MS instrument. Reverse-phase (RP) chromatography was performed at 300 μL / min using a MabPac RP HPLC column, analytical 4 μm particle size, 2.1 × 100 mm (Thermo Scientific). The eluents were LC water, 0.1% formic acid (A) and 90% acetonitrile, 10% LC water, 0.1% formic acid (B). 2 μg of protein was injected onto the column and eluted using a 12-minute linear gradient from 0% to 95% B. Data analysis was performed using Expressionist software 13.0.3 (Genedata). Molecular masses were calculated based on the amino acid sequences of the proteins using GPMAW software version 10.32b1 (Lighthouse data).

[0189] Surface Plasmon Resonance (SPR) Antigen binding to the antibody constructs was measured using surface plasmon resonance (SPR) with a BIAcore 3000 instrument (GE Healthcare) and HBS-EP buffer (GE Healthcare). Human IL4 (IL004, Millipore) and human IL13 (IL012, Millipore) were used as antigens. Anti-human Fc capture antibodies (Human Antibody Capture Kit, GE Life Sciences) were immobilized using standard techniques via primary amine groups (11,000 RU) on a research-grade CM5 chip (GE Life Sciences). Ligands were captured at a flow rate of 10 μl / min with an adjusted RU value resulting in a maximum analyte binding of 30 RU. The tested antibody constructs were used as analytes and injected at 100 nM concentration for 240 seconds, with a dissociation time of 300 seconds at a flow rate of 30 μL / min. Serial dilutions of human IL4 and IL13 were used, ranging from 0.1 nM to 3 nM and 0.8 nM to 25 nM, respectively. The chip surface was regenerated with a 2-minute injection of the regeneration buffer provided with the capture kit. Sensorgrams were run with a blank chip surface and a HBS-EP buffer blank as duplicate standards. Data analysis was performed using BIAevaluation software v4.1. [Example]

[0190] Expression and in vivo processing of protease-cleavable tandem-IgG in HEK293-FS cells Bispecific tandem-IgG (Figure 1B; Table 1) constructs were expressed in HEK293-FS cells after transient transfection of two plasmids encoding the heavy chain (HC) and single light chain (sLC), with the VLa-Cκ and VLb-Cκ chains connected via different protease-cleavable linker sequences. As a control, we used tandem-(anti-IL4xanti-IL13)-human IgG1 (tandem-IgG control), in which VLa-Cκ and VLb-Cκ are expressed as two separate chains. The sLC tandem-IgG showed expression and purification characteristics comparable to those of the control tandem-IgG (Figure 3A-B; selected constructs in bold in Table 1).

[0191] [Table 1]

[0192] SDS-PAGE analysis under reducing conditions of the in vivo processing of tandem-IgG constructs with protease-cleavable linkers revealed that sLCs containing linker sequences with a modified protease recognition / cleavage site HRRRKRSVDE (PCS2; SEQ ID NO: 43; "PCL2" refers to PCL containing PCS2) displayed two separate light chains (Figure 3C) as seen for the tandem-IgG control. Similar results were obtained with a modified protease recognition / cleavage site HRRQQRSVDE (PCS2a; SEQ ID NO: 44; "PCL2a" refers to PCL containing PCS2a). In contrast, all constructs containing the minimal protease recognition sequence (PCS1; SEQ ID NO: 49; "PCL1" refers to the PCS1-containing PCL), different linker sequences, or deleted recognition sequences (delta PCS / PCL) were not processed in vivo during expression in HEK293-FS cells and showed the same profile of single LCs and HCs under reducing conditions (Figure 3C: exemplary profile shown for sLC PCL1 tandem-IgG).

[0193] The protein integrity and in vivo processing of the tandem-IgG constructs were further analyzed by LC-mass spectrometry (LC-MS). Samples were measured under reducing and non-reducing conditions after deglycosylation. Molecular masses were calculated based on the amino acid sequences of the proteins (Table 2). LC-MS analysis confirmed complete in vivo processing of the linker sequence in tandem-IgG constructs containing the PCS2 recognition site. In contrast, no processing was detected in protein samples of tandem-IgGs with deleted (delta PCS / PCL) or minimal PCS (PCS1) recognition sites. Furthermore, control tandem-IgGs consisting of two separate LCs showed LC to HC mispairing (3*LC1 + 1*LC2 + 2*HC), which was not observed in sLC tandem-IgG-PCL2 after processing of the sLC to LC1 and LC2. Thus, the sLC tandem-IgG construct reduces / avoids LC-HC mispairing in this bispecific IgG format. Furthermore, in the fully processed sample, further processing of the C-terminal charged amino acids containing the protease recognition site RRKR by the carboxy-terminal protease was observed (RRKR loss). In contrast, no further N-terminal processing was detected by Edman sequencing (data not shown).

[0194] [Table 2] [Example]

[0195] Expression and in vivo processing of protease-cleavable tandem-IgG in ExpiCHO cells The sLC constructs selected for transient expression of tandem-IgG in ExpiCHO cells are shown in Table 3. sLC-tandem-IgGs with various protease recognition sites showed expression and purification characteristics comparable to those of the control tandem-IgG (Figure 4A-B). However, the expression levels were lower compared to transient transfection and expression in HEK293-FS cells.

[0196] [Table 3]

[0197] SDS-PAGE analysis of in vivo processing of tandem-IgG under reducing conditions revealed that sLC containing a linker sequence with a modified protease recognition site, HRRRKRSVDE (PCS2; SEQ ID NO: 43), exhibited two separate light chains (Figure 4C). Constructs containing a minimal protease recognition sequence (PCS1; SEQ ID NO: 49) or a deleted recognition sequence (delta PCS / PCL) were not processed in vivo by endogenously expressed proteases in ExpiCHO cells. Thus, expression of protease-cleavable sLC tandem-IgG in ExpiCHO cells confirmed the results from HEK293-FS cultures.

[0198] LC-MS analysis confirmed the SDS-PAGE results of fully processed sLC-PCL2 tandem-IgG in ExpiCHO cells, with a further processing profile of the C-terminal charged amino acids by a carboxy-terminal protease (RRKR loss).

[0199] [Table 4] [Example]

[0200] Co-transfection of proteases and protease-cleavable constructs in HEK293-FS cells A tandem-IgG construct with sLC containing PCS1 and delta-PCS recognition sites in the sLC linker sequence was cotransfected with PCSK family proteases in HEK293-FS cells (Table 5), and in vivo processing was induced by overexpression of the proteases. The sLC tandem-IgG construct exhibited expression and purification characteristics comparable to those of the single-transfected antibody (Figure 5A and B). The reduced expression yield was due to the lower amount of plasmid in the cotransfection (1:1 molar ratio with the individual protease-encoding plasmids). Analysis of in vivo processing by SDS-PAGE and LC-MS confirmed the processing of the sLC tandem-IgG containing the PCS1 recognition site by recombinantly expressed human furin and human furin with a KDEL retention sequence (Figure 5C and D). In contrast, overexpression of other protease family members did not result in sLC tandem-IgG processing. Comparable results were obtained with sC-IgG and tandem-like IgG constructs (Fig. 7A and B).

[0201] [Table 5]

[0202] For the generation of stable CHO cell pools, transposon-based plasmids encoding tandem-IgG constructs with sLC containing PCS1, PCS2, and delta-PCS recognition sites in the linker sequence of sLC, as well as a tandem-IgG control (two LC plasmids), were used for electroporation of CHO 9E4 cells with and without protease coexpression. The sLC tandem-IgG constructs showed expression and purification characteristics comparable to those of the tandem-IgG control. Analysis of in vivo processing by SDS-PAGE confirmed the processing of sLC tandem-IgG containing the PCS2 recognition site in samples expressed without protease coexpression, as well as the processed sLC of tandem-IgG with PCS1 and PCS2 recognition sites by recombinantly expressed human furin (Figure 6). [Example]

[0203] Assessment of antigen binding of bispecific sLC tandem-IgG with a protease-cleavable linker The binding of different bispecific tandem-IgG constructs expressed in HEK293-FS or ExpiCHO cells, as well as in vivo processed tandem-IgGs from cotransfection with furin variants, was measured by surface plasmon resonance (SPR). Binding to two human antigens, IL4 and IL13, was compared with the binding characteristics of individual monoclonal antibodies. The binding characteristics of monoclonal anti-IL4 and anti-IL13 mAbs and bispecific tandem-IgGs are shown in Table 6. Specific binding to IL4 was measured for all tandem-IgGs and was comparable to that of the anti-IL4 control antibody. In contrast, specific binding to IL13 was only detectable for in vivo processed tandem-IgGs and the tandem-IgG control containing two LCs. Unprocessed sLC tandem-IgGs showed no binding. Thus, processing of the linker sequence connecting the VLb (VL for IL13) in the internal position of the tandem IgG with the Cκ domain of the IL4-binding Fab in the external position is required for a functional IL13-binding site. In vivo processing can thereby be mediated by endogenously expressed proteases or by co-transfected furin variants.

[0204] [Table 6] [Example]

[0205] Expression and purification of sC-IgG and tandem-like IgG with protease-cleavable linkers in HEK293-FS and ExpiCHO cells Tables 6 and 7 show the expression and purification data of sC-IgG and tandem-like IgG constructs with protease-cleavable linker sequences from HEK293-FS and ExpiCHO cultures, respectively, after transient transfection.

[0206] After Protein A purification and preparative SEC, sC-IgG showed high purity with a monomer peak fraction of 83-99%. Protein yields were (G4S) n (SEQ ID NO: 51) decreased for sC-IgG with decreasing length of the linker sequence.

[0207] In contrast to sC-IgG, the tandem-like IgG format exhibited only low protein purity (23-75% monomer peak fractions) accompanied by high aggregation potential as seen in high molecular weight species in preparative SEC.

[0208] [Table 7]

[0209] [Table 8]

[0210] In both cell lines, only sC-IgG and tandem-like IgG with PCS2 recognition sites were cleaved into their respective chain fragments by endogenous proteases during protein expression, as shown for the sLC tandem-IgG format (Fig. 7A and B). [Example]

[0211] Co-transfection of proteases and protease-cleavable constructs in HEK293-FS cells Single-chain Ig and tandem-like IgG constructs with sLC containing PCS1 and delta-PCS recognition sites in the linker sequence were cotransfected with PCSK family proteases in HEK293-FS cells to induce in vivo processing by overexpression of the proteases. Analysis of in vivo processing by SDS-PAGE confirmed the induced processing of sC-IgG and tandem-like IgG containing the PCS1 recognition site by recombinantly expressed human furin and human furin with the KDEL retention sequence (Figures 8A and B). Furthermore, overexpression of PACE4 resulted in partially processed constructs. [Example]

[0212] Expression and in vivo processing of protease-cleavable CODV-scFab-IgG in HEK293-FS cells Trispecific CODV-scFab-IgG (Figure 2C; Table 8) constructs were expressed in HEK293-FS cells after transient transfection of three plasmids encoding the CODV HC, CODV LC, and single chain (sC), with the VLc-CK and VHc-CH1-CH2-CH3 chains connected via various protease-cleavable linker sequences. As a control, a trispecific CODV-Fab-IgG with the Fab-LC and HC expressed as two separate chains (classical CODV-Fab-IgG control) was used. The single-chain Fab CODV showed expression and purification characteristics comparable to the control, with a reduced aggregate peak in SEC (Table 9, Figure 9A).

[0213] [Table 9]

[0214] Electrophoretic analysis under reducing conditions showed that single chains of Fab arms containing either the PCS2 (SEQ ID NO: 43) or PCS2a (SEQ ID NO: 44) sequence were processed after expression in HEK293-FS cells to yield Fab-LC and Fab-HC moieties (Figure 9B).

[0215] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 10-9] [Table 10-10] [Table 10-11] [Table 10-12] [Table 10-13] [Table 10-14] Table 10-15 Table 10-16 Table 10-17 Table 10-18 Table 10-19 Table 10-20 Table 10-21 Table 10-22 Table 10-23 Table 10-24 Table 10-25 Table 10-26

Claims

1. A protein having the formula PP 1 -PCL-PP 2 {During the ceremony, PP 1 is the first polypeptide, PP 2 is a second polypeptide, PCL is a protease-cleavable linker comprising the amino acid sequence HRRRKRSVDE (SEQ ID NO: 43) or the amino acid sequence HRRQQRSVDE (SEQ ID NO: 44).

2. The protein of claim 1 , wherein protease cleavage of the linker results in a change in the activity of the protein.

3. The protein of claim 2 , wherein the altered activity is binding or increased binding to at least one antigen.

4. 4. The protein of claim 1, wherein the protease-cleavable linker comprises two protease cleavage sites, wherein the two protease cleavage sites are located at the N-terminus and C-terminus of the protease-cleavable linker, respectively, and the two protease cleavage sites may be the same or different.

5. PP 1 comprises at least one immunoglobulin constant region and / or at least one immunoglobulin variable region, and / or PP 2 The protein according to any one of claims 1 to 4, comprising at least one immunoglobulin constant region and / or at least one immunoglobulin variable region.

6. PP 1 comprises an immunoglobulin constant region and PP 2 comprises an immunoglobulin variable region, or PP 1 comprises an immunoglobulin variable region and PP 2 comprises an immunoglobulin constant region, or PP 1 comprises an immunoglobulin constant region and PP 2 comprises an immunoglobulin constant region. PP 1 comprises an immunoglobulin variable region and PP 2 comprises an immunoglobulin variable region.

7. The protein according to any one of claims 1 to 6, which is an antibody or an antibody derivative.

8. The protein described in claim 7, which is a single-chain antibody.

9. The protein described in claim 8, which is a multispecific single-chain antibody.

10. (a) VL-CL-PCL-VH-CH1-hinge-CH2-CH3 {During the ceremony, VL is an immunoglobulin light chain variable region; CL is an immunoglobulin light chain constant region; VH is an immunoglobulin heavy chain variable region; CH1 is the immunoglobulin CH1 heavy chain constant region; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; PCL is a protease-cleavable linker}; (b)VL a -CL a -PCL-VL b -CL b {During the ceremony, VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; CL a is a first immunoglobulin light chain constant region; CL b is a second immunoglobulin light chain constant region; PCL is a protease-cleavable linker; and (c) VL a -CL a -PCL-VL b -CL b -L-hinge-CH2-CH3 {During the ceremony, VL a is a first immunoglobulin light chain variable region; VL b is a second immunoglobulin light chain variable region; CL a is a first immunoglobulin light chain constant region; CL b is a second immunoglobulin light chain constant region; L is an optional linker; The hinge is an immunoglobulin hinge region; CH2 is the immunoglobulin CH2 heavy chain constant region; CH3 is the immunoglobulin CH3 heavy chain constant region; PCL is a protease-cleavable linker.

10. The protein of any one of claims 1 to 9, comprising a polypeptide chain having a formula selected from the group consisting of:

11. A nucleic acid or set of nucleic acids, said nucleic acid or set of nucleic acids encoding a protein according to any one of claims 1 to 10.

12. A vector or set of vectors comprising the nucleic acid or set of nucleic acids of claim 11.

13. A host cell comprising a protein according to any one of claims 1 to 10, a nucleic acid or set of nucleic acids according to claim 11, or a vector or set of vectors according to claim 12.

14. A host cell described in claim 13, which is a mammalian cell.

15. 1. A method for producing a protein, comprising: i) culturing the host cell of claim 13 or 14; ii) isolating the protein from the host cell; The method for producing a protein, comprising:

16. The protein of any one of claims 1 to 10, which is a therapeutically active protein.

17. 17. The protein of claim 16 for use in therapy.

18. Use of the protein of claim 16 for the manufacture of a medicament.

19. A pharmaceutical composition comprising the protein of claim 16.

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