Fusion with proteases and their use

Abstract

This specification provides a fusion protein comprising an antibody bound to a target fused to a protease that cleaves a substrate, the target being in close proximity to the substrate. The target and substrate are, in some cases, the same molecule. Methods for constructing and using such fusion proteins are also provided.

Description

[Technical Field]

[0001] This disclosure discloses a fusion protein comprising an antibody fused to a protease that binds to a target and cleaves a substrate when the target is in proximity to the substrate. Furthermore, this disclosure also provides polynucleotides encoding the disclosed fusion protein, as well as vectors and host cells containing such polynucleotides. This disclosure further provides methods for producing the fusion protein, pharmaceutical compositions containing the same, and uses thereof. [Background technology]

[0002] Protein-based therapeutics are a successful class of drugs that address major medical needs in various therapeutic areas. For example, monoclonal antibodies are a highly successful class of drugs that address major medical needs in various therapeutic areas. The success of antibodies is partly due to their high specificity, immune mobilization ability, long serum half-life, relatively low immunogenicity, and streamlined discovery methods. Despite these characteristics, an inherent limitation of antibodies is their general reliance on stoichiometric target binding to induce the desired therapeutic effect. This aspect of antibodies can hinder their effective application to some therapeutic targets, specifically high-abundance targets and targets where barriers to the site of action exist. Another factor hindering clinical success is insufficient exposure to systemically administered antibody-based drugs. For example, low exposure to systemic antibodies against the CNS (approximately 0.1%) (Wang et al., 2018, Fluids Barriers Cns. 15, 10; Lemere, 2013, Mol. Neurodegener. 8, 36-36; Yu et al., 2011, Sci Transl Med. 3, 84ra44) may have required very high doses of antibodies targeting pathogenic amyloid-β and tau proteins in the central nervous system, which may have hindered their clinical success (Lemere, 2013, Mol. Neurodegener. 8, 36-36; Kwan et al., 2021, Dement Geriatr Cogn. 49, 334-348; Dyck 2018, Biol Psychiat. 83, 311-319).

[0003] Enzymes are another class of proteins that have a quasi-stoichiometric mechanism of action. In contrast to antibodies, a single enzyme can react with many substrate molecules at high catalytic rates and turnover, thus allowing it to maintain sufficient activity at low doses. Enzymes are approved for the treatment of cancer, hematological disorders, lysosomal storage disorders, and metabolic deficiencies, among many other pathological conditions (Cioni et al., 2021 Curr Med Chem.; Tandon et al., 2021 J. Drug Deliv Sci Tec.; Fuente et al., 2021 Int J. Mol Sci. 22, 9181). However, several drawbacks, including short half-lives, lack of tissue specificity, broad substrate specificity, and high immunogenicity when not of human origin, limit the broader application of this therapeutic class.

[0004] Previous studies have combined antibodies with enzymes to circumvent the shortcomings of antibody- and enzyme-based therapeutics. However, these studies focused on the use of the former as a delivery vehicle and had varying preclinical and limited clinical successes (Zhou et al., 2019 Trends Mol. Med. 25, 1094-1109). Enzyme-mediated antibody delivery has been studied for three main applications. The first approach, called enzyme replacement therapy, involves replacing the function of an inactive native enzyme and has demonstrated clinical success with antibody-mediated delivery of enzyme activity to lysosomes, cytosols, and the brain (Zhou et al., 2019 Trends Mol. Med. 25, 1094-1109; Silver et al. 2021, Trends Pharmacol Sci. 42, 1064-1081; Yi et al., J. Mol Med. 95, 513-521; Giugliani et al. 2018, Orphanet J. Rare Dis. 13, 110). The second approach, called antibody-directed enzyme prodrug therapy (ADEPT), combines a tumor-targeting antibody-enzyme fusion with a systemically delivered inactive prodrug. Local enzyme activation of the prodrug at the tumor site is intended to minimize toxicity. Most ADEPT developments have been preclinical, with minimal success in early clinical studies (Zhou et al., 2019 Trends Mol. Med. 25, 1094-1109; Silver et al. 2021, Trends Pharmacol Sci. 42, 1064-1081; Sharma et al. 2017 Adv Drug Deliver Rev. 118, 2-7; Mayer et al. 2006 Clin Cancer Res. 12, 6509-6516). The third category is a broad one that includes any attempt to localize or induce enzymes to specific tissues, cell types, or intracellular locations.For example, antibodies that bind to human insulin receptors or transferrin receptors have been used to treat enzyme deficiencies by allowing enzyme-containing cargo to cross the blood-brain barrier (Zhou et al., 2019 Trends Mol. Med. 25, 1094-1109; Boado et al. 2013 Bioconjugate Chem. 24, 1741-1749). Cancer therapies combining tumor-targeted antibodies with cytotoxic enzymes such as RNases and various pro-apoptotic enzymes are being investigated (D'Avino et al. 2014 Protein Eng Des Sel. 27, 83-88; Xu et al 2004 J.Immunol. 173, 61-67; Andrady et al. 2011 Immunotherapy 3, 193-211). Therefore, there is an unmet need to provide novel therapeutic approaches to neutralize challenging therapeutic targets.

[0005] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference. [Overview of the project]

[0006] It should be understood that one, some, or all of the characteristics of the various embodiments described herein can be combined to form other embodiments of the Disclosure. These and other embodiments of the Disclosure will be apparent to those skilled in the art. These and other embodiments of the Disclosure are further described below in detail.

[0007] In one embodiment, the present application provides a fusion protein comprising an antibody that binds to a target, wherein the antibody is fused to a protease that cleaves a substrate, and the target is in proximity to the substrate. In some embodiments, the protease is fused to one or more polypeptide chains of the antibody. In some embodiments, the protease is fused to the N-terminus and / or C-terminus of one or more polypeptide chains of the antibody.

[0008] In some embodiments following any of the above fusion proteins, the antibody is a) A heavy chain variable domain (VH) and a light chain variable domain (VL), wherein a protease is fused to the N-terminus or C-terminus of VH or VL; or b) A full-length antibody comprising two heavy chains and two light chains, wherein a protease is fused to the N-terminus or C-terminus of one or both of the two heavy chains and / or two light chains.

[0009] In some embodiments relating to any of the fusion proteins described above, the antibody comprises a) a first polypeptide comprising a heavy chain variable (VH) domain and a first heavy chain constant (CH1) domain, and b) a second polypeptide comprising a light chain variable (VL) domain and a light chain constant (CL) domain, wherein the protease is fused to the N-terminus of the VL domain, the VH and VL domains form a binding domain to the target, and a disulfide bond is formed between the CH1 and CL domains. In some embodiments, the antibody does not contain an Fc fragment.

[0010] In some embodiments, the antibody comprises a) a first polypeptide containing a light chain variable (VL) domain and a light chain constant (CL) domain, b) a second polypeptide containing a heavy chain variable (VH) domain and a first heavy chain constant (CH1) domain, and c) a third polypeptide containing an Fc region, wherein the protease is fused to the N-terminus of the third polypeptide, and the VH and VL domains form a binding domain to the target.

[0011] In some embodiments, the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, with the protease fused to the N-terminus of the two light chains.

[0012] In some embodiments, the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, with the protease fused to the N-terminus of the two heavy chains.

[0013] In some embodiments, the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, with the protease fused to the N-terminuses of the two light chains and the N-terminuses of the two heavy chains.

[0014] In some embodiments, the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, with the protease fused to one C-terminus of the heavy chain.

[0015] In some embodiments, the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, with the protease fused to both C-terminuses of the heavy chains.

[0016] In some embodiments following any of the above fusion proteins, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable.

[0017] In some embodiments following any of the above fusion proteins, the dissociation rate constant (k) of the antibody against the target is off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies:

[0018] In some embodiments following any of the above fusion proteins, the equilibrium dissociation constant (k) of the antibody against the target is D ) has a range of approximately 0.1 nM to approximately 1000 nM.

[0019] In some embodiments following any of the fusion proteins described above, the target and substrate are expressed in the central nervous system (CNS).

[0020] In some embodiments following any of the fusion proteins described above, the target and substrate are the same molecule.

[0021] In some embodiments following any of the above fusion proteins, the protease is a metalloprotease.

[0022] In some embodiments following any of the above fusion proteins, the target and substrate are amyloid-β (Aβ). In some embodiments, the antibody and / or protease are Aβ 1-40 and Aβ 1-42The antibody targets both of the following: In some embodiments, the antibody is crenezumab or a variant thereof, or solanezumab or a variant thereof, optionally, the crenezumab variant contains G33S in its heavy chain, and further optionally, the crenezumab variant contains S56F in its light chain. In some embodiments, the protease is selected from the group consisting of neprolysin (NEP), neprolysin-2 (NEP2), endothelin-converting enzymes 1 and 2 (ECE1 and ECE2), angiotensin-converting enzyme (ACE), insulinase (IDE), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), and matryptase (MTSP1). In some embodiments, the protease is neprolysin. In some embodiments, the antibody comprises two heavy chains and two light chains, and the protease is fused to the C-terminus of one or both heavy chains of the antibody.

[0023] In some embodiments following any of the above fusion proteins, the target and substrate are IgG. In some embodiments, the protease is selected from the group consisting of matrix metalloproteinase 3 (MMP3), MMP7, cathepsin G, or variants thereof. In some embodiments, the protease is MMP3 or a variant thereof. In some embodiments, the antibody comprises a full-length antibody. In some embodiments, the antibody comprises an R335E mutation in the Fc fragment. In some embodiments, the full-length antibody comprises a hinge region that is resistant to the protease, and optionally, the hinge region comprises a (G4A)2 sequence. In some embodiments, the antibody comprises a Fab fragment. In some embodiments, the antibody comprises rheumatoid factor or a variant thereof.

[0024] In another embodiment, the present application provides isolated nucleic acids encoding any of the above-described fusion proteins.

[0025] In another embodiment, this application provides a host cell containing any of the above-mentioned nucleic acids.

[0026] In another embodiment, the present application provides a method for producing any of the fusion proteins or fragments thereof, comprising culturing the host cells described above under conditions suitable for the expression of the fusion protein or fragment thereof. In some embodiments, the method further comprises recovering the fusion protein or fragment thereof from the host cells.

[0027] In another aspect, this application provides a fusion protein produced by the method described above.

[0028] In another embodiment, this application provides a pharmaceutical composition comprising any of the fusion proteins and a pharmaceutically acceptable carrier.

[0029] In another aspect, this application provides any of the above-mentioned fusion proteins or any of the pharmaceutical compositions for use as a pharmaceutical.

[0030] In another aspect, the present application provides any of the above-mentioned fusion proteins or pharmaceutical compositions for use in treating a disease or pathological condition.

[0031] In another aspect, this application provides the use of either a fusion protein or any pharmaceutical composition in the manufacture of a pharmaceutical for treating a disease or pathological condition.

[0032] In another aspect, the present application provides a method for treating an individual having a disease or pathological condition, comprising administering to the individual an effective amount of either a fusion protein or a pharmaceutical composition. [Brief explanation of the drawing]

[0033] [Figure 1A] A schematic diagram of the antibody guide protease design is shown.

[0034] [Figure 1B]This diagram shows schematic representations and naming conventions for the untargeted and targeted formats of antibody-enzyme fusion proteins. The first label indicates the molecular format (Fc: crystallizable fragment, Fab: fragment antigen binding, scIgG: monovalent single-chain immunoglobulin G, IgG: immunoglobulin G). Enz refers to the free enzyme. The middle label indicates the fusion format as either NTF (N-terminal fusion) or CTF (C-terminal fusion). Further clarification of the fusion domain is indicated in parentheses. The third and last labels indicate the number of proteases per molecule as either 1 or 2.

[0035] [Figure 2A] The results of the activity of the Aβ protease panel against control substrates are shown. The activity of nine proteases previously shown to cleave Aβ was tested with one of three fluorescence resonance energy transfer (FRET) control substrates. Substrate cleavage resulted in an increase in fluorescence, which was reported as a percentage change compared to no protease. The catalog name of each substrate is shown within each box.

[0036] [Figure 2B] This shows an in vitro protease screening assay for the cleavage of Aβ(1-40) (round) and Aβ(1-42) (square).

[0037] [Figure 2C] The chart shows the expression yields of various Fc and IgG protease fusion formats. Four different proteases were expressed in the context of eight formats, indicated by icons. The IgG fusions contained the crenezumab variable region of the anti-Aβ antibody crenezumab, with all heavy chain constant regions being human IgG1. The bar graphs show the expression yield from 293 expressions in 30 ml of replicates of each construct.

[0038] [Figure 2D]The size exclusion chromatography traces of the purified crenezumab NEP protease fusion formats are shown. Each NEP fusion format was expressed in HEK 293 cells and initially purified using protein A resin. Size exclusion chromatography (SEC) combined with the sample fractions was used for further purification. The SEC chromatograms revealed the presence of multiple species, with each sample containing 2-3 peaks.

[0039] [Figure 2E] The results for the cleavage activity of the central fraction are shown. The activity of the central fraction associated with each peak in the chromatogram above was tested by assaying for Aβ(1-40) cleavage.

[0040] [Figure 3] This paper compares the untargeted and targeted proteolytic degradation of Aβ in different fusion formats. All targeted formats contain the variable domain of the anti-Aβ antibody crenezumab. The same enzyme format, along with Fab titers and enzyme fusion sites, are compared in each plot, along with the enzyme alone. The table shows EC50 values, visually illustrating the targeted and untargeted formats.

[0041] [Figure 4] Panel A summarizes the kinetic parameters and EC50 values ​​of the anti-Aβ antibody offrate series, Panel B shows the results of an in vitro cleavage assay using Aβ(1-40) as a substrate, and Panel C shows a graph illustrating the relationship between the EC50 value of Aβ(1-40) cleavage and the offrate of the anti-Aβ targeted antibody.

[0042] [Figure 5A] A schematic diagram of human and bacterial protease cleavage sites within the hinge of human IgG is shown.

[0043] [Figure 5B]The structure of MMP3 is shown (PDB ID: 1SLM(63)). The prodomain (Pro-D) and catalytic domain (catalyst) are shown in cartoon form, while the signaling peptide (SP) and hemopexin domain (hemopexin) are not present in the published crystal structure. The four substitution sites of the factor Xa (IEGR) and enterokinase (DDDDK) cleavage sequences are shown in boxes and listed along with the corresponding MMP3 residues.

[0044] [Figure 5C] This shows an SDS-PAGE gel illustrating eight protease cleavage site insertion variants before and after activation by each external protease (factor Xa and enterokinase).

[0045] [Figure 5D] The results of the cleavage assay are shown. MMP3-D4K-4 (this represents a variant with an enterokinase cleavage site substituted within the 4-position of the MMP3 prodomain). This variant efficiently cleaves the fluorescent MMP3 peptide substrate after the prodomain is removed by enterokinase (black circles), while the intact form containing the prodomain cleaves the substrate minimally (white circles).

[0046] [Figure 5E] The SDS-PAGE gels show the cleavage of human IgG by MMP3-D4K-4 with and without a prodomain at different relative concentrations (10% and 1% w / w) at 37°C after 24 hours. The presence or absence of components in the reaction is indicated by + and -, respectively. MMP3-D4K-4 sequentially cleaves the lower hinge of intact human IgG, first producing a single cleavage product (SCP, half of Fc lost upon denaturation), and then, after a second cleavage, producing F(ab')2 and Fc (not shown). Enterokinase does not detectably cleave human IgG (lane 3).

[0047] [Figure 6A]The structure of two RF61 Fabs (LC: blue, HC: orange) bound to human Fc (red) is shown (PDB ID: 2J6E). The inset highlights residues identified by saturated mutagenesis, which are important for Fc binding.

[0048] [Figure 6B] A waterfall plot summarizing the affinity of RF61 variants to human IgG1 across three rounds of saturated mutagenesis and screening is shown. The affinity for wild-type RF61 could not be determined and was estimated to be greater than 1 μM. Affinity was measured by SPR against RF61 variants in a mouse IgG2a chimeric format against human IgG1 Fc (see Methods).

[0049] [Figure 6C] This section quantifies binding between eight high-affinity RF61 variants identified in the third round of affinity maturation and 38 binding-losing variants of human Fc. See Example 4 for specific RF61 variants. Binding-losing variants were evaluated using late analyte binding values ​​from SPR sensorograms representing the binding of each RF61 variant to human Fc, with complete binding loss shown in white and high binding levels in gray. Asterisks indicate that the Fc variant (R355E, variant 12) is used to eliminate self-binding in all RF61 IgG constructs.

[0050] [Figure 6D] This study demonstrates that mutations in the lower hinge sequence and N-terminal region of human IgG1 CH2 effectively inhibit cleavage by MMP3-D4K-4. SDS-PAGE gel images (top left) and densitometry (top right) confirm the resistance of the IgG1 hinge variant to proteolytic cleavage.

[0051] [Figure 7A] Cartoon representations of IgG-MMP3-D4K-4 and Fab-MMP3-D4K-4 fusion protein formats are shown.

[0052] [Figure 7B] Figures 7C to 7F show a summary of affinity and associated mutations for the anti-IgG antibody affinity series tested.

[0053] [Figure 7C] The results of an in vitro cleavage assay measuring the proteolytic activity of the anti-IgG affinity series of Fab-MMP3-D4K-4 fusion proteins against a fluorescence-generating MMP3 peptide substrate are shown. Substrate cleavage generated a fluorescence signal by dequenching of the fluorophore.

[0054] [Figure 7D] This report presents the results of an in vitro cleavage assay measuring the proteolytic activity of the anti-IgG affinity series of Fab-MMP3-D4K-4 fusion proteins against fluorescent DQ collagen, a type IV substrate. Substrate cleavage generated a fluorescence signal by dequenching of the fluorophores.

[0055] [Figure 7E] This report presents the results of an in vitro cleavage assay measuring the proteolytic activity of the anti-IgG affinity series of Fab-MMP3-D4K-4 fusion proteins against human IgG1 substrates. IgG cleavage was measured by ELISA.

[0056] [Figure 7F] This report presents the results of an in vitro cleavage assay measuring the proteolytic activity of the anti-IgG affinity series of IgG-MMP3-D4K-4 fusion proteins against a human IgG1 substrate. IgG cleavage was measured by ELISA.

[0057] [Figure 8]The results of a cleavage assay measuring the proteolytic activity of targeted (purple) and untargeted (blue) MMP3-D4K-4 in human serum against human IgG1 (A) and a fluorescent MMP3 peptide substrate (B) after 24 hours at 37°C are shown. The targeted MMP3-D4K-4 construct contains anti-IgG Fab RF61-DEF, while the untargeted construct contains anti-IgG Fab.

[0058] [Figure 9] This shows the dependence of targeted and untargeted IgG cleavage on human IgG subtypes. MMP3 does not cleave IgG2, but RF61 does not bind to IgG4.

[0059] [Figure 10] This shows the activity of various antibody-MMP3-D4K-4 fusion formats. After affinity column purification, eluates from each sample were incubated with enterokinase overnight at room temperature, and then MMP3-D4K-4 activity was tested using a fluorescence-generating peptide substrate (top) and IgG (bottom). The blue (left) and red (right) bars represent samples with and without incubation with enterokinase for prodomain cleavage, respectively. Background color shading distinguishes between IgG and Fab formats, as well as untargeted formats. Formats with and without antibody domain fusions are separated by dashed black lines. Activity against peptide substrates (top) indicates the presence of active MMP3-D4K-4. All samples containing MMP3 showed some level of enzymatic activity. Activity against IgG, as measured by ELISA, indicates antibody-targeted proteolytic activity. Little to no activity was observed for untargeted formats, while targeted MMP3-D4K-4 formats cleaved IgG. The varying degrees of activity can be explained by the different sample concentrations and purities, as the samples in this initial screening trial were not normalized or further purified by size exclusion chromatography. [Modes for carrying out the invention]

[0060] This application provides a fusion protein comprising an antibody that binds to a target, wherein the antibody is fused to a protease that cleaves a substrate and the target is in proximity to the substrate. In some embodiments, the protease is fused to one or more polypeptide chains of the antibody. In some embodiments, the target and the substrate are the same molecule. [[ID=​​​​​​​​​​​​​​​​​​-13 M, for example 10 -9 M~10 -13 The dissociation constant (K) of M D ) has an antibody with a K content of 1 μM or less. D When an antibody has this property, it is said to "specifically bind" to its target. In certain embodiments, antibodies bind to target epitopes that are conserved across targets from different species.

[0064] The term "antibody" as used herein is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0065] An "antibody fragment" refers to a molecule other than an intact antibody, which contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For an overview of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0066] The term "epitope" refers to a site on an antigen, either proteinaceous or nonproteinaceous, to which an antibody binds. Epitopes can be formed from a continuous stretch of amino acids (linear epitopes) or from discontinuous amino acids that are spatially adjacent, for example, due to the folding of the antigen, i.e., tertiary folding of a proteinaceous antigen (conventional epitopes). Linear epitopes are typically still bound to antibodies even after exposure of proteinaceous antigens to denaturants, while conformative epitopes are typically destroyed by treatment with denaturants. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial structure.

[0067] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) can be performed using methods common in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope cleavage analysis, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies," Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0068] Competitive binding can be used to determine whether an antibody competes for binding with a reference antibody that binds to the same target. For example, an antibody that "competes for binding" with a reference antibody is one that blocks the reference antibody's binding to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the antibody's binding to its antigen by 50% or more in a competitive assay. Alternatively, to determine whether an antibody competes for binding with a reference antibody, the reference antibody is bound to the target under saturated conditions. After removing the excess reference antibody, the ability of the antibody in question to bind to the target is evaluated. If the antibody can bind to the target after saturated binding of the reference antibody, it can be concluded that the antibody in question binds to a different epitope than the reference antibody. However, if the antibody in question cannot bind to the target after saturated binding of the reference antibody, it may bind to the same epitope to which the reference antibody binds. Conventional experiments can be used to determine whether the antibody in question is binding to the same epitope or whether binding is simply being hindered for steric reasons (e.g., peptide mutation analysis, or binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art). This assay should be performed in two setups, i.e., with both antibodies being saturated antibodies. If, in both setups, only the first (saturated) antibody can bind to the target, it can be concluded that the antibody in question and the reference antibody are competing for binding to the target.

[0069] In some embodiments, two antibodies are considered to bind to the same or overlapping epitopes if, as measured by competitive binding assays, a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, at least 75%, at least 90%, or even more than 99%. (See, for example, Junghans et al., Cancer Res. 50(1990) 1495-1502).

[0070] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody. Two antibodies are considered to have an "overlapping epitope" if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody also reduces or eliminates the binding of the other antibody.

[0071] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, while the rest of the heavy chain and / or light chain originates from a different source or species.

[0072] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is the IgG1 isotype. In certain embodiments, the antibody is the IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is the IgG2 isotype. In certain embodiments, the antibody is the IgG4 isotype with the S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0073] As used in this application, the terms “human-derived constant region” or “human constant region” refer to the constant heavy chain region and / or constant light chain kappa region or constant light chain lambda region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG4. Such constant regions are known in the art and are described, for example, in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, e.g., Johnson, G., and Wu, TT, Nucleic Acids Res. 28(2000) 214-218; Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72(1975) 2785-2788). Unless otherwise specified herein, the numbering of amino acid residues in the constant region follows the EU numbering system (also known as the Kabat EU index), as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0074] "Effector function" refers to the biological activity resulting from the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0075] The “effective amount” of a drug, such as a pharmaceutical composition, refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0076] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more amino acids from the C-terminus of the heavy chain, particularly one or two amino acids. Thus, antibodies produced by host cells by the expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain either the full-length heavy chain or a cleaved variant of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Thus, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the amino acid sequence of the heavy chain containing the Fc region is shown herein without the C-terminal glycine-lysine dipeptide. In one embodiment, the heavy chain containing the Fc region as specified herein, as contained in the antibody according to the present invention, includes a further C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one embodiment, the heavy chain containing the Fc region as specified herein, as contained in the antibody according to the present invention, includes a further C-terminal glycine residue (G446, numbering according to the EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0077] The "framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally appear in VH (or VL) in the following sequence: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0078] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein synonymously to refer to antibodies having a structure substantially similar to that of a native antibody or having a heavy chain containing an Fc region as defined herein.

[0079] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and cells including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring may contain mutations, although they may not be completely identical to the parent cells in terms of nucleic acid content. In this specification, mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included.

[0080] A "human antibody" is an antibody produced by a human or human cell, or an antibody that has an amino acid sequence corresponding to a non-human antibody that utilizes a sequence encoding a human antibody, such as the human antibody repertoire. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0081] The "Human Consensus Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I, as described in Kabat et al. above. In another embodiment, for VH, the subgroup is subgroup Kappa III, as described in Kabat et al. above.

[0082] A "humanized" antibody refers to a chimeric antibody containing amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody substantially contains all of at least one, typically two, variable domains, in which all or substantially all of the CDRs correspond to the CDRs of a non-human antibody and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0083] As used herein, the terms “hypervariable region” or “HVR” mean each of the regions of an antibody variable domain that are hypervariable within a sequence and determine antigen-binding specificity, such as “complementarity-determining regions” (CDRs).

[0084] An "immune conjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0085] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0086] An “isolated” antibody is one that has been separated from its natural environment. In some embodiments, antibodies are purified to a purity higher than 95% or 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J.Chromatogr.B 848:79-87 (2007).

[0087] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing polymers of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented 5' to 3'. In this specification, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. In addition, the term nucleic acid molecule includes both sense strands and antisense strands, as well as both single-stranded and double-stranded forms. Furthermore, nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone links or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may or may not be modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule so that the mRNA can be injected into a target in vivo to generate an antibody (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or European Patent No. 2101823B1).

[0088] "Isolated" nucleic acids are nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found in cells that contain nucleic acid molecules, but these nucleic acid molecules are located outside of chromosomes or in chromosomal locations different from their natural chromosomal locations.

[0089] "Isolated nucleic acids encoding a fusion protein or a fragment thereof" refers to one or more nucleic acid molecules encoding one or more polypeptides of a fusion protein or a fragment thereof, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations within a host cell.

[0090] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bound to the same epitope, except for variant antibodies that, for example, contain naturally occurring mutations or may arise during the production of a monoclonal antibody preparation, the presence of such variants generally in trace amounts. Typically, in contrast to polyclonal antibody preparations, which contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies according to the present invention can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, including but not limited to such methods and other exemplary methods for producing monoclonal antibodies described herein.

[0091] "Native antibodies" refer to naturally occurring immunoglobulin molecules with diverse structures. For example, a native IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant heavy domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light (CL) domain.

[0092] The term “packaging insert” is used to refer to the instructions typically included in the commercial packaging of a therapeutic product, which contain information about the indications, use, dosage, administration, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic product.

[0093] The terms "pharmaceutical composition" or "pharmaceutical preparation" refer to a preparation in which the biological activity of the active ingredient contained therein is effective, and which does not contain any additional components that are unacceptably toxic to the subject to which the pharmaceutical composition is to be administered.

[0094] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or preparation other than the active ingredient that is non-toxic to the target. Pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0095] As used herein, “treatment” (and its grammatical variations, e.g., “to treat” or “treating”) refers to a clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be carried out for preventive purposes or during the course of a clinicopathological disease. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological outcomes of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and achieving remission or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of the disease or to slow the progression of the disease.

[0096] A "variable region" or "variable domain" is a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (e.g., Kindt et al. Kuby Immunology, 6) th See ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH or VL domain of the antibody that binds to that antigen, and complementary libraries of VL or VH domains can be screened. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0097] The term “vector” as used herein refers to a nucleic acid molecule capable of replicating another nucleic acid it is linked to. This term includes not only vectors as self-replicating nucleic acid structures, but also vectors that have been incorporated into the genome of a host cell into which they have been introduced. Certain vectors can direct the expression of a operably linked nucleic acid. Such vectors are referred to herein as “expression vectors.”

[0098] II. Compositions and Methods In one embodiment, the present invention is based in part on the remarkable finding that a fusion protein having an antibody bound to a target fused with a protease that cleaves a substrate can catalyze cleavage in a much more potent manner. In some embodiments, the target and substrate are in close proximity. In some embodiments, the target and substrate are the same molecule. In some embodiments, the fusion protein is an antibody-enzyme fusion that improves catalytic activity and modulates selectivity by providing preferred selectivity of the antibody against the enzyme. In another embodiment of the present invention, the present invention is based on manipulating a fusion protein (e.g., an antibody-enzyme fusion) to broaden the therapeutic range of a protease. In another embodiment of the present invention, the antibody-enzyme fusion provides a novel therapeutic approach for neutralizing therapeutic targets (e.g., those in high abundance). In another embodiment of the present invention, the antibody-enzyme fusion provides improved delivery of proteases, for example, in hard-to-reach intracellular tissue sites. In another embodiment of the present invention, the antibody-enzyme fusion provides enrichment of a protease at one or more sites. In another embodiment of the present invention, the antibody-enzyme fusion reduces the nonspecific activity of a protease (e.g., protease activity at off-target sites). In another aspect of the present invention, the present invention is based on targeting proteins having a high protein content in order to effectively reduce their amount therapeutically. The fusion proteins of the present invention are useful in treating various diseases or pathological conditions, for example, involving therapeutic substrates that are to be removed. The fusion proteins are also effective in several systems and tissue types, including hard-to-reach locations, including the central nervous system.

[0099] A. Fusion protein The fusion proteins described herein combine a protease with a substrate that is a target of proteolysis. To facilitate the proteolysis of the target, the fusion protein comprises an antibody that binds to the target and an enzyme that catalyzes the hydrolysis of the substrate. These groups can be fused directly or via a linker. This molecular construct allows the enzyme to be specifically brought into contact with the target so that it is specifically proteolytic by the enzyme.

[0100] This specification provides a fusion protein comprising an antibody that binds to a target, wherein the antibody is fused to a protease that cleaves a substrate, and the target is in close proximity to the substrate. In some embodiments, the target and substrate are the same molecule. The fusion proteins described herein have one or more advantages: a) higher specificity for the desired substrate, b) increased potency for the substrate, c) longer half-life, and d) more efficient removal of the substrate. These advantages enable the fusion protein to more effectively inhibit the therapeutic target.

[0101] In some embodiments, a fusion protein is provided that contains an antibody that binds to a target, the antibody is fused to a protease that cleaves a substrate, the target and substrate being the same molecule, and the antibody comprises a) a first polypeptide containing a heavy chain variable (VH) domain and a first heavy chain constant (CH1) domain, and b) a second polypeptide containing a light chain variable (VL) domain and a light chain constant (CL) domain, the protease being fused to the N-terminus of the VL domain, the VH and VL domains forming a binding domain to the target, and a disulfide bond being formed between the CH1 and CL domains. In some embodiments, the antibody does not contain an Fc fragment. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k) of the antibody against the target is used. off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies. In some embodiments, the equilibrium dissociation constant (k) of the antibody against the target is used. D The target has a concentration ranging from approximately 0.1 nM to approximately 1000 nM. In some embodiments, the target and substrate are expressed in the central nervous system (CNS). In some embodiments, the target and substrate are the same molecule. In some embodiments, the protease is a metalloproteinase.

[0102] In some embodiments, a fusion protein is provided that contains an antibody that binds to a target, the antibody is fused to a protease that cleaves the substrate, the target and substrate are the same molecule, and the antibody comprises a) a first polypeptide containing a light chain variable (VL) domain and a light chain constant (CL) domain, b) a second polypeptide containing a heavy chain variable (VH) domain and a first heavy chain constant (CH1) domain, and c) a third polypeptide containing an Fc region, the protease being fused to the N-terminus of the third polypeptide, the VH and VL domains forming a binding domain to the target. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k) of the antibody against the target is provided. off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies. In some embodiments, the equilibrium dissociation constant (k) of the antibody against the target is used. D The target has a concentration ranging from approximately 0.1 nM to approximately 1000 nM. In some embodiments, the target and substrate are expressed in the central nervous system (CNS). In some embodiments, the target and substrate are the same molecule. In some embodiments, the protease is a metalloproteinase.

[0103] In some embodiments, a fusion protein is provided containing an antibody that binds to a target, the antibody is fused to a protease that cleaves the substrate, the target and substrate are the same molecule, the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, the protease is fused to the N-terminuses of the two light chains. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k) of the antibody against the target is provided. off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies. In some embodiments, the equilibrium dissociation constant (k) of the antibody against the target is used. D The target has a concentration ranging from approximately 0.1 nM to approximately 1000 nM. In some embodiments, the target and substrate are expressed in the central nervous system (CNS). In some embodiments, the target and substrate are the same molecule. In some embodiments, the protease is a metalloproteinase.

[0104] In some embodiments, a fusion protein is provided containing an antibody that binds to a target, the antibody is fused to a protease that cleaves the substrate, the target and substrate are the same molecule, the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, the protease is fused to the N-terminus of the two heavy chains. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k) of the antibody against the target is provided. off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies. In some embodiments, the equilibrium dissociation constant (k) of the antibody against the target is used. D The target has a concentration ranging from approximately 0.1 nM to approximately 1000 nM. In some embodiments, the target and substrate are expressed in the central nervous system (CNS). In some embodiments, the target and substrate are the same molecule. In some embodiments, the protease is a metalloproteinase.

[0105] In some embodiments, a fusion protein is provided containing an antibody that binds to a target, the antibody is fused to a protease that cleaves the substrate, the target and substrate are the same molecule, the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, and the protease is fused to the N-terminuses of the two light chains and the N-terminuses of the two heavy chains. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k) of the antibody against the target is specified. off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies. In some embodiments, the equilibrium dissociation constant (k) of the antibody against the target is used. D The target has a concentration ranging from approximately 0.1 nM to approximately 1000 nM. In some embodiments, the target and substrate are expressed in the central nervous system (CNS). In some embodiments, the target and substrate are the same molecule. In some embodiments, the protease is a metalloproteinase.

[0106] In some embodiments, a fusion protein is provided containing an antibody that binds to a target, the antibody is fused to a protease that cleaves the substrate, the target and substrate are the same molecule, the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, the protease is fused to one C-terminus of the heavy chain. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k) of the antibody against the target is provided. off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies. In some embodiments, the equilibrium dissociation constant (k) of the antibody against the target is used. D The target has a concentration ranging from approximately 0.1 nM to approximately 1000 nM. In some embodiments, the target and substrate are expressed in the central nervous system (CNS). In some embodiments, the target and substrate are the same molecule. In some embodiments, the protease is a metalloproteinase.

[0107] In some embodiments, a fusion protein is provided containing an antibody that binds to a target, the antibody is fused to a protease that cleaves the substrate, the target and substrate are the same molecule, the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, the protease is fused to both C-terminuses of the heavy chains. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k) of the antibody against the target is provided. off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies. In some embodiments, the equilibrium dissociation constant (k) of the antibody against the target is used. D The target has a concentration ranging from approximately 10 nM to approximately 1000 nM. In some embodiments, the target and substrate are expressed in the central nervous system (CNS). In some embodiments, the target and substrate are the same molecule. In some embodiments, the protease is a metalloproteinase.

[0108] In some embodiments, a fusion protein is provided that contains an antibody that binds to a target, the antibody is fused to a protease that cleaves the substrate, and the target and substrate are in close proximity to each other or are the same molecule expressed in the central nervous system (CNS). In some embodiments, the antibody contains a full-length antibody. In some embodiments, the protease is fused to the N-terminus and / or C-terminus of one or more polypeptide chains of the antibody. In some embodiments, the antibody contains an antibody fragment. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k) of the antibody against the target is provided. off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies. In some embodiments, the equilibrium dissociation constant (k) of the antibody against the target is used. D The target has a concentration ranging from approximately 0.1 nM to approximately 1000 nM. In some embodiments, the target and substrate are expressed in the central nervous system (CNS). In some embodiments, the target and substrate are the same molecule. In some embodiments, the protease is a metalloproteinase.

[0109] In some embodiments, a fusion protein is provided containing an antibody that binds to a target, the antibody fused to a protease that cleaves the substrate, and the target and substrate are in close proximity to each other or are the same molecule expressed in circulation. In some embodiments, the antibody comprises a full-length antibody. In some embodiments, the protease is fused to the N-terminus and / or C-terminus of one or more polypeptide chains of the antibody. In some embodiments, the antibody comprises an antibody fragment. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k) of the antibody against the target is provided. off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies. In some embodiments, the equilibrium dissociation constant (k) of the antibody against the target is used. D The target has a concentration ranging from approximately 0.1 nM to approximately 1000 nM. In some embodiments, the target and substrate are expressed in the central nervous system (CNS). In some embodiments, the target and substrate are the same molecule. In some embodiments, the protease is a metalloproteinase.

[0110] In some embodiments, a fusion protein is provided containing an antibody that binds to a target, the antibody fused to a protease that cleaves a substrate, and the target and substrate are in close proximity to each other or are the same molecule expressed in cancer cells. In some embodiments, the antibody comprises a full-length antibody. In some embodiments, the protease is fused to the N-terminus and / or C-terminus of one or more polypeptide chains of the antibody. In some embodiments, the antibody comprises an antibody fragment. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k) of the antibody against the target is provided. off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies. In some embodiments, the equilibrium dissociation constant (k) of the antibody against the target is used. D The target has a concentration ranging from approximately 0.1 nM to approximately 1000 nM. In some embodiments, the target and substrate are expressed in the central nervous system (CNS). In some embodiments, the target and substrate are the same molecule. In some embodiments, the protease is a metalloproteinase.

[0111] In some embodiments, a fusion protein is provided that contains an antibody that binds to a target, the antibody is fused to a protease that cleaves a substrate, and the target and substrate are in close proximity to each other or are the same molecule expressed in diseased tissue (e.g., with inflammation). In some embodiments, the antibody contains a full-length antibody. In some embodiments, the protease is fused to the N-terminus and / or C-terminus of one or more polypeptide chains of the antibody. In some embodiments, the antibody contains an antibody fragment. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k) of the antibody against the target is provided. off ) is approximately 5 x 10 -3 , 1 x 10 -3 , 5×10 -4 , or 1 × 10 -4 The following applies. In some embodiments, the equilibrium dissociation constant (k) of the antibody against the target is used. D The target has a concentration ranging from approximately 0.1 nM to approximately 1000 nM. In some embodiments, the target and substrate are expressed in the central nervous system (CNS). In some embodiments, the target and substrate are the same molecule. In some embodiments, the protease is a metalloproteinase.

[0112] In some embodiments, a fusion protein is provided that includes an antibody that binds to a target, the antibody is fused to a protease that cleaves a substrate, and the target and the substrate are in proximity to each other or are the same molecule expressed in fibrotic tissue. In some embodiments, the antibody includes a full-length antibody. In some embodiments, the protease is fused to the N-terminus and / or C-terminus of one or more polypeptide chains of the antibody. In some embodiments, the antibody includes an antibody fragment. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker, and optionally, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is cleavable by a protease different from the protease included in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the dissociation rate constant (k off ) of the antibody to the target is about 5×10 -3 , 1×10 -3 , 5×10 -4 , or 1×10 -4 or less. In some embodiments, the equilibrium dissociation constant (k D ) of the antibody to the target has a range of about 0.1 nM to about 1000 nM. In some embodiments, the target and the substrate are expressed in the central nervous system (CNS). In some embodiments, the target and the substrate are the same molecule. In some embodiments, the protease is a metalloprotease.

[0113] In some embodiments, a fusion protein is provided that includes an antibody that binds to a target, the antibody is fused to a protease that cleaves a substrate, and the target and the substrate are associated with a neurological disease. In some embodiments, the target and the substrate are amyloid-β (Aβ). In some embodiments, the antibody and / or protease is Aβ 1-40 and Aβ 1-42The antibody targets both of the following: In some embodiments, the antibody is crenezumab or a variant thereof, or solanezumab or a variant thereof, optionally, the crenezumab variant contains G33S in its heavy chain, and further optionally, the crenezumab variant contains S56F in its light chain. In some embodiments, the protease is selected from the group consisting of neprolysin (NEP), neprolysin-2 (NEP2), endothelin-converting enzymes 1 and 2 (ECE1 and ECE2), angiotensin-converting enzyme (ACE), insulinase (IDE), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), and matryptase (MTSP1). In some embodiments, the protease is neprolysin. In some embodiments, the antibody comprises two heavy chains and two light chains, and the protease is fused to the C-terminus of one or both heavy chains of the antibody.

[0114] In some embodiments, a fusion protein is provided that includes an antibody that binds to a target, the antibody being fused to a protease that cleaves a substrate, and the target and substrate are IgG. In some embodiments, the protease is selected from the group consisting of matrix metalloproteinase 3 (MMP3), MMP7, cathepsin G, or variants thereof. In some embodiments, the protease is MMP3 or a variant thereof. In some embodiments, the antibody includes a full-length antibody. In some embodiments, the antibody includes an R335E mutation in the Fc fragment. In some embodiments, the full-length antibody includes a hinge region that is resistant to the protease, and optionally, the hinge region includes (G4A)2. In some embodiments, the antibody includes a Fab fragment. In some embodiments, the antibody includes rheumatoid factor or a variant thereof.

[0115] antibody The antibodies described herein may be monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological activity (e.g., binding to a target).

[0116] In some embodiments, the antibody is a full-length antibody or comprises a full-length antibody. In some embodiments, the antibody is an intact IgA, IgG, IgM, IgD, IgE antibody or another antibody class or isotype as defined herein.

[0117] In some embodiments, the antibodies are chimeric, human, partially humanized, fully humanized, or semi-synthetic. The described antibodies and / or antibody fragments may originate from mouse antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camel antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0118] In some embodiments, the antibody comprises an Fc fragment. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments derived from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from human IgG. In some embodiments, the Fc fragment comprises the Fc region of human IgG1, IgG2, IgG3, IgG4, or combinations or hybrids of IgG.

[0119] In some embodiments, the antibody binds to a target or a fragment of the substrate that is identical to the substrate. In some embodiments, the antibody binds to an isoform of the target (e.g., a protein variant). Thus, in some embodiments, the antibody recognizes one isoform. In some embodiments, the antibody recognizes one or more isoforms. In some embodiments, the antibody prefers one isoform. This preference may be demonstrated by enhanced binding and other parameters that can be measured by biochemical assays such as those described herein. In some embodiments, the antibody prefers one or more isoforms. In some embodiments, the antibody is not isoform-specific. In some embodiments, the antibody binds to all isoforms. In some embodiments, the antibody does not exhibit superior binding to any isoform of the target.

[0120] In some embodiments, the target is an extracellular molecule. In some embodiments, the target is a molecule in a tissue. In some embodiments, the target is a molecule in a tissue within the central nervous system (e.g., the brain), and optionally, the antibody penetrates the blood meningeal membrane. See, for example, Fluids Barriers CNS. 2022 Dec 12;19(1):99. In some embodiments, the target is circulating. In some embodiments, the target is located within an organ (e.g., liver, spleen, lungs, heart, etc.).

[0121] In some embodiments, the target is an intracellular molecule.

[0122] In some embodiments, the target is a cell surface molecule.

[0123] In some embodiments, the target is a peptide or a fragment thereof. In some embodiments, the peptide has a length of about 0 to 100 amino acids, 10 to 80 amino acids, 20 to 70 amino acids, or 30 to 60 amino acids.

[0124] In a further embodiment, the target is amyloid beta (aβ or abeta).

[0125] In a further embodiment, the target is immunoglobulin G (IgG).

[0126] In a further embodiment, an antibody that binds to a target according to any of the above embodiments may incorporate any of the features described in Sections 1 to 7 below, either individually or in combination:

[0127] 1. Antibody affinity In certain embodiments, the antibodies provided herein are ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13It has a dissociation constant (KD) of (M).

[0128] In certain embodiments, the antibodies provided herein have a dissociation constant (KD) of 100 nM to 1 μM, 10 nM to 100 nM, 1 nM to 10 nM, 0.1 nM to 1 nM, 0.01 nM to 0.1 nM, or 0.001 to 0.1 nM.

[0129] In certain embodiments, the antibodies provided herein have one or more modifications that promote a weaker dissociation constant for the target.

[0130] In certain embodiments, the antibodies provided herein have a dissociation rate constant (k -2 s -1 ), ≤ 5×10 -3 s -1 ), ≤ 10 -3 s -1 ), ≤ 5×10 -4 s -1 ), ≤ 10 -4 s -1 ), ≤ 5×10 -5 s -1 ), or ≤ 10 -5 s<000011 "0>). In certain embodiments, the antibodies provided herein have an off-rate constant (k off ) of 10 -2 s -1 to 5×10 -3 s -1 , 10<0000 "16>s -1 to 5×10 -3 s -1 , 5×10 -4 s -1 to 10 -3 s -1 , 10 -4 s -1 [[ID=7 "0]] to 5×10 -4 s -1 , 10 -4 s -1 to 5×10 -4 s -1 , 5×10 -5 s -1 to 10 -4 s -1 or 10 -5 s -1 to 5×10-5 s -1 Off rate (k off ) has.

[0131] In certain embodiments, the antibodies provided herein have one or more modifications that promote a faster off-rate constant against the target.

[0132] In one aspect, K D BIACORE (登録商標) It is measured using a surface plasmon resonance assay. For example, BIACORE (登録商標) -2000 or BIACORE (登録商標) The assay using -3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C for approximately 10 response units (RUs) using an immobilized antigen CM5 chip. In one embodiment, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate at pH 4.8 and injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RUs) of the coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For dynamic measurement, 2x serial dilutions of Fab (0.78 nM to 500 nM) were injected at a flow rate of approximately 25 μL / min at 25°C in 0.05% polysorbate 20 (TWEEN-20). (商標) Inject into PBS containing surfactant (PBST). Association rate (k on ) and dissociation rate (k off By simultaneously fitting the association sensorgram and dissociation sensorgram, a simple one-to-one Langmuir coupled model (BIACORE) can be achieved. (登録商標) The calculation is performed using Evaluation Software version 3.2. The equilibrium dissociation constant (K D ) is k off / k onIt is calculated as a ratio. For example, see Chen et al. J.Mol.Biol.293:865-881(1999). The ON velocity is 10 by the above surface plasmon resonance assay. 6 M -1 s -1 If this exceeds the limit, this ON speed will be measured using a stop-flow equipped spectrophotometer with a stirred cuvette (Aviv Instruments) or the 8000 series SLM-AMINCO. (商標) This can be determined by using a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band passthrough) of a 20 nM anti-antigen antibody (Fab type) in PBS (pH 7.2) at 25°C in the presence of gradually increasing concentrations of the antigen, using a spectrophotometer such as a ThermoSpectronic.

[0133] Alternatively, K D This is measured by radiolabeled antigen-binding assay (RIA). In one embodiment, the RIA is performed using the antibody of interest and its Fab version of the antigen. For example, the solution binding affinity of Fab to the antigen is measured in the presence of a titration series of unlabeled antigens at the lowest concentration ( 125 I) Fab is equilibrated with labeled antigen, and then the bound antigen is captured by a plate coated with anti-Fab antibody (see, for example, Chen et al., "J.Mol.Biol." Vol. 293, pp. 865-881 (1999)). To establish the assay conditions, use a MICROTITER (登録商標) Multiwell plates (Thermo Scientific) are coated overnight with 5 μg / mL of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2–5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc#269620), 100 pM or 26 pM [ 125Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, but incubation can be extended for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. Then transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Next, remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®, Packard) and count the plate on a TOPCOUNT® gamma counter (Packard) for 10 minutes. Select the concentration of each Fab that yields less than 20% of the maximum binding for use in competitive binding assays.

[0134] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments.

[0135] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, particularly a Fab fragment. Papain digestion of an intact antibody produces two identical antigen-binding fragments (so-called "Fab" fragments), each containing the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, in addition to the variable domains of the heavy and light chains (VH and VL, respectively). Therefore, the term "Fab fragment" refers to an antibody fragment containing a light chain with the VL and CL domains, and a heavy chain fragment containing the VH and CH1 domains. A "Fab' fragment" differs from a Fab fragment by the addition of a residue at the carboxyl terminus of the CH1 domain, containing one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues (multivalent) of the constant domain retain a free thiol group. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a description of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have a longer in vivo half-life, see U.S. Patent No. 5,869,046.

[0136] In another embodiment, the antibody fragment is a bispecific, triplicate, or quadruplicate antibody. A diabody is an antibody fragment having two antigen-binding sites that may be bivalent or bispecific. See, for example, European Patent No. 404,097, WO1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0137] In a further embodiment, the antibody fragment is a single-chain Fab fragment. The "single-chain Fab fragment" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following orders from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In particular, the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-stranded Fab fragments can be further stabilized by the formation of interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain, according to Kabat numbering).

[0138] In another embodiment, the antibody fragment is a single-chain variable fragment (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy chain (VH) and light chain (VL) of an antibody, linked by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids, usually rich in glycine for flexibility and serine or threonine for solubility, and can be linked at either the N-terminus of VH or the C-terminus of VL. This protein can retain the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. For a review of scFv fragments, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also International Publication No. 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458.

[0139] In another embodiment, the antibody fragment is a single-domain antibody. A "single-domain antibody" is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In a particular embodiment, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA; e.g., U.S. Patent No. 6,248,516 B1).

[0140] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, protein digestion of intact antibodies and recombinant production by recombinant host cells (e.g., Escherichia coli), as described herein.

[0141] 3. Chimeric antibodies and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from mouse, rat, hamster, rabbit, or non-human primate, e.g., monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass is changed from those of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0142] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce their immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. Humanized antibodies usually contain one or more variable domains (CDRs or parts thereof) derived from the non-human antibody, and FRs (or parts thereof) derived from the human antibody sequence. Humanized antibodies also optionally contain at least a portion of the human constant region. In some embodiments, several FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve antibody specificity or affinity, for example.

[0143] Humanized antibodies and methods for their production are outlined, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods. Further information is available in 36:25-34 (2005) (describes specificity-determining region (SDR) grafts); padlan, mol.immunol. 28:489-498 (1991) (describes "resurfacing"); dall'acqua et al., methods 36:43-60 (2005) (describes "FR shuffling"); and osbourn et al., methods 36:61-68 (2005) and klimka et al., br.j.cancer, 83:252-260 (2000) (describes "guided selection" in FR shuffling).

[0144] Human framework regions that may be used for humanization include, but are not limited to, the following: framework regions selected using the “best fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germ cell framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. See al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0145] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0146] Human antibodies may be prepared by administering an immunogen to a transgenic animal modified to produce intact human antibodies or intact antibodies with a human variable region in response to an antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or that is extrachromosomal or randomly incorporated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HuMab® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication 2007 / 0061900 describing VelociMouse® technology. Such human variable regions derived from intact antibodies produced by animals may be further modified, for example, by combining them with different human constant regions.

[0147] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human xenomyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology have also been described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies derived from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0148] Human antibodies can also be generated by isolating variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0149] 5. Antibodies derived from libraries In certain embodiments, the antibodies provided herein are obtained from a library. The antibodies of the present invention can be isolated by screening a combinatorial library for antibodies having a desired activity(s). Methods for screening combinatorial libraries are reviewed, for example, in Nature Reviews 16:498-508 (2016) by Lerner et al. For example, various methods for generating phage display libraries and screening such libraries for antibodies having desired binding properties are known in the art. Such methods are outlined, for example, in Frenzel et al., mAbs 8:1177-1194 (2016); Bazan et al., Human Vaccines and Immunotherapeutics 8:1817-1828 (2012); Zhao et al., Critical Reviews in Biotechnology 36:276-289 (2016); Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001); and Marks and Bradbury in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0150] In certain phage display methods, the repertoire of VH and VL genes are individually cloned by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages, as described by Winter et al. in Annual Review of Immunology 12:433-455 (1994). The phages typically present antibody fragments either as single-stranded Fv (scFv) fragments or as Fab fragments. Libraries from immunization sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, a naive repertoire can be cloned (e.g., from humans) to obtain a single antibody source against a wide range of non-self to self antigens without immunization, as described by Griffiths et al. in EMBO Journal 12:725-734 (1993). Furthermore, as described by Hoogenboom and Winter, Journal of Molecular Biology 227:381-388 (1992), naive libraries can also be synthetically created by cloning an unrearranged V gene segment derived from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and achieving in vitro rearrangement. Examples of patent publications describing human antibody phage libraries include: U.S. Patent Nos. 5,750,373, 7,985,840, 7,785,903 and 8,679,490, and U.S. Patent Application Publications 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936.

[0151] Further examples of methods known in the art for screening combinatorial libraries for antibodies with desired activity(s) include ribosome and mRNA display, as well as methods for antibody display and selection in bacterial, mammalian, insect, or yeast cells. Methods for yeast surface display are outlined, for example, in Scholler et al. in Methods in Molecular Biology 503:135-56 (2012), Cherf et al. in Methods in Molecular Biology 1319:155-175 (2015), and Zhao et al. in Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, for example, in He et al. in Nucleic Acids Research 25:5132-5134 (1997) and Hanes et al. in PNAS 94:4937-4942 (1997).

[0152] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification.

[0153] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecific antibody has three or more binding specificities. In certain embodiments, one binding specificity is for a target and another specificity is for any other antigen. In certain embodiments, a bispecific antibody may bind to two (or more) different epitopes of a target. Multispecific (e.g., bispecific) antibodies may also be used to localize cytotoxic agents or cells to cells expressing a target. Multispecific antibodies may be prepared as full-length antibodies or antibody fragments.

[0154] Techniques for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and the "knob-in-hole" operation (see, for example, U.S. Patent No. 5,731,168 and Atwell et al., J.Mol.Biol.270:26 (1997)). Multispecific antibodies are also produced by manipulating the electrostatic steering effect to create antibody Fc heterodimer molecules (see, e.g., International Publication No. 2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992) and International Publication No. 2011 / 034605); using common light chain techniques to avoid light chain mispairing problems (see, e.g., International Publication No. 98 / 50431); and using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Hollinger et al.) It can be prepared by using single-stranded Fv(sFv) dimers (see, for example, Gruber et al., J.Immunol., 152:5368 (1994)); and by preparing a trispecific antibody as described, for example, Tutt et al. J.Immunol. 147:60 (1991).

[0155] For example, this also includes manipulated antibodies having three or more antigen-binding sites, such as "octopus antibodies," or DVD-Ig (see, for example, International Publication No. 2001 / 77342 and International Publication No. 2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in International Publication No. 2010 / 115589, International Publication No. 2010 / 112193, International Publication No. 2010 / 136172, International Publication No. 2010 / 145792, and International Publication No. 2013 / 026831. Bispecific antibodies or their antigen-binding fragments also include "dual-acting FAbs" or "DAFs" that include antigen-binding sites that bind to a target, as well as to another different antigen or two different epitopes of the target (see, for example, U.S. Patent Application Publication No. 2008 / 0069820 and International Publication No. 2015 / 095539).

[0156] Multispecific antibodies may also be provided in an asymmetric form having a domain crossover in one or more binding arms of the same antigen specificity, i.e., by exchanging a VH / VL domain (see, e.g., International Publication 2009 / 080252 and International Publication 2015 / 150447), a CH1 / CL domain (see, e.g., International Publication 2009 / 080253), or a complete Fab arm (see, e.g., International Publication 2009 / 080251, International Publication 2016 / 016299, and also Schaefer et al, PNAS, 108(2011)1187-1191, and Klein et al., MAbs 8(2016)1010-20). In one embodiment, the multispecific antibody comprises a cross-Fab fragment. The terms "cross-Fab fragment," "xFab fragment," or "crossover Fab fragment" refer to Fab fragments in which either the variable or constant regions of the heavy and light chains are exchanged. A cross-Fab fragment includes a polypeptide chain consisting of a light chain variable region (VL) and heavy chain constant region 1 (CH1), and a polypeptide chain consisting of a heavy chain variable region (VH) and light chain constant region (CL). Asymmetric Fab arms can also be manipulated by introducing charged or uncharged amino acid mutations into the domain interface to direct correct Fab pairing. See, for example, International Publication 2016 / 172485.

[0157] Various further molecular formats of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67(2015) 95-106).

[0158] Certain types of multispecific antibodies, similarly included herein, are bispecific antibodies designed to simultaneously bind to a target cell, such as a surface antigen on a tumor cell, and to an activated invariant component of the T cell receptor (TCR) complex, such as CD3, for retargeting T cells and killing the target cells. Thus, in certain embodiments, the antibodies provided herein are multispecific antibodies, in particular bispecific antibodies, where one binding specificity is to a target and the other is to a different target (e.g., another antigen).

[0159] Examples of bispecific antibody formats that may be useful for this purpose include so-called "BiTE" (bispecific T cell engager) molecules in which two scFv molecules are fused by a flexible linker (e.g., International Publication Nos. 2004 / 106381, 2005 / 061547, 2007 / 042261 and 2008 / 119567, see Nagarsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAb"; Kipriyanov et al., J Mol Biol This includes, but is not limited to, the "DART" (Dual Affinity Retargeting) molecules based on the Diabody format but characterized by a C-terminal disulfide crosslink for stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), as well as the so-called triomab, which is a whole-hybrid mouse / rat IgG molecule (Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Specific T-cell bispecific antibody formats included herein are described in International Publication Nos. 2013 / 026833, 2013 / 026839, and 2016 / 020309; Bacac et al., Oncoimmunology 5(8)(2016)e1203498.

[0160] 7. Antibody variants In certain embodiments, amino acid sequence variants of antibodies provided herein are intended. For example, it may be desirable to alter the binding affinity and / or other biological properties of the antibody. In some embodiments, the antibody is mutated to have a faster offrate compared to the parent antibody. In some embodiments, the antibody has a higher KD value compared to the parent antibody. In some embodiments, the antibody has a lower affinity for the substrate. In some embodiments, the antibody has a weaker affinity for the target. In some embodiments, the antibody has both a faster offrate and lower affinity for the target. In some embodiments, the antibody has a lower KD value compared to the parent antibody. In some embodiments, the antibody has a stronger binding affinity for the target compared to the parent antibody.

[0161] Antibody amino acid sequence variants may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from residues in the antibody amino acid sequence, and / or insertions into residues in the antibody amino acid sequence, and / or substitutions of residues in the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to reach the final construct, insofar as the final construct possesses the desired characteristics (e.g., antigen binding).

[0162] (i) Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial substitutions are provided in Table 1 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1]

[0163] Amino acids can be classified according to their general side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0164] Non-conservative substitution involves exchanging a member of one class with a member of another class.

[0165] One type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant(s) selected for further study have altered (e.g., improved) certain biological properties (e.g., increased affinity, decreased immunogenicity) and / or substantially retain certain biological properties of the parent antibody compared to the parent antibody. Exemplary substitution variants are affinity-mature antibodies, which can be readily generated using, for example, phage display-based affinity maturation techniques as described herein. In short, one or more CDR residues are mutated, and the variant antibody displayed on a phage is screened for specific biological activity (e.g., binding affinity).

[0166] To improve antibody affinity, modifications (e.g., substitutions) may be made in the CDR, for example. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that frequently undergo mutations during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that come into contact with the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by secondary library construction and reselection from there is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some aspects of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. Next, this library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed methods in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are often targeted.

[0167] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such alterations do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative alterations that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within a CDR. Such alterations may, for example, be outside the antigen-contact residue in the CDR. In the specific variant VH and VL sequences described above, each CDR is either unaltered or has one, two, or three or fewer amino acid substitutions.

[0168] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called “alanine scanning mutagenesis,” as described by Cunningham and Wells (1989) Science, 244:1081-1085. This method identifies target residues or groups of charged residues (e.g., arg, asp, his, lys, and glu) and substituted them with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively, or in addition to this, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues may be targeted as candidates for substitution or removed. Variants may be screened to determine whether they contain desired properties.

[0169] Amino acid sequence insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of an antibody to an enzyme (e.g., ADEPT (for antibody-directed enzyme prodrug therapy)) or polypeptide, which increases the serum half-life of the antibody.

[0170] (ii) Glycosylated variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

[0171] If the antibody contains an Fc region, the oligosaccharide attached to the antibody may be modified. Native antibodies produced by mammalian cells typically contain branched, bibranched oligosaccharides that are commonly bound to Asn297 of the CH2 domain of the Fc region by an N-bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to the GlcNAc of the "stem" of the bibranched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of the present invention may be carried out to generate antibody variants having specific improved properties.

[0172] In one embodiment, an antibody variant is provided that has an oligosaccharide structure lacking a non-fucosylated oligosaccharide, i.e., fucose binding (direct or indirect) to the Fc region. Such a non-fucosylated oligosaccharide (also called "afucosylated" oligosaccharide) is in particular an N-linked oligosaccharide lacking a fucose residue to which a first GlcNAc is bound in the stem of a branched oligosaccharide structure. In one embodiment, an antibody variant is provided in which the proportion of non-fucosylated oligosaccharides in the Fc region is increased compared to the native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or possibly about 100% (i.e., no fucosylated oligosaccharides are present). The proportion of non-fucosylated oligosaccharides is the (average) amount of fucose-less oligosaccharides relative to the total of all oligosaccharides bound to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in International Publication No. 2006 / 082515. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues); however, due to minor sequence changes in the antibody, Asn297 may be located upstream or downstream of position 297, i.e., approximately ±3 amino acids between positions 294 and 300. Such antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108; and No. 2004 / 0093621.

[0173] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application Publication No. 2003 / 0157108; and International Publication No. 2004 / 056312, particularly Example 11), and knockout cell lines, such as FUT8 of the alpha-1,6-fucosyltransferase gene, and knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al.). Examples include cells in which GDP-fucose synthesis or transporter protein activity is reduced or lost (see, for example, U.S. Patent Application Publications No. 2004259150, 2005031613, 2004132140, and 2004110282).

[0174] In a further embodiment, the antibody variant is provided with a bifurcated oligosaccharide, for example, in which a bifurcated oligosaccharide bound to the Fc region of the antibody is bifurcated by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); International Publication No. 99 / 54342, International Publication No. 2004 / 065540, and International Publication No. 2003 / 011878.

[0175] Antibody variants are also provided that have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in International Publications 1997 / 30087; 1998 / 58964; and 1999 / 22764.

[0176] (iii) Fc region variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies presented herein, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgG1Fc region, a human IgG2Fc region, a human IgG3Fc region, or a human IgG4Fc region) that includes one or more amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0177] In certain embodiments, the present invention conceives antibody variants that, by possessing some, but not all, effector functions, are desirable candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (e.g., complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or harmful. In vitro and / or in vivo cytotoxic assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that an antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96). (登録商標)Non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition to the above, the desired ADCC activity can be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody cannot bind to C1q and lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J.Immunol.Methods202:163(1996); Cragg, MS et al., Blood101:1045-1052(2003); and Cragg, MS and MJ Glennie, Blood103:2738-2743(2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, S B et al., Int'l.Immunol.18(12):1759-1769(2006); WO2013 / 120929Al).

[0178] Antibodies with reduced effector function include those having one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include the so-called "DANA" Fc mutant, which has substitutions at residues 265 and 297 of alanine, as well as Fc mutants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581).

[0179] Specific antibody variants exhibiting improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; International Publication No. 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

[0180] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0181] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., Fc region positions 234 and 235 (residues in EU numbering). In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further includes D265A and / or P329G in the Fc region derived from the human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A and P329G (LALA-PG) in the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2012 / 130831.) In another embodiment, the substitutions are L234A, L235A and D265A (LALA-DA) in the Fc region derived from the human IgG1 Fc region.

[0182] In some embodiments, modifications are made in the Fc region that result in alterations (e.g., reductions) to C1q binding and / or complement-dependent cell-mediated cytotoxicity (CDC), as disclosed, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0183] Antibodies having an increased half-life and improved binding to the neonatal Fc receptor (FcRn), which are responsible for the transfer of maternal IgG into the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)), are described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions in one or more of the Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substitution of Fc region residue 434 (e.g., U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0184] The Fc region residues crucial to the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU numbering of the residues) are involved in the interaction (Medesan, C. et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M. et al., Int. Immunol. 13 (2001) 993; Kim, JK et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be decisive in the interaction between human Fc and mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are decisive in the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, YA, et al. (J. Immunol 182 (2009) 7667-7671) reported and investigated various mutants of residues 248-259, 301-317, 376-382, and 424-437.

[0185] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at Fc region positions 253 and / or 310 and / or 435 (residues in EU numbering). In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A within the Fc region derived from the human IgG1 Fc region. See, for example, Grevys, A., et al., J.Immunol. 194 (2015) 5497-5508.

[0186] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at positions 310 and / or 433 and / or 436 (residues in EU numbering) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 310, 433, and 436. In one embodiment, the substitutions are H310A, H433A, and Y436A in the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2014 / 177460.)

[0187] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., substitutions at positions 252 and / or 254 and / or 256 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in the Fc region derived from the human IgG1 Fc region. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and International Publication No. 94 / 29351.

[0188] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce self-recognition, for example, substitutions at positions R355, E356, K414, E438, K439, and S440 (residue EU numbering) in the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in the Fc region derived from the human IgG1 Fc region. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and International Publication No. 94 / 29351.

[0189] The C-terminus of the heavy chain of an antibody as reported herein may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus, with one or two of the C-terminal amino acid residues removed. In some embodiments, the C-terminus of the heavy chain is PG ending with a shortened C-terminus. In one embodiment of all embodiments reported herein, an antibody containing a heavy chain with the C-terminal CH3 domain specified herein contains a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid position). In one embodiment of all embodiments reported herein, an antibody containing a heavy chain with the C-terminal CH3 domain specified herein contains a C-terminal glycine residue (G446, EU index numbering of amino acid position).

[0190] (iv) Cysteine-modified antibody variant In certain embodiments, it may be desirable to produce cysteine-modified antibodies, such as THIOMAB®, in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at easily accessible sites on the antibody. By substituting these residues with cysteine, the reactive thiol group is positioned at an accessible site on the antibody and can be used to create an immunoconjugate by conjugating the antibody to a drug site or other site, such as a linker drug site, as further described herein. Cysteine-modified antibodies can be produced, for example, as described in U.S. Patents 7,521,541, 8,30,930, 7,855,275, 9,000,130, or International Publication 2016040856.

[0191] 8. Antibody derivatives In certain embodiments, the antibodies provided herein may be further modified to include further non-proteinoid moieties that are known in the art and readily available. Suitable sites for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody varies, and if multiple polymers are attached, they may be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used for therapeutic purposes under defined conditions, although this is not limiting.

[0192] protease Proteases (proteinases) are catalytic enzymes that cleave polypeptides through hydrolysis into shorter polypeptides or single amino acids. They are highly regulated biologically and are involved in several diseases (Bond, Journal of Biological Chemistry, Volume 294, Issue 5, 1643-1651). Proteolysis triggers post-translational processing of polypeptides. Proteases are diverse and can be highly specific to their substrate or non-specific, capable of cleaving at consensus sequences. Proteases are currently classified into seven broad groups: serine proteases, cysteine ​​proteases, threonine proteases, aspartate proteases, glutamate proteases, metalloproteases, and asparagine peptides. A list of useful proteases has been identified in the MEROPS database (Rawlings, ND, Barrett, AJ, Thomas, PD, Huang, X., Bateman, A. & Finn, RD (2018) The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res 46, D624-D632).

[0193] Proteases are a very promising extended class of drugs. See, for example, Biochem J. 2011 Apr 1;435(1):1-16. The proteases provided herein cleave substrates, such as those involved in disease or pathological conditions.

[0194] In some embodiments, the protease is a metalloprotease. In some embodiments, the protease is a zinc metalloprotease.

[0195] Proteases are found in many organisms, including those of prokaryotic and eukaryotic origin. However, proteins of non-human origin can also be immunogenic in the human system, interfering with repeated administration and thus limiting therapeutic application. In some embodiments, proteases are of eukaryotic origin. In some embodiments, proteases are of human origin. In some embodiments, proteases are of prokaryotic origin. In some embodiments, proteases are not of human origin.

[0196] Protease selection can also be guided by a combination of reasons such as specificity and / or potency for the desired substrate, yield, and immunogenicity. Methods for evaluating these characteristics include those described in the examples.

[0197] Protease selection can also be guided by the number of substrates that the protease can cleave. In some embodiments, the protease has indiscriminate activity. In some embodiments, the protease hydrolyzes a wide variety of peptide bonds. In some embodiments, the protease has one substrate. In some embodiments, the protease has one or more substrates. In some embodiments, the protease does not have indiscriminate activity. In some embodiments, the protease hydrolyzes limited or specific peptide bonds. In some embodiments, the protease is specific to a substrate or a class of substrates. In some embodiments, the protease has one substrate. The protease may be substrate isoform specific. Thus, in some embodiments, the protease recognizes one isoform. In some embodiments, the protease prefers one isoform. The preference may be indicated by binding, enhancement of substrate binding, enhancement of catalytic activity, and other enzyme and / or binding parameters that can be measured by biochemical assays such as those described herein. In some embodiments, the protease recognizes one or more isoforms. In some embodiments, the protease prefers one or more isoforms. In some embodiments, the protease is not isoform-specific. In some embodiments, the protease catalyzes the hydrolysis of all isoforms equally well. In some embodiments, the protease catalyzes the hydrolysis of one or more isoforms better than other isoforms.

[0198] Protease selection can also be guided by the number of sites and / or types of sequences that the protease can cleave. In some embodiments, the protease recognizes a consensus sequence. In some embodiments, the protease cleaves a consensus sequence. In some embodiments, the protease recognizes a specific sequence. In some embodiments, the protease cleaves a specific sequence.

[0199] Protease selection can also be guided by the rate of substrate catalysis. In some embodiments, the protease causes rapid hydrolysis of the substrate. In some embodiments, the protease does not cause rapid hydrolysis of the substrate. In some embodiments, the protease is engineered (e.g., mutated) to cause rapid hydrolysis of the substrate. In some embodiments, the protease is engineered (e.g., mutated) to slow the rate of substrate hydrolysis.

[0200] Proteases may also contain other regulatory mechanisms that act as switches to modulate their catalytic activity. Other mechanisms that modulate enzyme activity include temperature (e.g., thermally stable proteases), the addition of organic mercury or organic reagents, or partial proteolysis. Proteases may contain prodomains in addition to their catalytic domains. Prodomains are protein domains that modulate the catalytic activity of a protease. For example, prodomains can inhibit activity if the conditions are not correct for protease function. In some embodiments, successful protease expression may require prodomain selection. In some embodiments, prodomains are protectively removed to induce activity.

[0201] Protease selection can also be guided by functional assays. For example, the activity of several proteases can be tested with substrates (such as fluorescent substrates). Candidate proteases can then be selected based on their comparative performance.

[0202] Target and Substrate In some embodiments, the target bound by the antibody described herein and the substrate cleaved by the protease described herein are located in close proximity to each other such that the affinity / binding activity of the antibody to the target facilitates access of the substrate to the protease.

[0203] In some embodiments, the substrate is located in the CNS. In some embodiments, the substrate is in circulation. In some embodiments, the substrate is located in cancerous tissue. In some embodiments, the substrate is located in fibrous tissue. In some embodiments, the substrate is located in diseased tissue (e.g., with inflammation). In some embodiments, the substrate is located in extracellular tissue. In some embodiments, the level of the substrate is at least 5, 10, 20, 50, 100, or 1000 times higher than the reference level of the substrate (e.g., the level of the substrate in a healthy individual at the same location). In some embodiments, the substrate is in circulation and has a concentration of at least about 0.1 mg / ml, 0.5 mg / ml, 1 ml / ml, 5 mg / ml, or 10 mg / ml.

[0204] In some embodiments, the target and substrate are located on adjacent cells. In some embodiments, the target and substrate are located on the same cell. In some embodiments, the target and substrate are in the same complex (e.g., a polymer complex). In some embodiments, the target is a fragment or part of the substrate. In some embodiments, the substrate is a fragment or part of the target. In some embodiments, the target and substrate are the same molecule (e.g., the same polypeptide). In some embodiments, the antibody binds to an epitope of the target, and the epitope does not overlap with the site where the protease binds to or cleaves the molecule. In some embodiments, the antibody binds to an epitope of the target, and the epitope overlaps with the site where the protease binds to or cleaves the molecule.

[0205] In some embodiments, the target and substrate are A-beta. In some embodiments, the antibody and / or protease are Aβ 1-40 and Aβ 1-42Both are targeted. In some embodiments, the antibody is crenezumab or a variant thereof, or solanezumab or a variant thereof, optionally the crenezumab variant containing G33S in its heavy chain, and further optionally the crenezumab variant containing S56F in its light chain. In some embodiments, the protease is selected from the group consisting of neprolysin (NEP), neprolysin-2 (NEP2), endothelin-converting enzymes 1 and 2 (ECE1 and ECE2), angiotensin-converting enzyme (ACE), insulinase (IDE), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), and matryptase (MTSP1). In some embodiments, the protease is neprolysin.

[0206] In some embodiments, the target and substrate are IgG. In some embodiments, the target and substrate are isotypes of IgG. In some embodiments, the isotypes are IgG1, IgG2, IgG3, or IgG4. In some embodiments, the protease is selected from the group consisting of matrix metalloproteinase 3 (MMP3), MMP7, cathepsin G, or variants thereof. In some embodiments, the protease is MMP3 or a variant thereof. In some embodiments, the antibody comprises a full-length antibody, and optionally, the antibody contains an R335E mutation in the Fc fragment. In some embodiments, the full-length antibody comprises a hinge region that is resistant to the protease, and optionally, the hinge region contains a (G4A)2 sequence. In some embodiments, the antibody comprises a Fab fragment. In some embodiments, the antibody comprises rheumatoid factor or a variant thereof.

[0207] Fusion protein format / structure The antibodies and proteases discussed herein can be fused in various ways to form fusion proteins.

[0208] In some embodiments, the antibody has one or more polypeptide chains, and the protease is fused to one or more polypeptide chains of the antibody.

[0209] In some embodiments, the protease is fused to the N-terminus and / or C-terminus of one or more polypeptide chains of the antibody.

[0210] In some embodiments, the antibody a) a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the protease is fused to the N-terminus or C-terminus of VH or VL; or b) a full-length antibody comprising two heavy chains and two light chains, wherein the protease is fused to the N-terminus or C-terminus of one or both of the two heavy chains and / or the two light chains.

[0211] In some embodiments, the antibody comprises a first polypeptide comprising a heavy chain variable (VH) domain and a first heavy chain constant (CH1) domain, and a second polypeptide comprising a light chain variable (VL) domain and a light chain constant (CL) domain, wherein the protease is fused to the N-terminus of the VL domain, the VH domain and the VL domain form a binding domain for the target, and a disulfide bond is formed between the CH1 domain and the CL domain. In some embodiments, the antibody does not comprise an Fc fragment.

[0212] In some embodiments, the antibody comprises a first polypeptide comprising a heavy chain variable (VH) domain and a first heavy chain constant (CH1) domain, and a second polypeptide comprising a light chain variable (VL) domain and a light chain constant (CL) domain, wherein the protease is fused to the N-terminus of the VH domain, the VH domain and the VL domain form a binding domain for the target, and a disulfide bond is formed between the CH1 domain and the CL domain. In some embodiments, the antibody does not comprise an Fc fragment.

[0213] In some embodiments, the antibody comprises: a) a first polypeptide comprising a variable light (VL) domain and a constant light (CL) domain; b) a second polypeptide comprising a variable heavy (VH) domain and a first constant heavy (CH1) domain; and c) a third polypeptide comprising an Fc region, wherein the protease is fused to the N-terminus of the third polypeptide, and the VH domain and the VL domain form a binding domain for the target.

[0214] In some embodiments, the antibody comprises two heavy chains and two light chains, each heavy chain comprises a variable heavy (VH) domain and a constant heavy (CH) domain, each light chain comprises a variable light (VL) domain and a constant light (CL) domain, and the protease is fused to the N-terminus of one or both of the light chains.

[0215] In some embodiments, the antibody comprises two heavy chains and two light chains, each heavy chain comprises a variable heavy (VH) domain and a constant heavy (CH) domain, each light chain comprises a variable light (VL) domain and a constant light (CL) domain, and the protease is fused to the N-terminus of one or both of the heavy chains.

[0216] In some embodiments, the antibody comprises two heavy chains and two light chains, each heavy chain comprises a variable heavy (VH) domain and a constant heavy (CH) domain, each light chain comprises a variable light (VL) domain and a constant light (CL) domain, and the protease is fused to the N-terminus of both of the light chains and the N-terminus of both of the heavy chains.

[0217] In some embodiments, the antibody comprises two heavy chains and two light chains, each heavy chain comprises a variable heavy (VH) domain and a constant heavy (CH) domain, each light chain comprises a variable light (VL) domain and a constant light (CL) domain, and the protease is fused to the C-terminus of one of the heavy chains.

[0218] In some embodiments, the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, with the protease fused to both C-terminuses of the heavy chains.

[0219] Considerations for identifying the optimal geometric shape of a fusion protein may be evaluated by one or more parameters. See, for example, the examples of this application. In some embodiments, the geometric shape of the fusion protein is partially determined, for example, by in vitro expression and purification. In some embodiments, the geometric shape is partially determined, for example, by protein yield. In some embodiments, the geometric shape is partially determined, for example, by target (e.g., substrate) binding and / or recognition. In some embodiments, the geometric shape is partially determined, for example, by activity measured or observed in biochemical assays. In some embodiments, the geometric shape of the fusion protein is determined, for example, by the accessibility of the protease cleavage site. In some embodiments, the antibody epitope and the protease cleavage site are not the same. In some embodiments, the fusion protein is manipulated with one protease per molecule. In some embodiments, the fusion protein is manipulated with two or more (e.g., two, three, or four) proteases per molecule.

[0220] Linker In some embodiments, the antibody and protease are fused to each other via a linker.

[0221] The length, degree of flexibility, and / or other properties of the linker used in the fusion proteins described herein may have some effect on properties including, but not limited to, the affinity, specificity, or binding activity of the antibody or protease, and / or the affinity, specificity, or binding activity to one or more specific antigens or epitopes present on the target or substrate. For example, a longer linker may be chosen to ensure that the antibody and protease portions do not sterically interfere with each other. In some embodiments, the linker (e.g., a peptide linker) contains flexible residues (e.g., glycine and serine) so that adjacent portions can move freely relative to each other. For example, a glycine-serine double helix may be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an inescapable linker. In some embodiments, the linker is a cleavable linker (but not cleavable by the protease in the fusion protein).

[0222] Other linker considerations include their impact on the physical or pharmacokinetic properties of the resulting fusion protein, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (higher or lower stability and planned degradation), rigidity, flexibility, immunogenicity, regulation of antibody binding, and ability to be incorporated into micelles or liposomes.

[0223] a. Non-peptide linker Any or all of the linkers described herein can be achieved by any chemical reaction that binds two molecules, insofar as the components or fragments retain their respective activity. This binding can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complexation. In some embodiments, the binding is covalent. Covalent bonding can be achieved either by direct condensation of existing side chains or by incorporating external crosslinking molecules. Many divalent or polyvalent linkers are useful for linking protein molecules, such as Fc fragments, to the antibodies of the present invention. For example, typical coupling agents can include organic compounds such as thioesters, carbodiimides, succinimides, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This enumeration is not intended to cover all types of coupling agents known in the art, but rather to be an example of more general coupling agents (see Killen and Lindstrom, Jour.Immun.133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987), the whole of which is incorporated for all purposes by reference).

[0224] The linkers applicable in this application are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester), which is incorporated in its entirety by reference for all purposes). In some embodiments, non-peptide linkers used herein include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridinedithio)-toluene) (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamide]hexanoate) (Pierce Chem. Co., Cat. #21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propianamide]hexanoate) (Pierce Chem. Co., Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfosuccinimide) (Pierce Chem. Co., Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfosuccinimide) Chem.Co., Cat.#24510) (Conjugate to EDC).

[0225] The linkers described above contain components with different attributes and therefore result in fusion proteins with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT can contain sterically hindered disulfide bonds and form highly stable fusion proteins. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in less usable fusion protein. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. When carbodiimide coupling (such as EDC) is used in combination with sulfo-NHS, it forms esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0226] b. Peptide linker Any or all of the linkers described herein may be peptide linkers. Peptide linkers may have sequences that are naturally occurring or sequences that are not naturally occurring. For example, a sequence derived from the hinge region of a heavy-chain-only antibody may be used as a linker. See, for example, International Publication No. 1996 / 34103, which is incorporated by reference in its entirety for all purposes. In some embodiments, the peptide linker comprises the amino acid sequence of CPPCP (a sequence found in the native IgG1 hinge region).

[0227] The peptide linker may be of any suitable length. In some embodiments, the length of the peptide linker is one of the following: about 1aa to about 10aa, about 1aa to about 20aa, about 1aa to about 30aa, about 5aa to about 15aa, about 10aa to about 25aa, about 5aa to about 30aa, about 10aa to about 30aa, about 30aa to about 50aa, about 50aa to about 100aa, or about 1aa to about 100aa.

[0228] An essential technical feature of such peptide linkers is that they do not exhibit polymerization activity. Features of peptide linkers, including the absence of secondary structure promotion, are known in the art, for example, as described by Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80, the whole of which is incorporated for all purposes by reference). A particularly preferred amino acid in relation to "peptide linkers" is glycerin. Furthermore, peptide linkers that do not promote secondary structure are preferred. Linking of molecules to each other can be provided, for example, by genetic engineering. Methods for preparing fused and functionally linked antibody constructs and expressing them in mammalian cells or bacteria are well known in the art (see, for example, International Publication No. 99 / 54440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY 1989 and 1994, or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001, the whole of which is incorporated for all purposes by reference, respectively).

[0229] In some embodiments, the peptide linker is a stable linker that is not cleaved by proteases, such as matrix metalloproteinases (MMPs).

[0230] In some embodiments, the peptide linker does not tend to adopt a rigid three-dimensional structure, but rather tends to provide flexibility to the polypeptide (e.g., the first and / or second component), such as providing flexibility between the antibody and the protease. In some embodiments, the peptide linker is a flexible linker. As a flexible linker, glycine polymer (G) n , glycine-serine polymer (e.g., (GS) n (GSGGS) n (Sequence 7), (GGGGS) n (Sequence ID 2), and (GGGS) n Examples include glycine-alanine polymers (including SEQ ID NO: 8, where n is at least an integer of 1), alanine-serine polymers, and other flexible linkers known in the art. Because glycine and glycine-serine polymers are relatively unstructured, they may be able to function as neutral tethers between components. Glycine has significantly more access to the phi-pse space than alanine and is less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Those skilled in the art will recognize that the design of a fusion protein may include all or part flexible linkers, and consequently, the linker may include one or more parts that confer a more inflexible structure to provide a flexible linker portion, as well as a more inflexible structure to provide a desired fusion protein structure.

[0231] In some embodiments, the antibody and substrate are linked together by a linker of sufficient length to allow the fusion protein to fold in a manner that enables binding to the target and cleavage of the substrate. In some embodiments, the linker is (GGGGS) n (Sequence ID 2) is an array, or contains one, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0232] Natural linkers adopt various conformations in secondary structures such as helices, β-strands, coils / bends, and turns to perform their functions. Linkers in the α-helix structure function as rigid spacers to effectively separate protein domains and thus can reduce their unfavorable interactions. Non-helical linkers with Pro-rich sequences can function to increase the rigidity of the linker and reduce domain interference.

[0233] In some embodiments, the protease is directly fused to the antibody without a linker. In some embodiments, the protease is fused to the antibody via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the linker is at least about 5 amino acids. In some embodiments, the linker is at least about 10 amino acids.

[0234] In some embodiments, the linker is cleavable by a protease different from the protease contained in the fusion protein. In some embodiments, the linker is not cleavable. In some embodiments, the linker is not cleavable by the protease in the fusion protein.

[0235] In some embodiments, the linker is a proline-rich linker. In some embodiments, the linker is a GGGGA (SEQ ID NO: 3) linker or a variant thereof. In some embodiments, the linker is a (G4A)2 (SEQ ID NO: 4) linker.

[0236] The linker arrangement can be identified based on an evaluation of the optimal geometric shapes disclosed herein. In some embodiments, the linker is arranged following the hinge disulfide (e.g., the lower hinge and N-terminal region of the CH2 domain of an antibody).

[0237] The linker configuration and linker length of the fusion protein can also be altered to affect substrate capture and / or enzymatic cleavage. These parameters can be evaluated by biochemical assays, such as those disclosed herein.

[0238] B. Recombination methods and compositions Fusion proteins can be produced using recombinant methods and compositions, for example, as described in the examples. For these methods, one or more isolated nucleic acids encoding the fusion protein are provided. If two or more isolated nucleic acids are used, these nucleic acids may be on the same expression vector or different expression vectors, and typically these nucleic acids are located on two or three expression vectors, i.e., one vector may contain more than one of these nucleic acids. An example of such bispecific antibodies is CrossMab (see, e.g., Schaefer, W. et al, PNAS, 108(2011) 11187-1191). For example, one heteromonomer heavy chain contains a so-called "knob mutation" (T366W and optionally one of S354C or Y349C), while the other contains a so-called "hole mutation" (T366S, L368A, and Y407V and optionally Y349C or S354C) (see, for example, Carter, P. et al., Immunotechnol. 2 (1996) 73) (following EU index numbering).

[0239] In one embodiment, an isolated nucleic acid encoding the fusion protein described herein is provided.

[0240] In one embodiment, a method for producing a fusion protein as described herein is provided, which includes culturing host cells containing nucleic acids encoding the fusion protein provided above under conditions suitable for the expression of the fusion protein, and optionally recovering the fusion protein from the host cells (or host cell culture medium).

[0241] For recombinant production of fusion proteins, for example, the nucleic acids encoding the fusion proteins described above are isolated, inserted into one or more vectors, and further cloned and / or expressed in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of antibodies), or they can be produced by recombinant methods or obtained by chemical synthesis.

[0242] Suitable host cells for cloning or expressing vectors encoding fusion proteins include prokaryotic or eukaryotic cells as described herein. For example, fusion proteins can be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of fusion proteins and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (Also see Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the fusion protein can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0243] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable as cloning or expression hosts for vectors encoding fusion proteins, including strains of fungi and yeast in which the glycosylation pathway has been "humanized," resulting in the production of fusion proteins with a partially or completely human glycosylation pattern. See Gerngross, TU, Nat. Biotech. 22(2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24(2006) 210-215.

[0244] Host cells suitable for the expression of (glycosylated) fusion proteins can also be induced from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified, and these may be used in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0245] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0246] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspensions may be useful. Other examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed by SV40 (COS-7), human embryonic kidney cells (e.g., 293 cells or 293T cells as described in Graham et al., J Gen Virol 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), and TRI cells (e.g., Mather et al., Annals). These include MRC 5 cells and FS4 cells (described in NYAcad.Sci.383(1982)44-68). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (Urlaub, G. et al., Proc. Natl. Acad.Sci. USA 77(1980)4216-4220), including DHFR-CHO cells, and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol.248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp.255-268.

[0247] In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cells).

[0248] C. Assay The fusion proteins provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0249] 1. Antibody binding assays and other assays in fusion proteins In one embodiment, the antibody in the fusion protein is tested for its antigen-binding activity by known methods such as ELISA or Western blotting.

[0250] In another embodiment, a competitive assay may be used to identify antibodies that compete with a reference antibody for binding to a desired target. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) bound by the reference antibody. Detailed illustrative methods for mapping the epitopes to which antibodies bind are provided in Morris (1996), "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0251] In an exemplary competition assay, an immobilized target is incubated in a solution containing a first labeled antibody that binds to the target (e.g., a reference antibody) and a second unlabeled antibody being tested for its ability to compete with the first antibody for binding to the target. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized target is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to the target, excess unbound antibody is removed and the amount of labeling associated with the immobilized target is measured. If the amount of labeling associated with the immobilized target is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to the target. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0252] 2. Activity assay In one embodiment, an assay is provided for identifying an antibody or its protease that has biological activity. Biological activity may include, for example, a protease that cleaves a substrate. Proteases having such biological activity in vivo and / or in vitro are also provided.

[0253] In certain embodiments, the fusion proteins described herein are tested for such biological activity.

[0254] D. Pharmaceutical Compositions In further embodiments, pharmaceutical compositions are provided, for example, for use in any of the following therapeutic methods, comprising one of the fusion proteins provided herein. In one embodiment, the pharmaceutical composition comprises one of the fusion proteins provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises one of the fusion proteins provided herein and at least one additional therapeutic agent, for example, as described below.

[0255] Pharmaceutical compositions (formulations) of fusion proteins described herein can be prepared by combining the fusion protein with a pharmaceutically acceptable carrier or excipient known to those skilled in the art. See, for example, International Publication No. 2019 / 224842, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), Shire S., Monoclonal Antibodies: Meeting the Challenges in Manufacturing, Formulation, Delivery and Stability of Final Drug Product, 1st Ed., Woodhead Publishing (2015), §4 and Falconer RJ, Biotechnology Advances (2019), 37, 107412. Exemplary pharmaceutical compositions of fusion proteins described herein include lyophilized, aqueous, and frozen formulations.

[0256] Pharmacochemically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used and include, but are not limited to, buffers such as histidine, phosphates, citrates, acetates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-crezo Polypeptides with low molecular weight (less than approximately 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0257] The pharmaceutical compositions described herein may also include multiple active ingredients required for the specific symptom being treated, preferably having complementary activities that do not adversely affect one another. Such active ingredients are appropriately combined in amounts effective for the intended purpose.

[0258] Pharmaceutical compositions used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration using a sterile filtration membrane.

[0259] E. Treatment methods and routes of administration Any of the fusion proteins provided herein can be used in therapeutic methods.

[0260] In one embodiment, a fusion protein for use as a pharmaceutical is provided. In a further embodiment, a fusion protein for use in the treatment of a disease or pathological condition is provided. In a particular embodiment, a fusion protein for use in a therapeutic method is provided. In a particular embodiment, the present invention provides a fusion protein for use in a method of treating an individual having a disease or pathological condition (e.g., a disease or pathological condition involving a substrate), comprising administering an effective amount of the fusion protein to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents) to the individual, as described, for example below. In a further embodiment, the present invention provides a fusion protein for use, for example, to enhance the specificity / potency of a protease to a substrate and to facilitate the access of the protease to a substrate. In a particular embodiment, the present invention provides a fusion protein for use in a method of enhancing the specificity / potency of a protease to a substrate and facilitating the access of the protease to a substrate in an individual, comprising administering an effective amount of the fusion protein to the individual. The "individual" in any of the above embodiments is preferably a human being.

[0261] In a further embodiment, the present invention provides the use of a fusion protein in the manufacture or preparation of a pharmaceutical. In one embodiment, the pharmaceutical is for the treatment of a disease or pathological condition involving or caused by abnormal deposition of a substrate. In a further embodiment, the pharmaceutical is for use in a method of treating a disease, comprising administering an effective amount of the pharmaceutical to an individual having the disease. In such an embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, e.g., one described below, to the individual. In a further embodiment, the pharmaceutical is for use in a method of enhancing the specificity / potency of a protease to a substrate and / or facilitating access of the protease to a substrate in the individual, comprising administering an effective amount of the pharmaceutical to the individual. The “individual” in any of the above embodiments may be a human.

[0262] In further embodiments, the present invention provides a method for treating a disease or pathological condition (e.g., a disease or pathological condition involving or caused by abnormal deposition of a substrate). In one embodiment, the method comprises administering an effective amount of a fusion protein to an individual having such a disease or pathological condition. In such an embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, as described below.

[0263] An "individual" in any of the above-described manner may be a human being.

[0264] In further embodiments, the present invention provides a method for enhancing the specificity / potency of a protease to a substrate and / or facilitating access of the protease to a substrate in an organism. In one embodiment, the method comprises administering an effective amount of fusion protein to an organism. In one embodiment, “organism” is a human.

[0265] In further embodiments, the present invention provides a pharmaceutical composition comprising one of the fusion proteins provided herein for use, for example, in any of the therapeutic methods described above. In one embodiment, the pharmaceutical composition comprises one of the fusion proteins provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises one of the fusion proteins provided herein and at least one additional therapeutic agent, for example, described below.

[0266] The antibodies of the present invention may be administered alone or used in combination therapy. For example, combination therapy may include administering the fusion protein of the present invention and administering at least one additional therapeutic agent (e.g., additional therapeutic agents 1, 2, 3, 4, 5, or 6). In certain embodiments, combination therapy may include administering the fusion protein of the present invention and administering at least one additional therapeutic agent.

[0267] Such combination therapies described above encompass combined administration (where two or more therapeutic agents are contained in the same or separate pharmaceutical composition) and separate administration, in which case the administration of the antibody of the present invention may be performed prior to, simultaneously with, and / or subsequently to the administration of the additional therapeutic agent or drug. In one embodiment, the administration of the fusion protein and the administration of the additional therapeutic agent are performed within about one month of each other, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days. In one embodiment, the antibody and the additional therapeutic agent are administered to the patient on day one of treatment. The fusion protein of the present invention can also be used in combination with radiotherapy.

[0268] The fusion protein of the present invention (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if desired, intralesional administration in topical treatment. Parenteral administrations include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any preferred route, e.g., intravenous or subcutaneous injection, depending in part whether the administration is short-term or long-term. Various dosing schedules, including but not limited to single doses, multiple doses at various time points, bolus doses, and pulse infusions, are contemplated herein.

[0269] The fusion protein for use in the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to the healthcare professional. The fusion protein does not need to be formulated together with one or more reagents currently used to prevent or treat the disease, but may be formulated together as such. The effective amount of such other agents depends on the amount of antibody present in the pharmaceutical composition, the type of disorder or treatment, and the other factors mentioned above. These are generally used by the same dosages and routes of administration as described herein, or at about 1–99% of the dosages described herein, or by any dosage and route that is empirically / clinically deemed appropriate.

[0270] For the prevention or treatment of disease, the appropriate dosage of the fusion protein of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the fusion protein is administered for preventive or therapeutic purposes, previous treatments, the patient's medical history and response to the fusion protein, and the discretion of the attending physician. The fusion protein is administered appropriately to the patient in a single dose or over a series of treatments. In repeated administrations over several days or more, treatment is usually continued, depending on the pathological condition, until the desired suppression of disease symptoms occurs. However, other drug regimens may be useful. The progress of this treatment is readily monitored by conventional techniques and assays.

[0271] F.Manufactured products In another aspect of the present invention, a product is provided comprising a material useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders. The product comprises a container and a label or accompanying documentation on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and IV solution bags. The container may be formed from a variety of materials, such as glass or plastic. The container holds the composition to be used alone or in combination with another composition effective for treating, preventing, and / or diagnosing a pathological condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous injection needle). At least one activator in the composition is the antibody of the present invention. The label or accompanying documentation indicates that the composition is used to treat a selected pathological condition. Furthermore, the product may comprise (a) a first container containing the composition comprising the antibody of the present invention, and (b) a second container containing the composition further comprising a cytotoxic agent or other therapeutic agent. The product in this aspect of the present invention may further include accompanying documentation indicating that the composition may be used to treat a particular pathological condition. Alternatively, or in addition thereto, the product may further comprise a second (or third) container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The product may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. [table] TIFF2026509243000003.tif252170TIFF2026509243000004.tif255170TIFF2026509243000005.tif255170TIFF202 6509243000006.tif255170TIFF2026509243000007.tif226170TIFF2026509243000008.tif255170TIFF20265092430 00009.tif255170TIFF2026509243000010.tif255170TIFF2026509243000011.tif255170TIFF2026509243000012.t if255170TIFF2026509243000013.tif255170TIFF2026509243000014.tif252170TIFF2026509243000015.tif201170 [Examples]

[0272] Example 1: Expression and evaluation of antibody-guided protease activity. This example demonstrates that the antibody-enzyme fusion possesses antibody specificity and a long serum half-life, while the enzyme exhibits high substrate turnover and low dose requirements, and that antibody co-targeting of the enzyme against Aβ substrates improves Aβ inhibition.

[0273] We manipulated a series of Aβ-targeting fusion formats, investigating the geometric shapes and titers of both N-terminal and C-terminal fusions containing one or two enzymes per molecule (Figure 1A-B). The N-terminal enzyme fused to the antibody light chain (LC) or fragment crystallizable region (Fc), while the C-terminal fusion fused to the CH3 domain (HC) of the heavy chain. We constructed Fc-fusion versions lacking a target arm or IgG format targeting herpes simplex virus glycoprotein D (gD) and tested them as untargeted controls.

[0274] Aβ proteolysis is a crucial and natural elimination process to avoid pathogen accumulation, and consequently, a diverse set of proteases has been found to play a biological role in endogenous Aβ elimination. To select the optimal protease for our targeted catalytic approach, we screened a set of nine proteases previously involved in Aβ degradation, including neprolysin (NEP), neprolysin-2 (NEP2), endothelin-converting enzymes 1 and 2 (ECE1 and ECE2), angiotensin-converting enzyme (ACE), insulinase (IDE), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), and matryptase (MTSP1).

[0275] The activity of each protease was first tested with a commercially available fluorescence-generating substrate (Figure 2A). All enzymes were active against the control substrate, with the exception of ECE2, which is known to have optimal activity at low pH. The activity of each enzyme was then determined for two isoforms of Aβ, 1-40 and 1-42, using an optimized ELISA-based assay that utilized capture and detection antibodies specific to the N-terminus and C-terminus of the Aβ peptide, respectively. The efficiency of Aβ cleavage was variable, and similar levels of activity were observed between the two isoforms (Figure 2B). The most active enzymes for Aβ were IDE (EC50 approx. 0.1 nM) and NEP (EC50 approx. 12 nM), while ECE2 and ACE were completely inactive for both Aβ isoforms.

[0276] To identify the optimal geometric shape of Aβ-degrading targeted protease fusions in terms of expression and activity, several constructs with different proteases and fusion orientations against the anti-Aβ antibody crenezumab, known to bind to Aβ monomers, oligomers, and fibrils with nanomolar affinity, were constructed. After purification using Protein A resin, the NEP fusions consistently showed the highest yield compared to the other proteases tested and were selected for further purification (Figure 2C). For each NEP fusion format, the Protein A purified material was fractionated using size exclusion chromatography (SEC) (Figure 2D), and the fractions from each of the major peaks were tested for Aβ cleavage activity to identify the fraction of interest (Figure 2E).

[0277] As shown, both IgG-CTF-1 and IgG-CTF-2 exhibited dramatically enhanced potency, on average 15-fold against Aβ(1-40) and 9-fold against Aβ(1-42) compared to their respective untargeted controls (Figure 3). Importantly, all Fc-fusion controls showed activity comparable to the unfused free enzyme, demonstrating that neither N-terminal nor C-terminal fusion impairs enzymatic activity.

[0278] method:

[0279] Molecular Cloning: Gene fragments encoding all in-house derived constructs with human codon optimization were synthesized and cloned into pRK mammalian expression vectors. The pRK vectors contain a cytomegalovirus (CMV) enhancer and promoter to regulate gene expression, an N-terminal secretory signal (MGWSCIILFLVATATGVHS; SEQ ID NO: 1), a C-terminal Simianvirus 40 (SV40) polyA sequence, and an ampicillin resistance gene for bacterial selection. For Aβ protease constructs, NEP (Y52-W750), NEP2 (R74-W770), IDE (M42-L1019), or MTSP1 (G596-V855) were fused to either Fc (D221-K447, EU numbering) or full-length human IgG1 via the GGGGS (SEQ ID NO: 2) linker. For C-terminal protease fusions, the C-terminal lysine of Fc was removed. For all monovalent protease fusions, a knob-in-hole mutation was introduced into the Fc to enable heterodimerization (Merchant et al. (1998) An efficient route to human bispecific IgG. Nat Biotechnol. 16, 677-681).

[0280] Protein Expression and Purification: Protein expression was performed using a standard protocol by transfecting HEK293 cells with 30 μg of DNA per 30 ml of cell culture at a 1:1 heavy-chain:light-chain DNA ratio. Some formats required transfection with only a single DNA, while others required simultaneous transfection with separate DNAs encoding, for example, heavy and light chains or knob and hole constructs. Affinity chromatography was performed using MabSelect® SuRe® resin (Cytiva, 17543803) for Fc-containing proteins and CaptureSelect® CH1-XL resin (Thermo, 194346201L) for Fab proteins. For most antibody-enzyme fusion proteins, analytical size exclusion chromatography (SEC) revealed the presence of multiple species, possibly representing various cleavage products, mispaired antibody subunits, and aggregates. To isolate the appropriate species within each sample, fractions were tested for cleavage activity from each major peak in the SEC chromatogram. Further SEC purification using a HiLoad 16 / 600 Superdex 200 column was required to isolate the desired monomer species. Protein quality was determined by analytical SEC and SDS-PAGE using a Waters xBridge BEH200A SEC 3.5 μm (7.8 x 300 mm) column (Waters, 176003596).

[0281] Aβ protease panel: For initial Aβ cleavage activity screening, NEP (R&D, 1182-ZNC-010), NEP2 (R&D, 2340-ZN-010), ECE-1 (R&D, 1784-ZN-010), ECE-2 (R&D, 1645-ZN-010), ACE (R&D, 929-ZN-010), IDE (R&D, 2496-ZN-010), MMP2 (R&D, 902-MP-010), MMP9 (R&D, 911-MP-010), and MTSP1 (R&D, 3946-SEB-010) were commercially purchased. Activation of a 100 μg / mL solution of MMP2 or MMP9 was performed by incubation in 1 μM APMA (Sigma, A9563) for 1 hour or 24 hours, respectively. The catalytic activity of each protease was confirmed using one of three control fluorescence-generating substrates: Mca-RPPGFSAFK(Dnp)-OH (R&D, ES005), Mca-PLGL-Dpa-AR-NH2 (R&D, ES001), or Boc-QAR-AMC (R&D, ES014). For each protease in Aβ assay buffer, a 3-fold dilution series was constructed starting at 100 nM: 50 mM HEPES pH 7.4, 150 mM NaCl, and 0.05% Brij-35, a nonionic surfactant known to enhance protein solubility (Sigma, B4184). Each dilution series was incubated for 10 minutes with either 220 μM Mca-RPPGFSAFK(Dnp)-O, 60 μM Mca-PLGL-Dpa-AR-NH2, or 50 μM Boc-QAR-AMC in a black 96-well plate (Corning, 3356). Fluorescence was measured using a Molecular Devices SpectraMax® M2 microplate reader, with excitation at 320 nm and emission at 460 nm for Mca-RPPGFSAFK(Dnp)-O and Mca-PLGL-Dpa-AR-NH2, and excitation at 380 nm and emission at 460 nm for Boc-QAR-AMC.

[0282] Aβ cleavage assay: Aβ(1-40) (Anaspec, AS-24236) and Aβ(1-42) (Anaspec, AS-20276) substrates were resuspended in 1% ammonium hydroxide (Anaspec, AS-61322) to a concentration of 1 mg / mL. The solution was sonicated twice on ice for 30 seconds, divided equally, and stored at -80°C. Before each assay, Aβ was thawed on ice and a 200 nM working solution was prepared in Aβ assay buffer. A 3-fold dilution series of each protease or protease fusion was produced in Aβ assay buffer, starting at 1.8 μM. 5 μL of the protease dilution was added to 5 μL of Aβ and incubated at 37°C for 1 hour. The reaction was stopped by adding 10 μL of 20 μM 1,10-phanthroline (Sigma, 131377). Next, each sample was diluted 10-fold with 180 μL of PBST (10 mM sodium phosphate, pH 7.4, 150 mM NaCl, and 0.05% Tween® 20), and the concentration of intact Aβ was evaluated as previously described (62). Biotinylated capture antibody anti-Aβ(1-16) clone 6E10 (Biolegend, 803009) was diluted to 1 μg / mL with PBST. Detection antibodies for Aβ(1-40) (in-house developed) and Aβ(1-42) (Thermo, 700254) were fluorescently labeled using the Alexa Fluor 647 antibody labeling kit (Thermo, A20186) and diluted to 25 nM with Rexxip® F buffer (Gyros Protein Technologies, P0004825). Standard curves for Aβ(1-40) or Aβ(1-42) were constructed using a 3-fold, 12-point dilution series starting at 50 μM in PBST. All samples, including capture and detection antibodies, Aβ standards, and diluted protease reactions, were loaded into 96-well plates (Thermo, AB0800) and run on a Gyrolab® xPand system using 1000 nL CD (Gyros Protein Technologies, P0004253) according to the manufacturer's standard protocol for a three-step ELISA with the following two wash buffers: PBST and pH 11 wash buffer (Gyros Protein Technologies, P0020096).Aβ concentrations were measured by fitting a standard curve using Gyros software.

[0283] Example 2: Evaluation of the catalytic activity of antibody-enzyme fusions. This example investigated the effect of antigen affinity on anti-Aβ antibody-enzyme activity and demonstrated that when fused to the enzyme, the antibody facilitates the rapid recycling of the target antigen for cleavage by the fusion protease.

[0284] Method: The cutting assay was performed as described in Example 1.

[0285] Aβ surface plasmon resonance: The solution affinity constant of anti-Aβ antibodies was evaluated using Biacore™ T200. Anti-Aβ fusions were diluted to 1 μg / mL with HBS-P+ (Cytiva, BR100671) and captured using a Series S Protein A tip (Cytiva, 29127555). A 3-fold 8-point dilution series of Aβ(1-28) (New England Peptide, 22360) was constructed in HBS-P+, injected for 5 minutes, and then dissociated for 5 minutes. Affinity constants were obtained by kinetic fitting using Biacore evaluation software.

[0286] result:

[0287] Crenezumab (Panel A in Figure 4), an anti-Aβ antibody with both fast on-rate and off-rate binding, was hypothesized to facilitate rapid recycling of the target antigen for cleavage by the fusion protease. To investigate the dependence of targeted catalysis on antibody binding kinetics and affinity, we also constructed bivalent NEP IgG-CTF-2 formats (G33S(HC) and G33S(HC) / S56F(LC)) with two variants of crenezumab having a slower off-rate and therefore stronger affinity. We also constructed a fusion of solanezumab, another anti-Aβ antibody that shares high sequence identity with crenezumab (93% identity in VH and 92% identity in VL) and binds to a similar epitope but has higher affinity. Finally, as an unbound control, we produced a fusion containing a variable region targeting an unrelated antigen (gD). The nucleotide sequences of the constructs are disclosed in Table 1. [Table 1]

[0288] This resulted in a panel of five constructs of IgG-CTF-2 with varying off-rates and affinities but similar on-rates (Panel A in Figure 4). Overall, higher Aβ cleavage activity correlated with faster off-rates across affinity variants, with the crenezumab-NEP fusion (fastest off-rate) having a 3-fold lower EC50 than the solanezumab-NEP fusion (slowest off-rate) (Panels B and C in Figure 4). These data suggest that a fast binding kinetics may be optimal, allowing the antibody to circulate through Aβ binding events for more effective enzyme turnover. Overall, the results suggest that faster off-rates and weaker Aβ affinity correlate with the catalytic efficacy of enzyme fusions.

[0289] Example 3: Protease manipulation for immunoglobulin G cleavage. We investigated the applicability of an antibody-guided protease platform for targeting and turning over high-abundance serum IgG. Therapeutic IgG suppression has been studied for a wide range of autoimmune and inflammatory diseases with clinical success. Due to the very high concentration of IgG in human serum (approximately 10 mg / ml), catalytic turnover is essential. The IgG-degrading enzyme IdeS is effective in depleting IgG in vitro and in vivo and has been clinically tested. However, due to its bacterial origin, IdeS is highly immunogenic, hindering repeated dosing, and consequently, its therapeutic application is limited to acute therapeutic indications such as kidney transplantation. While several human enzymes with a lower immunogenicity risk have been shown to cleave IgG (Figure 5A), they are not IgG-selective and lack the efficiency necessary to remove the high levels of substrate present in serum. Targeted catalysis was investigated as a means to enhance the cleavage efficiency and selectivity of human proteases for degrading serum IgG.

[0290] method:

[0291] Molecular Cloning: Gene fragments encoding all in-house derived constructs with human codon optimization were synthesized and cloned as described in Act 1. For IgG protease constructs, MMP3 (Y18-C477) was fused via the (GGGGA)2 (SEQ ID NO: 4) linker to the N-terminus of either Fc (D221-K447, EU numbering), full-length human IgG1 heavy chain, Fab heavy chain (Q1-S113, Kabat numbering), or full-length human kappa light chain. Only the MMP3 gene (Y18-C477) was synthesized with a C-terminal FLAG tag for purification, and enterokinase (DDDDK; SEQ ID NO: 5) and factor Xa (IEGR; SEQ ID NO: 6) protease cleavage sites were inserted by site-directed mutagenesis using a standard protocol (Qiagen®, 210513). Genes introducing Fc mutations for RF61 binding loss (Figure 6C) and alternative hinge sequences for MMP3 resistance (Figure 6D) were synthesized within the human IgG1 framework (Wuxi).

[0292] Protein expression and purification were carried out as described in Example 1.

[0293] result:

[0294] Three human proteases (matrix metalloproteinase 3 (MMP3), MMP7, and cathepsin G) were selected and screened for expression based on their human origin and IgG cleavage characteristics. MMP3 was chosen for further development. MMP3 is a zinc matrix metalloproteinase expressed by a wide variety of cell types, exhibiting indiscriminate activity towards the matrix and bioactive substrates. Its structure consists of an N-terminal signaling sequence linked by a proline-rich linker, a prodomain, a catalytic domain, and a hemopexin domain (Figure 5B). Successful expression of MMP3 requires fusion to an inhibitory N-terminal prodomain, which is then proteolytically removed to induce activity. While various approaches, including heat, addition of organic mercury reagents, or partial proteolysis, have been reported to induce activation in vitro, these strategies also posed significant risks to the structure and stability of potential therapeutic agents.

[0295] To avoid the problematic activation step (removal of the inhibitory N-terminal prodomain), we designed MMP3 variants capable of prodomain cleavage by the highly specific proteases enterokinase (EK) and factor Xa (Xa). To determine the optimal cleavage site for achieving complete and selective MMP3 activity, we substituted the recognition sequence for EK or Xa within the prodomain of MMP3 at four unstructured positions to allow maximum EK or Xa protease accessibility and to minimize structural perturbation of the prodomain (Figure 5B). SDS-PAGE analysis and MMP3 activity assays were performed on eight variants with and without the addition of EK or Xa. The variant with an EK site insertion at position 4 showed the best combination of low catalytic activity and high stability in the native state (with prodomain) while resulting in efficient removal of the prodomain in the presence of EK (Figures 5C and 5D). Therefore, we selected the original variant, called MMP3-D4K-4, in which an EK site was inserted at position 4, for further study. At high concentrations, and after EK cleavage of the prodomain, MMP3-D4K-4 cleaved the IgG hinge (Figure 5E).

[0296] Example 4: Evaluation of the catalytic activity of an anti-IgG antibody-enzyme fusion. In this example, an antibody that weakly recognizes IgG was affinity-matured and then fused with a protease. The antibody-enzyme fusion was then tested for its ability to cleave targeted IgG.

[0297] Example 4A: Manipulation of RF61 to increase binding affinity to IgG. Manipulation of non-autoselective anti-IgG antibodies for endogenous IgG targeting. To target MMP3 to IgG, the use of rheumatoid factor (RF), a naturally occurring human autoantibody that binds to IgG, was investigated. A well-characterized RF called RF61, initially isolated from a rheumatoid arthritis patient and binding to the antibody Fc region, was used (Harindranath, N. et al. (1991) Complete sequence of the genes encoding the VH and VL regions of low-and high-affinity monoclonal lgM and lgA1 rheumatoid factors produced by CD5+B cells from a rheumatoid arthritis patient. Int Immunol. 3, 865-875). Importantly, the crystal structure of RF61 in complex with Fc has been elucidated (Duquerroy et al. (2007) Crystal Structure of a Human Autoimmune Complex between IgM Rheumatoid Factor RF61 and IgG1 Fc Reveals a Novel Epitope and Evidence for Affinity Maturation. J Mol Biol. 368, 1321-1331), suggesting the possibility of non-self-selective manipulation (see below).

[0298] method:

[0299] RF61 Affinity Maturation: Affinity maturation of RF61 was performed using a two-step PCR protocol with PrimeSTAR® Max DNA polymerase (Takara, R045B) according to a standard protocol. Each residue in the heavy chain and light chain CDR was mutated to one of 18 other possible residues (excluding cysteine), generating 18 single-point mutants per CDR residue. For rounds 1 and 2, the heavy chain template DNA contained only the VH and CH1 domains to produce recombinant Fab protein. Fab protein variants were expressed in HEK293 cells by simultaneous transfection of heavy chain and light chain DNA on a 1 mL scale and purified with CaptureSelect® CH1-XL resin for affinity screening. In round 3, since RF61 does not bind to mouse IgG2a, the heavy chain and light chain variable domains of RF61 were fused to the mouse IgG2a constant domain to create chimeric full-length antibodies that do not bind to their own Fc domains. Protein expression was performed as described above, followed by purification using MabSelect® SuRe® resin.

[0300] RF61 surface plasmon resonance: RF61 affinity to human IgG was evaluated using Biacore 8K+ or T200. For the first and second rounds of affinity maturation, the Fc domain of human IgG1 was captured on Series S Protein A chips according to the manufacturer's protocol. Serial dilutions of RF61 Fab variants were prepared in HBS-P+ buffer. The dilutions were passed over the chip for 4 minutes, followed by a 5-minute dissociation step. The variants were evaluated using response units at the late analyte binding point normalized to the Fc capture level. For the third round of affinity maturation, RF61 chimeric antibody variants were captured on Series S CM5 chips (Cytiva, 29104988) containing immobilized anti-mouse antibodies from a mouse antibody capture kit (Cytiva, 29215281). Serial dilutions of human IgG1 Fc in HBS-P+ were passed over the chip for 10 minutes, followed by a 6-minute dissociation step. The affinity constant was obtained by kinetic fitting using Biacore evaluation software (GE). To evaluate Fc mutations for RF61 binding loss, the third round of affinity maturation was performed (R50D LC; R50N LC, L95N D99H T100aA HC; R50N LC, S62P L95N D99H T100aA D100cE M100eF HC; R50N Y34F LC, S62P D99H D100cE M100eF HC; R50D LC, S62P L95N T100aA D100cE M100eF HC; R50D Y34F LC, D100cE HC; R50D Y34F LC, S62P D99H M100eF HC; R50D Y34F LC, S62P L95N D100cE M100eF Eight RF61 chimeric antibody variants, ranging from a range of binding intensities from HC, were selected and captured as described above. Each debinding variant at a single 1000 nM concentration was passed through the chip for 10 minutes, followed by a 6-minute dissociation step. Binding was quantified by normalizing the late analyte binding signal (RU) against the antibody capture level, and the signals from the eight RF61 variants are shown in Figure 6C.To characterize the affinity of the antibody-MMP3 fusion construct, IgG1 subtype 4D5 of the anti-HER2 antibody was captured on Series S Protein L chips (Cytiva, 29205138) according to the manufacturer's protocol. Serial dilutions of the RF61-MMP3 fusion construct were prepared in HBS-P+ buffer. The dilutions were flowed on the chip for 3 minutes, followed by dissociation for 8 minutes. Since RF61 contains a lambda light chain, it does not bind to the Protein L chip. The affinity constants were determined as described above.

[0301] result:

[0302] RF61 was initially identified as IgM, which weakly binds to IgG1Fc with a KD of approximately 600 nM (including binding activity). The crystal structure shows a stoichiometry of two RF61 Fabs per Fc, with each Fab in contact with residues from both CH3 domains (Figure 6A). To improve the affinity of RF61 to IgG Fc, saturation mutagenesis (excluding cysteine) was performed at each residue in the complementarity-determining regions (CDRs) of both the heavy chain (HC, 38 residues) and the light chain (LC, 31 residues), yielding a total of 1242 single RF61 point mutants. Although we were unable to detect binding of wild-type RF61 Fabs to Fc in a monovalent surface plasmon resonance (SPR) binding format, screening of mutants using the same technique identified four promising mutants (R50N, R50D, Y34F, and Y34N) all located in the LC and exhibiting significantly increased affinity. A second round of screening was performed, pairing R50N mutant LCs with the same HC single-point mutant library (a total of 684 variants). SPR screening revealed six HC mutations that showed improved binding when combined with R50 NLC (S62P, L95N, D99H, T100aA, D100cE, and M100eF). For the third and final rounds, 320 variants were produced with selected LC and HC mutation combinations, ranging from 1 to 8 mutations per variant. Our third round of saturated mutagenesis screening yielded RF61 variants spanning 2 log with affinities >1 μM to approximately 10 nM (Figure 6B).

[0303] Further manipulation of the RF61 IgG format was necessary for effective endogenous IgG target antibodies. First, the Fc needed to be modified to avoid self-recognition. Using the crystal structure of RF61 bound to IgG1 Fc as a guide, Fc variants containing 1 to 4 mutations were designed to eliminate RF61 binding. The Fc variants were screened against a panel of eight affinity-improving RF61 variants using SPR (Figure 6C). The single mutant R355E showed sophisticated loss of RF61 binding, with minimal improvement from additional mutations, and was therefore selected. The second requirement was that the hinge had to be resistant to proteolysis by MMP3 to avoid self-cleavage by fusion enzymes. Resistance to MMP3 cleavage was conferred by replacing the 10 residues following the hinge disulfide, effectively the lower hinge and N-terminal region of the CH2 domain, with the (G4A)2 linker (Figure 6D). In summary, the IgG-targeted antibody contains a Fab arm with improved RF61 affinity, an Fc with an R355E mutation to avoid self-binding, and a mutated lower hinge to avoid self-cleavage by MMP3.

[0304] Example 4B: Fusion Format and Activity method:

[0305] MMP3 activity assay: All MMP3-D4K-4 and MMP3-D4K-4 fusion protein samples were replaced with cleavage buffer (10 mM HEPES, 150 mM NaCl, and 10 mM CaCl2 (pH 7.5)). MMP3-D4K-4 was activated with 16 units of enterokinase (NEB, P8070L) per 25 μg of protein by incubation at room temperature for 16 hours. To inactivate the enterokinase, 0.1 mg / ml of soy trypsin inhibitor (Sigma, 17075029) was added to the protein solution. 50 μL of 2.5 μM fluorescence-generating MMP3 peptide substrate (R&D Systems, ES002) or 50 μg / ml of DQ-collagen-IV (Invitrogen, D12052) in cleavage buffer was combined with the desired concentration of activated MMP3-D4K-4 sample in a 96-well black flat-bottom plate (Corning, CLS3925). Fluorescence signals were measured for the peptide substrate and DQ-collagen-IV using a Molecular Devices SpectraMax® M2 microplate reader (Molecular Devices) with excitation / emission at 320 / 405 nm and 485 / 535 nm, respectively. Concentration-dependent assays were performed in double-strand configurations using 2.5-fold dilutions starting from 400 nM.

[0306] IgG cleavage assay: The MMP3-D4K-4 fusion protein was activated with enterokinase as described above. Seven 2.5-fold dilutions of the activated fusion protein were prepared in cleavage buffer, starting at 800 nM. 4 μL of each dilution was mixed with 4 μL of antibody substrate (800 nM 1-arm anti-gD IgG1 antibody). The cleavage reaction was incubated at 37°C for 24 hours. The degree of antibody cleavage was evaluated by ELISA as follows: 100 μL of 1.2 μg / mL Affinipure goat anti-human Fc antibody (Jackson ImmunoResearch, 109-005-098) was added to each well of a Maxisorp® 96-well plate (Thermo, 44-2404-21). The plate was incubated at room temperature for 1 hour and then washed three times with PBST. The wells were blocked with SuperBlock® buffer (Thermo, 37515) at room temperature for 1 hour, and then washed three times with PBST. Each cleavage reaction product was diluted 400-fold in 100 ng / mL of 1-arm anti-gD substrate in PBST, and then 100 μL of the dilution was added to the block wells. The plate was incubated at room temperature for 1 hour, and then washed five times with PBST. 100 μL of a 1:40,000-fold dilution of goat anti-human Fab HRP conjugate antibody (Sigma, A0293) was added to each well and incubated at room temperature for 1 hour. The wells were washed five times with PBST. 100 μL of TMB substrate (Thermo, N301) was added to each well, and the reaction was allowed to proceed at room temperature for 15 minutes, followed by quenching with stop solution (Thermo, N600). Absorbance was measured at 405 nm.

[0307] IgG and fluorescence-generating peptide substrate cleavage assay in human serum: The MMP3-D4K-4 Fab fusion protein was activated with enterokinase as described above. To observe the effect of endogenous protease inhibitors in serum, soy trypsin inhibitors were not added to the fusion protein. Eight 2-fold dilutions of the fusion protein, starting at 600 nM, were prepared in the cleavage buffer described above. Serum was prepared from a single human donor blood sample by centrifugation and supernatant collection. Before use, serum was diluted 1:1 with cleavage buffer. 25 μL of serum was combined with 25 μL of antibody dilution to obtain a peak fusion concentration of 300 nM. The cleavage reaction was incubated at 37°C for 24 hours. IgG1 cleavage was detected by the ELISA assay described above with two important modifications. First, the antibody used to coat the Maxisorp 96-well plate was 1.0 μg / mL mouse anti-human IgG1 antibody in PBS (Thermo, MH1015). This modification ensured that the ELISA would detect only IgG1 cleavage, rather than all subtypes of IgG in serum. Secondly, the cleavage reaction was diluted 100-fold with PBST before being added to the Maxisorp plate. The optimal dilution factor was determined by performing the ELISA assay using a serum dilution series. The remaining assays were performed as described above. For the fluorescence-generating peptide substrate cleavage assay, 25 μL of the serum cleavage reaction was combined with 25 μL of 5 μM fluorescence-generating MMP3 peptide substrate (R&D Systems, ES002) in cleavage buffer, and the samples were added to the wells of a 384-well black flat-bottom transparent plate (Thermo, 242764). The final peak protease fusion concentration for the peptide cleavage assay was 150 nM, which was obtained from seven further 2-fold dilutions. After sealing the plates and incubating at room temperature for 1 hour, the fluorescence signals were read using a Perkin-Elmer EnVision® plate reader.

[0308] result:

[0309] The two manipulated modalities described above were combined into a single targeted protease. Using a similar approach to Aβ, various antibody-enzyme fusion formats were screened for expression, stability, affinity, and activity. Since C-terminal fusion of MMP3 to antibodies results in proteolysis and separation of the targeted modality during prodomain cleavage necessary to activate MMP3, we focused on N-terminal fusion. Sufficient purified material was obtained by fusing MMP3 and its prodomain to the N-terminus of antibody HC, LC, or Fc. Activity screening against both fluorescence-generating peptide substrates and IgG substrates revealed the most promising IgG format as MMP3 fusion to the LC N-terminus via a (G4A)2 linker, called IgG-NTF(LC)-2 (Figures 7A and 10). Both IgG and Fab formats of the MMP3 LC N-terminal fusion were scaled up and purified for further study (data not shown).

[0310] To determine whether RF61-mediated IgG targeting of MMP3 can enhance its activity, we designed three antibody-MMP3 fusion proteins with varying affinities to endogenous human IgG (protease fusions to the N-terminus of the light chain, listed in Table 2; Figure 7A, bottom panel). [Table 2]

[0311] Although the binding of wild-type RF61 to Fc was not a detectable SPR, it was still included, based on previous reports that it is a weak binder to IgG. Two RF61 mutants from the saturated mutagenesis screening described above were also included: RF61-D (104 nM) and RF61-DEF (32 nM) (Figure 7B). A gD control format was also produced as a true unbound control. Each affinity variant and control was produced using MMP3 fused to both Fab and full-length IgG LCs. The monovalent affinity of each targeting arm is summarized in Figure 7B. First, non-target activity was measured against two non-IgG substrates: a fluorescence-generating peptide substrate and DQ collagen IV representing an endogenous off-target substrate (Figure 7C). As expected, the activity curves of each member of the affinity series overlap well, indicating the non-selectivity of MMP3 against non-IgG substrates. To determine the effect of IgG targeting, Fab fusion proteins and IgG fusion proteins were incubated with human IgG1 substrates at various concentrations, and IgG cleavage was determined by ELISA-based assays (Figures 7C–7F). EC50 values ​​correlated remarkably well with IgG affinity of the targeted arm, with the Fab fusion format distinguishing two higher affinity RF61 variants, while the IgG fusion format did not (Figure 7C). The untargeted anti-gD construct showed little to no cleavage at the maximum test concentration. A quantitative measure of enhancement over the untargeted construct could not be determined due to the lack of IgG cleavage from the anti-gD control; however, the results suggest that antibody targeting provides at least a 1-log enhancement of protease activity.

[0312] Interestingly, while the EC50 values ​​were consistent with the Fab and IgG fusion formats of the RF61-DEF affinity variant (Figure 7C), the RF 61-DIgG fusion had a three-fold lower EC50 than the Fab fusion. These data suggest that binding activity in IgG fusion may contribute to enhanced activity. Furthermore, the lack of enhanced activity upon increased affinity for the two most closely bound IgG fusion formats indicates that the maximum beneficial effect of IgG targeting was achieved, with MMP3-D4K-4 protease activity being a limiting factor.

[0313] To evaluate the activity of RF61-mediated IgG targeting by MMP3 in a more biologically relevant environment, we measured cleavage of IgG1 in human serum from both targeted (RF61-DEF) and untargeted (anti-gD) Fab fusion formats (Panel A, Figure 8). The overall activity (EC50) of the targeted protease was reduced compared to the results for IgG1 in vitro, but a clear enhancement of activity still exists compared to the untargeted control. The reduction in activity may be attributable to various factors, including the presence of endogenous protease inhibitors as well as higher levels of additional MMP3 substrates (e.g., many matrix and bioactive substrates including other immunoglobulin subtypes) and / or RF61 antigens (e.g., IgG2 and IgG3) in human serum compared to IgG1-containing buffer. The cleavage activity against fluorescent MMP3 peptide substrates suggests that endogenous protease inhibitors in human serum do not affect the activity of these fusion constructs (Panel B, Figure 8). The promising results described herein suggest that an accessible therapeutic window for in vivo IgG degradation may exist.

[0314] IgG1 accounts for approximately two-thirds of all human IgG, but three other human IgG subclasses are present in serum: IgG2, IgG3, and IgG4. To evaluate the subtype dependence of our targeted protease, we measured the ability of targeted (RF61-DEF) and untargeted (anti-gD) antibody-MMP3 fusions to cleave all four human IgG subtypes (Figure 9). IgG1 and IgG3 substrates showed similarly enhanced target-dependent cleavage, with the RF61-DEF fusion completely cleaving IgG into Fc and F(ab')2 fragments. In contrast, the anti-gD control fusion was only capable of single hinge cleavage events for the same two subtypes. No cleavage of IgG2 was observed, which is consistent with the lack of an MMP3 cleavage site in its hinge sequence, as previously reported. No difference between targeted and untargeted cleavage was observed for the IgG4 substrate, which was expected based on the lack of RF61 binding to IgG4. (Data not shown.) Overall, the results are consistent with the enhanced selectivity provided by targeted catalysis.

Claims

1. A fusion protein comprising an antibody that binds to a target, wherein the antibody is fused to a protease that cleaves a substrate, and the target is in close proximity to the substrate.

2. The fusion protein according to claim 1, wherein the protease is fused to one or more polypeptide chains of the antibody.

3. The fusion protein according to claim 2, wherein the protease is fused to the N-terminus and / or C-terminus of one or more polypeptide chains of the antibody.

4. The aforementioned antibody a) A heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the protease is fused to the N-terminus or C-terminus of VH or VL; or b) A full-length antibody comprising two heavy chains and two light chains, wherein the protease is fused to the N-terminus or C-terminus of one or both of the two heavy chains and / or the two light chains. A fusion protein according to claim 1, comprising:

5. The fusion protein according to claim 1 or 2, wherein the antibody comprises a) a first polypeptide comprising a heavy chain variable (VH) domain and a first heavy chain constant (CH1) domain, and b) a second polypeptide comprising a light chain variable (VL) domain and a light chain constant (CL) domain, wherein the protease is fused to the N-terminus of the VL domain, the VH domain and the VL domain form a binding domain to the target, and a disulfide bond is formed between the CH1 domain and the CL domain.

6. The fusion protein according to claim 5, wherein the antibody does not contain an Fc fragment.

7. The fusion protein according to claim 1 or 2, wherein the antibody comprises a) a first polypeptide comprising a variable light chain (VL) domain and a constant light chain (CL) domain, b) a second polypeptide comprising a variable heavy chain (VH) domain and a first constant heavy chain (CH1) domain, and c) a third polypeptide comprising an Fc region, wherein the protease is fused to the N-terminus of the third polypeptide, and the VH domain and the VL domain form a binding domain to the target.

8. The fusion protein according to claim 1 or 2, wherein the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, and the protease is fused to the N-terminuses of the two light chains.

9. The fusion protein according to claim 1 or 2, wherein the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, and the protease is fused to the N-terminuses of the two heavy chains.

10. The fusion protein according to claim 1 or 2, wherein the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, and the protease is fused to the N-terminuses of the two light chains and the N-terminuses of the two heavy chains.

11. The fusion protein according to claim 1 or 2, wherein the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, and the protease is fused to one C-terminus of the heavy chain.

12. The fusion protein according to claim 1 or 2, wherein the antibody comprises two heavy chains and two light chains, each heavy chain comprising a variable heavy chain (VH) domain and a constant heavy chain (CH) domain, and each light chain comprising a variable light chain (VL) domain and a constant light chain (CL) domain, and the protease is fused to the C-terminus of both heavy chains.

13. The fusion protein according to any one of claims 1 to 12, wherein the protease is fused to the antibody via a linker, and optionally the linker includes SEQ ID NO: 2 or SEQ ID NO:

3.

14. The fusion protein according to claim 13, wherein the linker is a peptide linker, and optionally the linker has a length of at least about 5, 10, 15, 20, 25, or 30 amino acids.

15. The fusion protein according to claim 13 or 14, wherein the linker can be cleaved by a protease different from the protease contained in the fusion protein.

16. The fusion protein according to claim 13 or claim 14, wherein the linker is not cleavable.

17. The dissociation rate constant (k) of the antibody against the aforementioned target. ｏｆｆ ) is approximately 5 x 10 －３ , 1 x 10 －３ , 5 x 10 －４ , or 1 x 10 －４ The fusion protein according to any one of claims 1 to 16, which is as follows:

18. The equilibrium dissociation constant (k) of the antibody against the target. Ｄ The fusion protein according to any one of claims 1 to 17, wherein the ) has a range of about 0.1 nM to about 1000 nM.

19. The fusion protein according to any one of claims 1 to 18, wherein the target and the substrate are expressed in the central nervous system (CNS).

20. The fusion protein according to any one of claims 1 to 19, wherein the target and the substrate are the same molecule.

21. The fusion protein according to any one of claims 1 to 20, wherein the protease is a metalloprotease.

22. The fusion protein according to claim 20 or claim 21, wherein the target is amyloid-beta (Aβ).

23. The antibody and / or the protease is Aβ １－４０ and Aβ １－４２ The fusion protein according to claim 22, which targets both of the following.

24. The fusion protein according to claim 22 or 23, wherein the antibody is crenezumab or a variant thereof, or solanezumab or a variant thereof, and optionally, the variant of crenezumab contains G33S in its heavy chain, and further optionally, the variant of crenezumab contains S56F in its light chain.

25. The fusion protein according to any one of claims 22 to 24, wherein the protease is selected from the group consisting of neprolysin (NEP), neprolysin-2 (NEP2), endothelin-converting enzyme 1 and 2 (ECE1 and ECE2), angiotensin-converting enzyme (ACE), insulin-degrading enzyme (IDE), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), and matryptase (MTSP1).

26. The fusion protein according to claim 25, wherein the protease is neprolysin.

27. The fusion protein according to claim 26, wherein the antibody comprises two heavy chains and two light chains, and the protease is fused to the C-terminus of one or both heavy chains of the antibody.

28. The fusion protein according to claim 20 or claim 21, wherein the target is IgG.

29. The fusion protein according to claim 28, wherein the protease is selected from the group consisting of matrix metalloproteinase 3 (MMP3), MMP7, cathepsin G, or a variant thereof.

30. The fusion protein according to claim 29, wherein the protease is MMP3 or a variant thereof.

31. The fusion protein according to any one of claims 28 to 30, wherein the antibody comprises a full-length antibody.

32. The fusion protein according to claim 31, wherein the antibody contains an R335E mutation in the Fc fragment.

33. The full-length antibody includes a hinge region that is resistant to the protease, and optionally, the hinge region has a (G ４ A) ２ The fusion protein according to claim 31 or 32, comprising the sequence.

34. The fusion protein according to any one of claims 28 to 30, wherein the antibody comprises a Fab fragment.

35. The fusion protein according to any one of claims 28 to 34, wherein the antibody comprises rheumatoid factor or a variant thereof.

36. An isolated nucleic acid encoding a fusion protein or a fragment thereof according to any one of claims 1 to 35.

37. A host cell comprising the nucleic acid described in claim 36.

38. A method for producing a fusion protein or fragment according to any one of claims 1 to 35, comprising culturing the host cell according to claim 37 under conditions suitable for the expression of the fusion protein or fragment thereof.

39. The method according to claim 38, further comprising recovering the fusion protein or a fragment thereof from the host cell.

40. A fusion protein produced by the method described in claim 39.

41. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 35 and a pharmaceutically acceptable carrier.

42. A fusion protein according to any one of claims 1 to 35 or a pharmaceutical composition according to claim 41, for use as a pharmaceutical.

43. A fusion protein according to any one of claims 1 to 35 or a pharmaceutical composition according to claim 41, for use in treating a disease or pathological condition.

44. Use of a fusion protein according to any one of claims 1 to 35 or a pharmaceutical composition according to claim 41 in the manufacture of a pharmaceutical for treating a disease or pathological condition.

45. A method for treating an individual having a disease or pathological condition, comprising administering to the individual an effective amount of a fusion protein according to any one of claims 1 to 35 or a pharmaceutical composition according to claim 41.

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