Antibodies against liraglutide and their uses

Abstract

The present invention relates to specific antibodies and their uses, for example for the identification and / or quantification of liraglutide fibrils and / or semaglutide fibrils.

Description

[0001] The present invention relates to antibodies specific for fibrils of liraglutide or semaglutide and uses of such antibodies.

[0002] Sequence Listing

[0003] This application contains a Sequence Listing which has been submitted electronically in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on Mar. 30, 2020, is named 190042WO01Sequence Listing_ST25.txt and is 111 kilobytes in size.

[0004] Background

[0005] It is known that human GLP-1(7-37) and its analogs are prone to forming various types of aggregates in solution. A particular type of such aggregates, referred to herein as fibrils, are thought to form irreversibly and should be kept to a minimum in pharmaceutical products administered to patients in liquid form. To date, the preferred method for assaying (i.e., identifying and / or quantifying) such fibrils is based on thioflavin T (ThT), a fluorophore that changes its emission spectrum when binding to fibrils, see, e.g., Assay (V) herein. Assays for detecting peptide fibrils by ThT typically involve first applying pressure to the sample to amplify the amount of fibrils to allow detection, and such pressure application is undesirable and time-consuming. There is a need for means to identify such peptide fibrils with higher sensitivity and in mixtures containing the soluble form of the peptide. SUMMARY OF THE INVENTION

[0006] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the fibrils are prepared according to Assay (I) herein. In some embodiments, the present invention relates to an antibody that binds to semaglutide fibrils, wherein the fibrils are prepared according to Assay (II) herein. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the fibrils are optionally prepared according to Assay (I) herein, and the binding level of the antibody to liraglutide fibrils is at least 10-fold the binding level of the antibody to soluble liraglutide, wherein the binding level is determined at a liraglutide fibril concentration of at least 25 μM according to Assay (III). In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the fibrils are optionally prepared according to Assay (I) herein, and the detection limit of the antibody for liraglutide fibrils is at least 10-fold lower in concentration than the detection limit of liraglutide fibrils in the ThT assay, wherein the detection limit is determined at a liraglutide fibril concentration of at least 1 μM according to Assay (VI) herein. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the fibrils are optionally prepared according to Assay (I) herein, and the binding level of the antibody to liraglutide fibrils is at least 5-fold the binding level of the antibody to soluble liraglutide, wherein the antibody has a purity higher than 95% monomer, and wherein the binding level is determined at a liraglutide fibril concentration of at least 30 μM according to Assay (III-B) herein. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the fibrils are optionally prepared according to Assay (I) herein, and the detection limit of the antibody for liraglutide fibrils is at least 10-fold lower in concentration than the detection limit of liraglutide fibrils in the ThT assay, wherein the antibody has a purity higher than 95% monomer, and wherein the detection limit is determined at a liraglutide fibril concentration of at least 0.025 μM according to Assay (VI-B) herein.

[0007] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from SEQ ID NO: 115 and 121, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 37, 38, and 39; SEQ ID NO: 43, 44, and 45; SEQ ID NO: 49, 50, and 51; SEQ ID NO: 55, 56, and 57; SEQ ID NO: 61, 62, and 63; SEQ ID NO: 67, 68, and 69; SEQ ID NO: 73, 74, and 75; SEQ ID NO: 79, 80, and 81; SEQ ID NO: 85, 86, and 87; SEQ ID NO: 91, 92, and 93; SEQ ID NO: 97, 98, and 99; SEQ ID NO: 103, 104, and 105; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 115, 116, and 117; SEQ ID NO: 121, 122, and 123; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO:40, 41, and 42; SEQ ID NO:46, 47, and 48; SEQ ID NO:52, 53, and 54; SEQ ID NO:58, 59, and 60; SEQ ID NO:64, 65, and 66; SEQ ID NO:70, 71, and 72; SEQ ID NO:76, 77, and 78; SEQ ID NO:82, 83, and 84; SEQ ID NO:88, 89, and 90; SEQ ID NO:94, 95, and 96; SEQ ID NO:100, 101, and 102; SEQ ID NO:106, 107, and 108; or any of the foregoing sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO:118, 119, and 120; SEQ ID NO:124, 125, and 126; or any of the foregoing sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0008] In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the antibody comprises a heavy chain variable region as defined in any of the foregoing embodiments and a light chain variable region as defined herein. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the antibody comprises a sequence selected from the group consisting of: SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; or any of the foregoing sequences having at most 20, such as at most 15, or at most 10 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the antibody comprises a sequence selected from the group consisting of: SEQ ID NO:109 and 110; or any of the foregoing sequences having at most 20, such as at most 15, or at most 10 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the antibody has at least 80%, such as at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the antibody has at least 80%, such as at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO:109 and 110.

[0009] In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the antibody comprises a variable light chain (VL) sequence selected from the group consisting of: SEQ ID NO:13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 111, and 113; or any of said sequences having at most 20, such as at most 15 or at most 10 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the antibody comprises a variable heavy chain (VH) sequence selected from the group consisting of: SEQ ID NO:14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 112, and 114; or any of said sequences having at most 20, such as at most 15 or at most 10 amino acid substitutions, deletions, or insertions.

[0010] In some embodiments, the present invention relates to the use of the antibodies as defined herein for the identification of liraglutide fibrils or semaglutide fibrils. In some embodiments, the present invention relates to a method for identifying liraglutide fibrils or semaglutide fibrils, the method comprising the steps of: a) contacting an antibody as defined herein with liraglutide fibrils or semaglutide fibrils. In some embodiments, the present invention relates to a method for quantifying liraglutide fibrils or semaglutide fibrils, the method comprising the steps of: a) contacting an antibody as defined herein with liraglutide fibrils or semaglutide fibrils.

[0011] In some embodiments, the present invention relates to the use of the antibodies as defined herein for the purification of liraglutide or semaglutide by removing or reducing their fibrils, the removal or reduction of the fibrils being effected by immobilizing the antibody on a solid surface, e.g., a chromatographic or membrane surface creating an affinity surface, and exposing a mixture comprising the fibrils and the soluble form of liraglutide or semaglutide to the surface, thereby resulting in the separation of the fibrils or parts thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shows the analytical size exclusion chromatography analysis of selected antibody variants E, M, and N. DETAILED DESCRIPTION

[0013] The present invention relates to antibodies that specifically bind to fibrils of the GLP-1 receptor agonists liraglutide or semaglutide. Liraglutide and semaglutide are analogs of human GLP-1(7-37) and are therapeutic peptides commercially available in solution form. Fibrils of liraglutide or semaglutide are undesirable in pharmaceutical products. Thus, the antibodies of the present invention will allow the differentiation of fibrils of liraglutide or semaglutide from their soluble forms. Such antibodies of the present invention have several technical advantages, including allowing the identification and / or quantification of such fibrils, optionally in a mixture with their soluble forms, and providing a means to ensure the adequate quality of pharmaceutical products containing liraglutide or semaglutide. In some embodiments, the antibodies of the present invention allow the separation or partial separation of liraglutide fibrils from a mixture of soluble liraglutide. Such separation can be carried out by immobilization on a solid surface, such as a chromatography column, filter, or membrane. In some embodiments, the antibodies of the present invention allow for a sensitive assay for optionally detecting very low levels of peptide fibrils in the presence of a vast excess of the soluble form of the peptide. In some embodiments, the terms "fibril", "peptide fibril", also in relation to the specific peptides liraglutide or semaglutide, refer to a type of aggregate that can be obtained according to assay (I) for liraglutide herein or according to assay (II) for semaglutide herein, and such fibrils can be seen to be in the shape of thin threads using, for example, transmission electron microscopy.

[0014] The inventors have surprisingly found that the antibodies of the present invention are at least 100-fold, and possibly even at least 1000-fold, more sensitive in detecting fibrils compared to ThT assays, such as the ThT assay without shaking, such as assay (V) herein.

[0015] In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the fibrils are prepared according to Assay (I) herein. In some embodiments, the present invention relates to antibodies that bind to semaglutide fibrils. In some embodiments, the present invention relates to antibodies that bind to semaglutide fibrils, wherein the fibrils are prepared according to Assay (II) herein. In some embodiments, the detection limit of the antibody for liraglutide fibrils is at least 10-fold lower than the detection limit for liraglutide fibrils in the ThT assay, such as at least 100-fold or at least 1000-fold lower in concentration, and the detection limit is optionally determined according to Assay (VI) herein. In some embodiments, the binding level of the antibody to liraglutide fibrils is at least 10-fold, such as at least 20-fold or at least 50-fold, the binding level of the antibody to soluble liraglutide. In some embodiments, the detection limit of the antibody for semaglutide fibrils is at least 10-fold lower than the detection limit for semaglutide fibrils in the ThT assay, such as at least 100-fold or at least 1000-fold lower in concentration, and the detection limit is optionally determined according to Assay (VI) herein. In some embodiments, the binding level of the antibody to semaglutide fibrils is at least 10-fold, such as at least 20-fold or at least 50-fold, the binding level of the antibody to soluble semaglutide. In some embodiments, the binding level is determined according to Assay (IV) herein. In some embodiments, the binding level is determined according to Assay (III) herein. In some embodiments, the binding level is determined according to Assay (III-B) herein.

[0016] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the fibrils are optionally prepared according to Assay (I) herein, and the binding level of the antibody to the liraglutide fibrils is at least 10-fold the binding level of the antibody to soluble liraglutide, wherein the binding level is determined at a liraglutide fibril concentration of at least 25 μM according to Assay (III). In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the fibrils are optionally prepared according to Assay (I) herein, and the limit of detection of the antibody for liraglutide fibrils is at least 10-fold lower in concentration than the limit of detection of liraglutide fibrils in a ThT assay, wherein the limit of detection is determined at a liraglutide fibril concentration of at least 1 μM according to Assay (VI) herein. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the fibrils are optionally prepared according to Assay (I) herein, and the binding level of the antibody to the liraglutide fibrils is at least 5-fold the binding level of the antibody to soluble liraglutide, wherein the antibody has a purity of greater than 95% monomer, and wherein the binding level is determined at a liraglutide fibril concentration of at least 30 μM according to Assay (III-B) herein. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the fibrils are optionally prepared according to Assay (I) herein, and the limit of detection of the antibody for liraglutide fibrils is at least 10-fold lower in concentration than the limit of detection of liraglutide fibrils in a ThT assay, wherein the antibody has a purity of greater than 95% monomer, and wherein the limit of detection is determined at a liraglutide fibril concentration of at least 0.025 μM according to Assay (VI-B) herein.

[0017] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the antibody is capable of detecting liraglutide fibrils at a fibril concentration of 1 - 1000 ppm in solution, such as 1 - 10 ppm fibrils, or 10 - 100 ppm fibrils, or 100 - 1000 ppm fibrils.

[0018] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from SEQ ID NO: 115 and 121, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO: 37, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO: 43, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO: 49, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO: 55, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO: 61, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO: 67, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO: 73, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO: 79, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO:85, or any such sequence having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO:91, or any such sequence having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO:97, or any such sequence having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO:103, or any such sequence having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO:115, or any such sequence having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises the following CDR3 sequence: SEQ ID NO:121, or any such sequence having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0019] In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO:37, 38, and 39; SEQ ID NO:43, 44, and 45; SEQ ID NO:49, 50, and 51; SEQ ID NO:55, 56, and 57; SEQ ID NO:61, 62, and 63; SEQ ID NO:67, 68, and 69; SEQ ID NO:73, 74, and 75; SEQ ID NO:79, 80, and 81; SEQ ID NO:85, 86, and 87; SEQ ID NO:91, 92, and 93; SEQ ID NO:97, 98, and 99; SEQ ID NO:103, 104, and 105; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO:115, 116, and 117; SEQ ID NO:121, 122, 123; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO:37, 38, and 39; SEQ ID NO:43, 44, and 45; SEQ ID NO:49, 50, and 51; SEQ ID NO:55, 56, and 57; SEQ ID NO:61, 62, and 63; SEQ ID NO:67, 68, and 69; SEQ ID NO:73, 74, and 75; SEQ ID NO:79, 80, and 81; SEQ ID NO:85, 86, and 87; SEQ ID NO:91, 92, and 93; SEQ ID NO:97, 98, and 99; SEQ ID NO:103, 104, and 105; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO:115, 116, and 117; SEQ ID NO:121, 122, 123; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 37, 38, and 39; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 43, 44, and 45; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 49, 50, and 51; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 55, 56, and 57; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 61, 62, and 63; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 67, 68, and 69; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 73, 74, and 75; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 79, 80, and 81; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 85, 86, and 87; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 91, 92, and 93; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 97, 98, and 99; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 103, 104, and 105; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 115, 116, and 117; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 121, 122, 123; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0020] In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the light chain variable region of the antibody comprises CDR3, CDR2, and / or CDR1 sequences selected from the group consisting of: SEQ ID NO:40, 41, and 42; SEQ ID NO:46, 47, and 48; SEQ ID NO:52, 53, and 54; SEQ ID NO:58, 59, and 60; SEQ ID NO:64, 65, and 66; SEQ ID NO:70, 71, and 72; SEQ ID NO:76, 77, and 78; SEQ ID NO:82, 83, and 84; SEQ ID NO:88, 89, and 90; SEQ ID NO:94, 95, and 96; SEQ ID NO:100, 101, and 102; SEQ ID NO:106, 107, and 108; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the light chain variable region of the antibody comprises CDR3, CDR2, and / or CDR1 sequences selected from the group consisting of: SEQ ID NO:118, 119, and 120; SEQ ID NO:124, 125, and 126; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the light chain variable region of the antibody comprises CDR3, CDR2, and / or CDR1 sequences selected from the group consisting of: SEQ ID NO:40, 41, and 42; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the light chain variable region of the antibody comprises CDR3, CDR2, and / or CDR1 sequences selected from the group consisting of: SEQ ID NO:46, 47, and 48; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the light chain variable region of the antibody comprises CDR3, CDR2, and / or CDR1 sequences selected from the group consisting of: SEQ ID NO:52, 53, and 54; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to antibodies that bind to liraglutide fibrils, wherein the light chain variable region of the antibody comprises CDR3, CDR2, and / or CDR1 sequences selected from the group consisting of: SEQ ID NO:58, 59, and 60; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 64, 65, and 66; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 70, 71, and 72; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 76, 77, and 78; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 82, 83, and 84; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 88, 89, and 90; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 94, 95, and 96; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 100, 101, and 102; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the present invention relates to an antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of: SEQ ID NO: 106, 107, and 108; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of SEQ ID NOs: 118, 119, and 120; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of SEQ ID NOs: 124, 125, and 126; or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the antibody comprises a heavy chain variable region as defined herein and a light chain variable region as defined in any of the foregoing embodiments. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; or any of said sequences having at most 20, such as at most 15 or at most 10 amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 109 and 110; or any of said sequences having at most 20, such as at most 15 or at most 10 amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the antibody has at least 80%, such as at least 90% or at least 95% sequence identity with a sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, the invention relates to an antibody that binds to liraglutide fibrils, wherein the antibody has at least 80%, such as at least 90% or at least 95% sequence identity with a sequence selected from SEQ ID NOs: 109 and 110. In some embodiments, the antibody has at least 70%, such as at least 75% sequence identity with a sequence defined herein. In some embodiments, the antibody has at least 80%, such as at least 85% or at least 90% sequence identity with a sequence defined herein. In some embodiments, the antibody has at least 91%, such as at least 92% or at least 93% sequence identity with a sequence defined herein. In some embodiments, the antibody has at least 94%, such as at least 95% or at least 96% sequence identity with a sequence defined herein. In some embodiments, the antibody has at least 97%, such as at least 98% or at least 99% sequence identity with a sequence defined herein.

[0021] In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, wherein the antibody comprises a variable light chain (VL) sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 111, and 113; or any of said sequences having at most 20, such as at most 15 or at most 10 amino acid substitutions, deletions, or insertions. In some embodiments, the invention relates to antibodies that bind to liraglutide fibrils, wherein the antibody comprises a variable heavy chain (VH) sequence selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 112, and 114; or any of said sequences having at most 20, such as at most 15 or at most 10 amino acid substitutions, deletions, or insertions.

[0022] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a single-chain Fv fragment. In some embodiments, the antibody comprises an Fc domain. In some embodiments, the antibody is a single-chain Fv fragment that further comprises an Fc domain. In some embodiments, the antibody specifically binds to the liraglutide fibrils and / or semaglutide fibrils. In some embodiments, the antibody specifically binds to the liraglutide fibrils. In some embodiments, the antibody specifically binds to the semaglutide fibrils.

[0023] In some embodiments, the antibody that binds to liraglutide has a purity higher than 70%, or higher than 75%, or higher than 80%, or higher than 85%, or higher than 90%, or higher than 95% monomer. In some embodiments, the antibody that binds to liraglutide has a purity higher than 70% monomer. In some embodiments, the antibody that binds to liraglutide has a purity higher than 75% monomer. In some embodiments, the antibody that binds to liraglutide has a purity higher than 80% monomer. In some embodiments, the antibody that binds to liraglutide has a purity higher than 85% monomer. In some embodiments, the antibody that binds to liraglutide has a purity higher than 90% monomer. In some embodiments, the antibody that binds to liraglutide has a purity higher than 95% monomer. In some embodiments, the purity of the antibody that binds to liraglutide fibrils is determined according to the method described herein in the "Size Exclusion Chromatography" section, and subsequently, relative to the sum of the AUCs of all peaks, the area under the curve (AUC 280nm is determined for the peak of the monomeric antibody based on the absorbance at 280 nm 280nm ).

[0024] Liraglutide and Semaglutide

[0025] Liraglutide and semaglutide are analogs of human GLP-1(7-37) that contain covalently linked moieties. The antibodies of the present invention bind to liraglutide fibrils and / or semaglutide fibrils. As used herein, the term "fibril" in relation to liraglutide refers to liraglutide fibrils, and the term "fibril" in relation to semaglutide refers to semaglutide fibrils. In some embodiments, the antibodies of the present invention bind to liraglutide fibrils. In some embodiments, the antibodies of the present invention bind to semaglutide fibrils.

[0026] Liraglutide is Arg34,Lys26-(N-ε-(γ-L-glutamyl(N-α-hexadecanoyl)))-GLP-1(7-37) and can be prepared as described in Example 37 of WO98 / 08871. Example 37 of WO98 / 08871 is incorporated herein by reference. The structure of liraglutide has also been published in WHO Drug Information Vol.17, No.2, 2003. The structure of liraglutide has also been published in WHO Drug Information Vol.24, No.1, 2010. Liraglutide fibrils can be prepared as described in Test (I) herein. An example of soluble liraglutide is a commercially available solution manufactured by Novo Nordisk A / S, Denmark; for example, the trademark

[0027] Semaglutide is N-ε26-[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoyl amino)-4(S)-carboxybutanoyl amino]ethoxy)ethoxy]acetyl amino)ethoxy]ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37) peptide and can be prepared as described in Example 4 of WO2006 / 097537. Example 4 of WO2006 / 097537 is incorporated herein by reference. Semaglutide fibrils can be prepared as described in Test (II) herein. An example of soluble semaglutide is a commercially available solution manufactured by Novo Nordisk A / S, Denmark; trademark

[0028] Antibody

[0029] In some embodiments, the present invention relates to one or more of a series of antibodies, characterized by their functionality and / or the amino acid sequences of the CDRs, the heavy chain variable region, the light chain variable region, and / or the sequence of the Fc domain. In some embodiments, the term "CDR" as used herein is determined according to the Kabat antibody numbering scheme (Kabat, Elvin A. (1976). Structural Concepts in Immunology and Immunochemistry. New York, NY, USA: Holt, Rinehart & Winston). In some embodiments, the present invention relates to one or more of a series of antibodies, characterized by their functionality and / or the H-CDR3 amino acid sequence. In some embodiments, the present invention relates to one or more of a series of antibodies, characterized by their functionality and / or the CDR amino acid sequences (the CDR1, CDR2, and CDR3 of the heavy chain variable region may be referred to herein as H-CDR1, H-CDR2, and H-CDR3. Similarly, the CDR1, CDR2, and CDR3 of the light chain variable region may be referred to herein as L-CDR1, L-CDR2, and L-CDR3). In some embodiments, the present invention relates to one or more of a series of antibodies, characterized by their functionality and / or the amino acid sequences of the heavy chain variable region and the light chain variable region. In some embodiments, the present invention relates to one or more of a series of antibodies, characterized by their functionality and / or the amino acid sequences of the heavy chain variable region, the light chain variable region, and / or the sequence of the Fc domain. In some embodiments, the antibody comprises H-CDR3. In some embodiments, the antibody comprises H-CDR1, H-CDR2, and / or H-CDR3. In some embodiments, the antibody comprises H-CDR1, H-CDR2, and H-CDR3. In some embodiments, the antibody comprises L-CDR1, L-CDR2, and / or L-CDR3. In some embodiments, the antibody comprises L-CDR1, L-CDR2, and L-CDR3. In some embodiments, the antibody comprises the heavy chain variable region and / or the light chain variable region.

[0030] The antibodies of the present invention can be in any form, including intact antibodies and antigen-binding fragments (i.e., "antigen-binding portions") or single-chain antibodies.

[0031] In some embodiments, the antibody is a single-chain variable fragment (scFv) antibody. In some embodiments, the antibody is a single-chain variable fragment fused to an Fc domain (scFv-Fc) antibody. In some embodiments, the scFv or scFv-Fc antibody is composed of a sequence comprising a heavy chain variable region (V H ) and a light chain variable region (V Lcomposed of an amino acid sequence; the scFv-Fc antibody further comprises an Fc domain.

[0032] In some embodiments, the antibody is a full-length antibody comprising standard antibody domains and regions, as described herein, for example. A full-length antibody (or whole antibody) comprises four polypeptide chains, namely two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V H ) and a heavy chain constant region (C H ). Each light chain comprises a light chain variable region (V L ) and a light chain constant region (C L ). The heavy chain constant region comprises three domains, C H 1, C H 2, and C H 3. Each light chain comprises a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region comprises one domain, C L .

[0033] The heavy chain variable region and the light chain variable region each comprise a binding domain that interacts with an antigen. V H and V L regions can be further subdivided into hypervariable regions, known as complementarity determining regions (CDRs), interspersed with more conserved regions, known as framework regions (FRs). Each V H and V L can comprise three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0034] In some embodiments, the antibody is an antibody fragment, such fragments can be obtained using conventional recombinant or protein engineering techniques. The antibody fragments of the present invention can be prepared by truncation, for example, by removing one or more amino acids from the N-terminus and / or C-terminus of the polypeptide. Fragments can also be generated by one or more internal deletions. In some embodiments, the antibody of the present invention is, or comprises a fragment of any of the antibodies described herein. In some embodiments, the antibody of the present invention is, or comprises an antigen-binding portion or a variant thereof of one of the antibodies described herein. For example, the antibody of the present invention can be a Fab fragment or a variant thereof of one of the antibodies described herein, or the antibody of the present invention can be a single-chain antibody or a variant thereof derived from one of the antibodies described herein. Examples of antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, Fv (generally the V of a single arm of an antibody)L and V H ), single-chain Fv (scFv; see, e.g., Bird et al., Science 1988; 242:42S-426; and Huston et al., PNAS 1988; 85:5879-5883), Fd (generally V H and C H 1) and dAb (generally V H ); V H , V L , VhH and V-NAR; monovalent molecules comprising a single V H and a single V L chain; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10:949-57); camel IgG; IgNAR; and one or more isolated CDRs or functional complementarity-determining regions, wherein the isolated CDRs or antigen-binding residues or polypeptides can associate or be linked together to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed, e.g., in Holliger and Hudson, Nat Biotechnol 2005; 23:1126-1136, WO2005040219, and published U.S. patent applications 20050238646 and 20020161201.

[0035] As used herein, the term "complementary determining region" ("CDR") or "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. CDRs typically consist of CDR1, CDR2, and CDR3 in the light chain variable region and CDR1, CDR2, and CDR3 in the heavy chain variable region as defined by Kabat and / or those residues from "hypervariable loops" (Chothia and Lesk, J. Mol. Biol 1987; 196: 901-917). Generally, the numbering of amino acid residues in this region is performed by the method described by Kabat et al. (ibid.). The term "Kabat" as used herein refers to the numbering system for the heavy chain variable region and / or the light chain variable region, for example, as described in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242. By using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids, which correspond to the shortening or insertion into the framework (FR) of the variable region or the CDR. The Kabat numbering of residues of a given antibody can be determined by aligning the homologous regions of the antibody sequence with the "standard" Kabat-numbered sequence. The term "framework region" or "FR" residues refers to those V H or V L amino acid residues that are not within the CDRs as defined herein. The fragment crystallizable region ("Fc domain") of an antibody is the region of the antibody that is capable of interacting with cell surface receptors called Fc receptors and some proteins of the complement system.

[0036] The term "antibody derivative" refers to any modified form of an antibody, such as a conjugate of an antibody with another substance or an antibody.

[0037] The term "antigen" can refer to the molecular entity used to generate an antibody. However, herein, the term "antigen" generally refers to a target molecule that binds or specifically binds to an antibody; thus, it includes fragments or mimics of the molecular entity used to generate the antibody. Antibodies can be generated in any manner, including by animal immunization or display screening, such as phage display or yeast display.

[0038] As used herein, the term "epitope" is defined in the context of the molecular interaction between an "antigen-binding" polypeptide such as an antibody or a fragment thereof and its corresponding antigen. Generally, an "epitope" refers to a region or area on an antigen that is bound or specifically bound by an antibody, i.e., the region or area that physically contacts the antibody. An epitope can include amino acid residues in the antigen that are directly involved in binding to the antibody (also referred to as the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as antigen amino acid residues that are effectively blocked by the antibody (in other words, the amino acid residue is within the "solvent-excluded surface" and / or "footprint" of the antibody). A given antigen can contain many different epitopes, which can include, but are not limited to: linear peptide antigenic determinants, conformational antigenic determinants composed of one or more non-contiguous amino acids that are close to each other in the native (mature) conformation, and post-translational antigenic determinants composed in whole or in part of molecular structures covalently linked to the antigen, such as carbohydrate groups.

[0039] The terms "bind", "specifically bind", and "specificity" of an antibody are used herein to describe the selectivity of an antibody or an antigen-binding fragment thereof. The antibodies of the present invention can specifically bind to liraglutide fibrils or semaglutide fibrils, indicating that the binding level of the antibody to other antigens is significantly lower. In some embodiments, significantly lower means that the binding level is at least 10-fold lower, such as at least 15-fold lower or at least 20-fold lower. The binding level can be determined according to Assay (III) herein or according to Assay (IV) herein. The binding level can be determined according to Assay (III-B) herein.

[0040] As used herein, the term "sequence identity" refers to the degree of relatedness between polypeptide sequences, as determined by the number of matches between strings of two or more amino acid residues, and can be determined as the percentage of identical matches between two or more sequences with gap alignments (if any) obtained by a particular mathematical model or computer program (i.e., "algorithm"). The sequence identity of a polypeptide can be readily calculated by methods known in the art, including but not limited to those described in the following references: Computational Molecular Biology, Lesk, A.M. ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W. ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M. and Griffin, H.G. eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J. eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48 , 1073 (1988). Preferred methods for determining sequence identity are designed to give the maximum match between the sequences being tested. Methods for determining sequence identity are described in computer programs that are publicly available; such preferred computer program methods for determining sequence identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid Res. 12 , 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH, Bethesda, Md. 20894; Altschul et al., ibid.). The well-known Smith Waterman algorithm can also be used to determine sequence identity. For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis.), the best match of the amino acids of each of the two polypeptides for which the percent sequence identity is to be determined is aligned (the "span of the match", as determined by the algorithm). A gap opening penalty (which is calculated as 3 times the average diagonal; the "average diagonal" is the average of the diagonals of the comparison matrix used; the "diagonal" is the score or number assigned by a particular comparison matrix to each perfect amino acid match) and a gap extension penalty (which is typically 1 / 10 of the gap opening penalty) are used in combination with a comparison matrix such as PAM 250 or BLOSUM 62 with this algorithm. The algorithm also uses standard comparison matrices (for the PAM250 comparison matrix, see Dayhoff et al., Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978); for the BLOSUM 62 comparison matrix, see Henikoff et al., Proc. Natl. Acad. Sci USA 89, 10915-10919 (1992)). In some embodiments, sequence identity is determined using the following parameters, for example using the algorithm GAP: Algorithm: Needleman et al., J. Mol. Biol. 48 , 443-453 (1970); Comparison matrix: BLOSUM 62, from Henikoff et al., PNAS USA 89 , 10915-10919 (1992); and Gap penalty: 12, Gap length penalty: 4, Similarity threshold: 0, No penalty for terminal gaps.

[0041] In some embodiments, the antibodies of the invention comprise one or more amino acid substitutions or insertions. The amino acid substitutions can be in the form of conservative amino acid substitutions. A "conservative amino acid substitution" can involve substituting one amino acid residue with another residue such that there is no or little effect on the polarity or charge of the amino acid residue at that position. Conservative amino acid substitutions can be made within the following groups of amino acids: hydrophilic: Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr; aliphatic: Val, Ile, Leu, Met. basic: Lys, Arg, His; aromatic: Phe, Tyr, Trp; in addition, any residue can generally be substituted with alanine.

[0042] In some embodiments, one or more unnatural amino acids are introduced into the antibodies of the invention by substitution or insertion. Such unnatural amino acids include, but are not limited to, D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designed amino acids such as β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids.

[0043] Amino acid sequence variants of the antibodies of the invention can be prepared by introducing appropriate nucleotide changes into the nucleic acids of the invention, or by in vitro synthesis of the desired polypeptide. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence. Combinations of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final polypeptide product has the desired properties. Any technique known in the art can be used to prepare the variant (altered) polypeptide. For example, the polynucleotides of the invention can be mutagenized in vitro. Such in vitro mutagenesis techniques include subcloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as Escherichia coli XL-Ired (Stratagene), and propagating the transformed bacteria for a suitable number of generations. The products derived from the mutant / altered DNA can be readily screened using the techniques described herein to determine whether they have receptor binding and / or inhibitory activity. When designing amino acid sequence variants, the location of the mutation site and the nature of the mutation will depend on the property to be altered. The mutation site can be modified singly or sequentially, for example, by (1) first making a substitution with a conservative amino acid selection and then making a substitution with a more group selection based on the results obtained, (2) deleting the target residue, or (3) inserting additional residues adjacent to the located site. In some embodiments, the range of amino acid sequence deletions is from about 1 to 15 residues, more preferably from about 1 to 10 residues, and typically about 1 to 5 consecutive residues.

[0044] In some embodiments, the molecule consists essentially of a defined sequence. In some embodiments, the molecule consists of a defined sequence. In some embodiments, the antibody is an isolated antibody. The term "isolated antibody" refers to an antibody that has been separated and / or recovered from another / other component(s) in its natural environment, and / or purified from a mixture of components in its natural environment. The antibodies of the present invention can be from different species, including mammalian species such as mice, rats, rabbits, pigs or non-human primates. The antibody can be a rodent antibody, more particularly a mouse antibody. Alternatively, the antibody can be from a non-mammalian species such as a chicken. The antibody can also be a humanized antibody or a human antibody.

[0045] The antibodies of the present invention can be prepared according to methods known in the art such as recombinant protein, cell culture and immunological techniques. Such techniques are described and explained throughout the literature in sources such as: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (eds.) Current Protocols in Immunology, John Wiley and Sons (including all updates to date).

[0046] Single-chain antibodies, including scFv or scFv-Fc antibodies, can be prepared by inserting the DNA sequence corresponding to their amino acid sequence into a plasmid in a host cell and then expressing the antibody using the host cell by recombinant techniques, such as bacterial cell culture; such methods are well known in the art.

[0047] Monoclonal antibodies are typically prepared by fusing myeloma cells with spleen cells from a mouse that has been immunized with the desired antigen. Human monoclonal antibodies can be obtained from transgenic animals (such as mice or other suitable species) that encode human antibodies. Alternatively, recombinant monoclonal antibodies can be prepared using techniques known as repertoire cloning or phage display / yeast display. Recombinant antibody engineering involves using viruses or yeast to produce antibodies rather than mice.

[0048] Methods and uses of antibodies

[0049] In some embodiments, the present invention relates to the use of an antibody as defined herein for identifying and / or quantifying liraglutide fibrils or semaglutide fibrils. In some embodiments, the present invention relates to the use of an antibody as defined herein for separating from a solution containing soluble liraglutide or soluble semaglutide, including partially separating liraglutide fibrils or semaglutide fibrils. Such identification and / or quantification can be carried out by binding the antibody to the fibrils and then detecting the bound antibody, for example, by enzyme-linked immunosorbent assay (ELISA). ELISA can be carried out as known in the art. In some embodiments, the containers (such as microtiter plates) for ELISA are initially saturated. The saturation can be with a protein such as lysozyme or albumin, for example, bovine serum albumin (BSA) or ovalbumin. In some embodiments, the antibody of the present invention that binds to the fibrils will bind to a second antibody. If the antibody of the present invention contains an Fc domain, the second antibody can bind to the Fc domain. If a label is present on the second antibody, detection and / or quantification of the second antibody can be possible, and such a label can be a fluorophore that can be identified by spectroscopy. Quantification can be carried out using a standard of the fibrils that binds to the antibody of the present invention.

[0050] In some embodiments, the term "limit of detection" as used herein refers to the lowest detection limit, which is the lowest concentration of a substance that can be distinguished from the absence of that substance. In some embodiments, the term "limit of detection" as used herein refers to a mixture / soluble specific ratio of 3 as determined according to Test (IV) herein. The comparison of the limits of detection using the antibody and ThT can be carried out according to Test (VI) herein. The comparison of the limits of detection using the antibody and ThT can be carried out according to Test (VI-B) herein. In some embodiments, the term "limit of detection" as used herein in relation to an antibody refers to the limit of detection of an assay using the antibody in an ELISA, such as Test (III) or Test (IV) herein. In some embodiments, the term "limit of detection" as used herein in relation to an antibody refers to the limit of detection of an assay using the antibody in an ELISA, such as Test (III-B) herein. In some embodiments, the term "limit of detection" as used herein is three times the standard deviation of control samples tested in duplicate; the standard deviation can be determined by a Student t-test.

[0051] In some embodiments, the present invention relates to the use of an antibody as defined herein for the identification of liraglutide fibrils or semaglutide fibrils.

[0052] In some embodiments, the present invention relates to the use of an antibody as defined herein as an affinity ligand to remove fibrils from a mixture comprising (i) liraglutide fibrils and soluble liraglutide or (ii) semaglutide fibrils and soluble semaglutide.

[0053] In some embodiments, the present invention relates to a method for identifying liraglutide fibrils or semaglutide fibrils, the method comprising the steps of: a) binding an antibody as defined herein to the liraglutide fibrils or semaglutide fibrils.

[0054] In some embodiments, the present invention relates to a method for quantifying liraglutide fibrils or semaglutide fibrils, the method comprising the steps of: a) binding an antibody as defined herein to the liraglutide fibrils or semaglutide fibrils. According to the method of any of the foregoing embodiments, it further comprises the step of: b) detecting the antibody bound to the liraglutide fibrils or semaglutide fibrils. In some embodiments, the method further comprises the step of: c) quantifying the antibody bound to the liraglutide fibrils or semaglutide fibrils, optionally by using a standard of the fibrils. In some embodiments, the fibrils are in solution. In some embodiments, the fibrils are in a solution further comprising soluble liraglutide. In some embodiments, the fibrils are in a solution that does not contain other peptides or proteins other than liraglutide fibrils and optionally contains soluble liraglutide.

[0055] In some embodiments, the method comprises: (a) contacting a solid support with a sample under conditions such that one or more fibrils in the sample are immobilized on the solid support; (b) contacting the solid support with any one of the antibodies described herein or an antigen-binding fragment thereof under conditions such that the antibody binds to the one or more immobilized fibrils to form an antibody-fibril complex; and (c) contacting the antibody-fibril complex with a second antibody comprising a detectable label, wherein (i) the second antibody specifically binds to the antibody-fibril complex, and (ii) a signal from the detectable label is detected, indicating the presence of one or more fibrils in the sample.

[0056] In some embodiments, the method comprises: (a) contacting a solid support comprising a fibril-specific antibody with a sample such that fibrils (if present in the sample) bind to the antibody and are immobilized on the surface to form a complex; and (b) detecting the complex.

[0057] Any solid support known in the art can be used in the methods described herein, including but not limited to solid supports in the form of planar substrates or beads made of polymeric materials. For example, the solid support can be a glass slide, a microtiter plate (e.g., a 96-well plate), or a bead, such as a latex, agarose, sepharose, streptavidin, tosyl-activated, epoxy resin, polystyrene, amino bead, amine bead, carboxyl bead, etc. In certain embodiments, the bead can be a particle, such as a microparticle. The terms "bead" and "particle" are used interchangeably herein and refer to a substantially spherical solid support. The terms "microparticle" and "microbead" are used interchangeably herein and refer to a microbead or microparticle that is allowed to occupy or settle in a well array, such as a well array in a detection module. Many techniques known in the art can be used to attach a protein or peptide to a solid support, such as a plate or a microparticle. A variety of techniques for adding reactive moieties to proteins are known, such as the methods described in U.S. Patent 5,620,850. Methods for attaching proteins to surfaces are also described, for example, in Heller, Acc. Chem. Res., 23:128 (1990).

[0058] Any suitable method known in the art can be used to contact the solid support with a volume of the sample. As used herein, the term "contact" refers to any type of combined action that brings the solid support into sufficient proximity to one or more fibrils in the sample such that if one or more fibrils are present in the sample, a binding interaction will occur. Contact can be achieved in a variety of different ways, including combining the sample with a porous plate or microparticles. Contact can be repeated as needed. Incubation can be carried out in a binding buffer that promotes specific binding interactions, such as albumin (e.g., BSA), non-ionic detergents (Tween-20, Triton X-100), and / or protease inhibitors (e.g., PMSF). Other conditions for the binding interaction, such as temperature and salt concentration, can also be determined empirically or can be based on the manufacturer's instructions. For example, contact can be carried out at room temperature (21°C - 28°C, e.g., 23°C - 25°C), 37°C, or 4°C. As used herein, the terms "detectable label" and "label" refer to a moiety that can produce a signal detectable by visual or instrumental means. The detectable label can be, for example, a substance that produces a signal, such as a chromogen, a fluorescent compound, an enzyme, a chemiluminescent compound, a radioactive compound, etc. In one embodiment, the detectable label can be a fluorescent compound, such as a fluorophore. The presence or amount of fibrils in the sample can be determined (e.g., quantified) using any suitable method known in the art. Such methods include, but are not limited to, immunoassays, such as ELISA.

[0059] In some embodiments, the invention relates to an assay for detecting liraglutide fibrils relative to soluble and / or monomeric liraglutide, which comprises an antibody according to the invention, wherein the antibody is capable of detecting liraglutide fibrils at a fibril concentration of 1 - 1000 ppm in solution, such as 1 - 10 ppm fibrils, or 10 - 100 ppm fibrils, or 100 - 1000 ppm fibrils.

[0060] In some embodiments, "a" means "one or more". As used herein, the term "about" refers to a range from 10% less to 10% more than the indicated value. Unless otherwise specified in this specification, terms presented in the singular also include the plural.

[0061] Embodiments of the invention

[0062] Non-limiting embodiments of the invention include:

[0063] 1. An antibody that binds to liraglutide fibrils.

[0064] 2. An antibody that binds to liraglutide fibrils, wherein the fibrils are prepared according to Test (I) herein.

[0065] 3. Antibodies that bind to semaglutide fibrils.

[0066] 4. Antibodies that bind to semaglutide fibrils, wherein the fibrils are prepared according to Test (II) herein.

[0067] 5. The antibody according to any one of embodiments 1 or 2, wherein the limit of detection of the antibody for liraglutide fibrils is at least 10-fold lower, such as at least 100-fold or at least 1000-fold lower in concentration, than the limit of detection of liraglutide fibrils in the ThT assay, and the limit of detection is optionally determined according to Test (VI) herein.

[0068] 6. The antibody according to any one of embodiments 1 or 2, wherein the binding level of the antibody to liraglutide fibrils is at least 10-fold, such as at least 20-fold or at least 50-fold, the binding level of the antibody to soluble liraglutide.

[0069] 7. The antibody according to any one of embodiments 1 or 2, wherein the limit of detection of the antibody for liraglutide fibrils is at least 10-fold lower, such as at least 100-fold or at least 1000-fold lower in concentration, than the limit of detection of liraglutide fibrils in the ThT assay, and the limit of detection is optionally determined according to Test (VI-B) herein.

[0070] 8. The antibody according to any one of embodiments 1 or 2, wherein the binding level of the antibody to liraglutide fibrils is at least 5-fold, such as 10-fold, the binding level of the antibody to soluble liraglutide, and the binding level is optionally determined according to Test (III-B) herein.

[0071] 9. The antibody according to any one of embodiments 3 or 4, wherein the limit of detection of the antibody for semaglutide fibrils is at least 10-fold lower, such as at least 100-fold or at least 1000-fold lower in concentration, than the limit of detection of semaglutide fibrils in the ThT assay, and the limit of detection is optionally determined according to Test (VI) herein.

[0072] 10. The antibody according to embodiment 3 or 4, wherein the binding level of the antibody to semaglutide fibrils is at least 10-fold, such as at least 20-fold or at least 50-fold, the binding level of the antibody to soluble semaglutide.

[0073] 11. The antibody according to any one of embodiments 6 or 10, wherein the binding level is determined according to Test (IV) herein.

[0074] 12. The antibody according to any one of embodiments 6 or 10, wherein the binding level is determined according to Test (III) herein.

[0075] 13. An antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0076] 14. An antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of:

[0077] a. SEQ ID NO: 37, 38, and 39;

[0078] b. SEQ ID NO: 43, 44, and 45;

[0079] c. SEQ ID NO: 49, 50, and 51;

[0080] d. SEQ ID NO: 55, 56, and 57;

[0081] e. SEQ ID NO: 61, 62, and 63;

[0082] f. SEQ ID NO: 67, 68, and 69;

[0083] g. SEQ ID NO: 73, 74, and 75;

[0084] h. SEQ ID NO: 79, 80, and 81;

[0085] i. SEQ ID NO: 85, 86, and 87;

[0086] j. SEQ ID NO: 91, 92, and 93;

[0087] k. SEQ ID NO: 97, 98, and 99;

[0088] l. SEQ ID NO: 103, 104, and 105;

[0089] or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0090] 15. An antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of:

[0091] a. SEQ ID NO: 40, 41, and 42;

[0092] b. SEQ ID NO: 46, 47, and 48;

[0093] c. SEQ ID NO: 52, 53, and 54;

[0094] d. SEQ ID NO: 58, 59, and 60;

[0095] e. SEQ ID NO: 64, 65, and 66;

[0096] f. SEQ ID NO: 70, 71, and 72;

[0097] g. SEQ ID NO: 76, 77, and 78;

[0098] h. SEQ ID NO: 82, 83, and 84;

[0099] i. SEQ ID NO: 88, 89, and 90;

[0100] j. SEQ ID NO: 94, 95, and 96;

[0101] k. SEQ ID NO: 100, 101, and 102;

[0102] l. SEQ ID NO: 106, 107, and 108;

[0103] or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0104] 16. An antibody that binds to liraglutide protofibrils, wherein the antibody comprises a heavy chain variable region as defined in any of the foregoing embodiments and a light chain variable region as defined in any of the foregoing embodiments.

[0105] 17. An antibody that binds to liraglutide protofibrils, wherein the antibody comprises a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; or any of said sequences having at most 20, such as at most 15, or at most 10 amino acid substitutions, deletions, or insertions.

[0106] 18. An antibody that binds to liraglutide protofibrils, wherein the antibody has at least 80%, such as at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0107] 19. An antibody that binds to liraglutide fibrils, wherein the antibody comprises a variable light chain (VL) sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 111, and 113; or any of said sequences having at most 20, such as at most 15 or at most 10 amino acid substitutions, deletions, or insertions.

[0108] 20. An antibody that binds to liraglutide fibrils, wherein the antibody comprises a variable heavy chain (VH) sequence selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 112, and 114; or any of said sequences having at most 20, such as at most 15 or at most 10 amino acid substitutions, deletions, or insertions.

[0109] 21. The antibody according to any one of the preceding embodiments, wherein the antibody is an isolated antibody.

[0110] 22. The antibody according to any one of the preceding embodiments, wherein the antibody is a single-chain Fv fragment.

[0111] 23. The antibody according to any one of the preceding embodiments, wherein the antibody comprises an Fc domain.

[0112] 24. The antibody according to any one of the preceding embodiments, wherein the antibody is a single-chain Fv fragment further comprising an Fc domain.

[0113] 25. The antibody according to any one of the preceding embodiments, wherein the antibody specifically binds to the liraglutide fibrils and / or semaglutide fibrils.

[0114] 26. The antibody according to any one of the preceding embodiments, wherein the antibody specifically binds to the liraglutide fibrils.

[0115] 27. The antibody according to any one of the preceding embodiments, wherein the antibody specifically binds to the semaglutide fibrils.

[0116] 28. Use of the antibody as defined in any one of the preceding embodiments for the identification of liraglutide fibrils or semaglutide fibrils.

[0117] 29. Use of the antibody as defined in any one of the preceding embodiments as an affinity ligand for removing fibrils from a mixture comprising (i) liraglutide fibrils and soluble liraglutide or (ii) semaglutide fibrils and soluble semaglutide.

[0118] 30. A method for identifying liraglutide fibrils or semaglutide fibrils, the method comprising the following steps: a) binding an antibody as defined in any of the foregoing embodiments to the liraglutide fibrils or semaglutide fibrils.

[0119] 31. A method for quantifying liraglutide fibrils or semaglutide fibrils, the method comprising the following steps: a) binding an antibody as defined in any of the foregoing embodiments to the liraglutide fibrils or semaglutide fibrils.

[0120] 32. The method according to any of the foregoing embodiments, further comprising the following step: b) detecting the antibody bound to the liraglutide fibrils or semaglutide fibrils.

[0121] 33. The method according to any of the foregoing embodiments, further comprising the following step: c) quantifying the antibody bound to the liraglutide fibrils or semaglutide fibrils, optionally by using a standard of the fibrils.

[0122] 34. The method according to any of the foregoing embodiments, wherein the fibrils are in solution.

[0123] 35. The method according to any of the foregoing embodiments, wherein the fibrils are in a solution further comprising soluble liraglutide.

[0124] 36. The method according to any of the foregoing embodiments, wherein the fibrils are in a solution that does not contain other peptides or proteins other than liraglutide fibrils and optionally contains soluble liraglutide.

[0125] 37. An antibody that binds to liraglutide fibrils, wherein the fibrils are optionally prepared according to Test (I) herein, and the antibody

[0126] a. The binding level to liraglutide fibrils is at least 10 times, such as at least 20 times or at least 50 times, the binding level of the antibody to soluble liraglutide; and / or

[0127] b. The detection limit for liraglutide fibrils is at least 10 times lower, such as at least 100 times or at least 1000 times lower in concentration, than the detection limit for liraglutide fibrils in the ThT assay, and the detection limit is optionally determined according to Test (VI) herein.

[0128] 38. An antibody that binds to semaglutide fibrils, wherein the fibrils are optionally prepared according to Test (II) herein, and the antibody

[0129] c. The binding level to semaglutide fibrils is at least 10-fold, such as at least 20-fold or at least 50-fold, of the binding level of the antibody to soluble semaglutide; and / or

[0130] d. The limit of detection for semaglutide fibrils is at least 10-fold lower, such as at least 100-fold or at least 1000-fold lower in concentration, than the limit of detection for semaglutide fibrils in the ThT assay, and the limit of detection is optionally determined according to Assay (VI) herein.

[0131] 39. The antibody according to any one of embodiments 37 or 38, wherein the binding level is determined according to Assay (IV) herein.

[0132] 40. The antibody according to any one of embodiments 37 or 38, wherein the binding level is determined according to Assay (III) herein.

[0133] 41. An antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 115, and 121, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0134] 42. An antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3 sequence, and the CDR3 sequence is selected from SEQ ID NO: 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0135] 43. An antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of:

[0136] a. SEQ ID NO: 37, 38, and 39;

[0137] b. SEQ ID NO: 43, 44, and 45;

[0138] c. SEQ ID NO: 49, 50, and 51;

[0139] d. SEQ ID NO: 55, 56, and 57;

[0140] e. SEQ ID NO: 61, 62, and 63;

[0141] f. SEQ ID NO: 67, 68, and 69;

[0142] g. SEQ ID NO: 73, 74, and 75;

[0143] h. SEQ ID NO: 79, 80, and 81;

[0144] i. SEQ ID NO: 85, 86, and 87;

[0145] j. SEQ ID NO: 91, 92, and 93;

[0146] k. SEQ ID NO: 97, 98, and 99;

[0147] l. SEQ ID NO: 103, 104, and 105;

[0148] or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0149] 44. An antibody that binds to liraglutide fibrils, wherein the light chain variable region of said antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of:

[0150] a. SEQ ID NO: 40, 41, and 42;

[0151] b. SEQ ID NO: 46, 47, and 48;

[0152] c. SEQ ID NO: 52, 53, and 54;

[0153] d. SEQ ID NO: 58, 59, and 60;

[0154] e. SEQ ID NO: 64, 65, and 66;

[0155] f. SEQ ID NO: 70, 71, and 72;

[0156] g. SEQ ID NO: 76, 77, and 78;

[0157] h. SEQ ID NO: 82, 83, and 84;

[0158] i. SEQ ID NO: 88, 89, and 90;

[0159] j. SEQ ID NO: 94, 95, and 96;

[0160] k. SEQ ID NO: 100, 101, and 102;

[0161] l. SEQ ID NO: 106, 107, and 108;

[0162] or any of said sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0163] 45. An antibody that binds to liraglutide fibrils, wherein the antibody comprises a heavy chain variable region as defined in any of the foregoing embodiments and a light chain variable region as defined in any of the foregoing embodiments.

[0164] 46. An antibody that binds to liraglutide fibrils, wherein the antibody comprises a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; or any of said sequences having at most 20, such as at most 15 or at most 10 amino acid substitutions, deletions, or insertions.

[0165] 47. An antibody that binds to liraglutide fibrils, wherein the antibody has at least 80%, such as at least 90% or at least 95% sequence identity with a sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0166] 48. The antibody according to any of the foregoing embodiments, wherein the antibody is an isolated antibody.

[0167] 49. The antibody according to any of the foregoing embodiments, wherein the antibody is a single-chain Fv fragment.

[0168] 50. The antibody according to any of the foregoing embodiments, wherein the antibody comprises an Fc domain.

[0169] 51. The antibody according to any of the foregoing embodiments, wherein the antibody is a single-chain Fv fragment further comprising an Fc domain.

[0170] 52. The antibody according to any of the foregoing embodiments, wherein the antibody specifically binds to the liraglutide fibrils and / or the semaglutide fibrils.

[0171] 53. The antibody according to any of the foregoing embodiments, wherein the antibody specifically binds to the liraglutide fibrils.

[0172] 54. The antibody according to any of the foregoing embodiments, wherein the antibody specifically binds to the semaglutide fibrils.

[0173] 55. Use of the antibody as defined in any of the foregoing embodiments for identifying liraglutide fibrils or semaglutide fibrils.

[0174] 56. Use of an antibody as defined in any of the foregoing embodiments as an affinity ligand to remove fibrils from a mixture comprising (i) liraglutide fibrils and soluble liraglutide or (ii) semaglutide fibrils and soluble semaglutide.

[0175] 57. A method for identifying liraglutide fibrils or semaglutide fibrils, the method comprising the steps of: a) binding an antibody as defined in any of the foregoing embodiments to liraglutide fibrils or semaglutide fibrils.

[0176] 58. A method for quantifying liraglutide fibrils or semaglutide fibrils, the method comprising the steps of: a) binding an antibody as defined in any of the foregoing embodiments to liraglutide fibrils or semaglutide fibrils.

[0177] 59. The method according to any of the foregoing embodiments, further comprising the step of: b) detecting the antibody bound to liraglutide fibrils or semaglutide fibrils.

[0178] 60. The method according to any of the foregoing embodiments, further comprising the step of: c) quantifying the antibody bound to liraglutide fibrils or semaglutide fibrils, optionally by using a standard of the fibrils.

[0179] 61. The method according to any of the foregoing embodiments, wherein the fibrils are in solution.

[0180] 62. The method according to any of the foregoing embodiments, wherein the fibrils are in a solution further comprising soluble liraglutide.

[0181] 63. The method according to any of the foregoing embodiments, wherein the fibrils are in a solution that does not contain other peptides or proteins other than liraglutide fibrils and optionally contains soluble liraglutide.

[0182] 64. An antibody that binds to liraglutide fibrils, wherein the heavy chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of:

[0183] m. SEQ ID NO: 115, 116, and 117;

[0184] n. SEQ ID NO: 121, 122, 123;

[0185] or any of the foregoing sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0186] 65. An antibody that binds to liraglutide fibrils, wherein the light chain variable region of the antibody comprises a CDR3, CDR2, and / or CDR1 sequence selected from the group consisting of:

[0187] aa. SEQ ID NO: 118, 119, and 120;

[0188] bb. SEQ ID NO: 124, 125, and 126;

[0189] or any of the foregoing sequences having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

[0190] 66. An antibody that binds to liraglutide fibrils, wherein the antibody comprises a heavy chain variable region as defined in any of the foregoing embodiments and a light chain variable region as defined in any of the foregoing embodiments.

[0191] 67. An antibody that binds to liraglutide fibrils, wherein the antibody comprises a sequence selected from the group consisting of SEQ ID NO: 109 and 110; or any of the foregoing sequences having at most 20, such as at most 15, or at most 10 amino acid substitutions, deletions, or insertions.

[0192] 68. An antibody that binds to liraglutide fibrils, wherein the antibody has at least 80%, such as at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO: 109 and 110.

[0193] 69. The antibody according to any one of embodiments 37-68, wherein the antibody comprises a variable light chain (VL) sequence selected from the group consisting of SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 111, and 113; or any of the foregoing sequences having at most 20, such as at most 15, or at most 10 amino acid substitutions, deletions, or insertions.

[0194] 70. The antibody according to any one of embodiments 37-68, wherein the antibody comprises a variable heavy chain (VH) sequence selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 112, and 114; or any of the foregoing sequences having at most 20, such as at most 15, or at most 10 amino acid substitutions, deletions, or insertions.

[0195] 71. The antibody according to any one of embodiments 37-70, wherein the antibody is an isolated antibody.

[0196] 72. The antibody according to any one of embodiments 37-71, wherein the antibody is a single-chain Fv fragment.

[0197] 73. An antibody according to any one of embodiments 37 - 72, wherein the antibody comprises an Fc domain.

[0198] 74. An antibody according to any one of embodiments 37 - 73, wherein the antibody is a single-chain Fv fragment that further comprises an Fc domain.

[0199] 75. An antibody according to any one of embodiments 37 - 74, wherein the antibody specifically binds to the liraglutide fibrils and / or the semaglutide fibrils.

[0200] 76. An antibody according to any one of embodiments 37 - 75, wherein the antibody specifically binds to the liraglutide fibrils.

[0201] 77. An antibody according to any one of embodiments 37 - 76, wherein the antibody specifically binds to the semaglutide fibrils.

[0202] 78. An antibody according to any one of embodiments 37 - 77, wherein the fibrils are optionally prepared according to Test (I) herein, and the antibody

[0203] a. The binding level to the liraglutide fibrils is at least 10 - fold the binding level of the antibody to soluble liraglutide, wherein the binding level is determined according to Test (III) at a liraglutide fibril concentration of at least 25 μM; and / or

[0204] b. The limit of detection for the liraglutide fibrils is at a concentration that is at least 10 - fold lower than the limit of detection for the liraglutide fibrils in the ThT assay, wherein the limit of detection is determined according to Test (VI) herein at a liraglutide fibril concentration of at least 1 μM; and / or

[0205] c. The binding level to the liraglutide fibrils is at least 5 - fold the binding level of the antibody to soluble liraglutide, wherein the antibody has a purity of higher than 95% monomer, and wherein the binding level is determined according to Test (III - B) herein at a liraglutide fibril concentration of at least 30 μM; and / or

[0206] d. The limit of detection for the liraglutide fibrils is at a concentration that is at least 10 - fold lower than the limit of detection for the liraglutide fibrils in the ThT assay, wherein the antibody has a purity of higher than 95% monomer, and wherein the limit of detection is determined according to Test (VI - B) herein at a liraglutide fibril concentration of at least 0.025 μM.

[0207] 79. An antibody according to any one of embodiments 37 - 78, wherein the antibody is capable of detecting liraglutide fibrils at a fibril concentration of 1 - 1000 ppm in solution, such as 1 - 10 ppm fibrils, or 10 - 100 ppm fibrils, or 100 - 1000 ppm fibrils.

[0208] 80. An antibody according to any one of embodiments 37 - 79, wherein the antibody has a purity higher than 70%, or higher than 75%, or higher than 80%, or higher than 85%, or higher than 90%, or higher than 95% monomer.

[0209] 81. An antibody according to any one of embodiments 37 - 80, wherein the antibody has a purity higher than 95% monomer.

[0210] 82. Use of an antibody as defined in any one of embodiments 37 - 81 for identifying liraglutide fibrils or semaglutide fibrils.

[0211] 83. Use of an antibody as defined in any one of embodiments 37 - 81 as an affinity ligand to remove fibrils from a mixture comprising (i) liraglutide fibrils and soluble liraglutide or (ii) semaglutide fibrils and soluble semaglutide.

[0212] 84. A method for identifying liraglutide fibrils or semaglutide fibrils, the method comprising the steps of: a) binding an antibody as defined in any one of embodiments 37 - 81 to liraglutide fibrils or semaglutide fibrils.

[0213] 85. A method for quantifying liraglutide fibrils or semaglutide fibrils, the method comprising the steps of: a) binding an antibody as defined in any one of embodiments 37 - 81 to liraglutide fibrils or semaglutide fibrils.

[0214] 86. The method according to any one of embodiments 84 - 85, further comprising the step of: b) detecting the antibody bound to liraglutide fibrils or semaglutide fibrils.

[0215] 87. The method according to any one of embodiments 84 - 86, further comprising the step of: c) quantifying the antibody bound to liraglutide fibrils or semaglutide fibrils, optionally by using a standard of the fibrils.

[0216] 88. The method according to any one of embodiments 84 - 87, wherein the fibrils are in solution.

[0217] 89. The method according to any one of embodiments 84 - 88, wherein the fibrils are in a solution further comprising soluble liraglutide.

[0218] 90. The method according to any one of embodiments 84 - 89, wherein the fibrils are in a solution that does not contain other peptides or proteins other than liraglutide fibrils and optionally contains soluble liraglutide.

[0219] 91. An assay for selectively detecting liraglutide fibrils relative to soluble and / or monomeric liraglutide, comprising an antibody as defined in any one of embodiments 37 - 81, wherein the antibody is capable of detecting liraglutide fibrils at a fibril concentration of 1 - 1000 ppm in solution, such as 1 - 10 ppm fibrils, or 10 - 100 ppm fibrils, or 100 - 1000 ppm fibrils.

[0220] Examples

[0221] List of Abbreviations

[0222] · PBS: Phosphate Buffered Saline (aqueous solution of 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 、1.8 mM KH 2 PO 4 , adjusted to pH 7.4)

[0223] · PES: Polyethersulfone

[0224] · scFv - Fc: Single - chain variable fragment linked to an Fc domain

[0225] · ThT: Thioflavin T

[0226] Materials and Methods

[0227] Antibody Library Preparation, Sorting, and Cloning of Selected Antibody Variants

[0228] Antibodies were isolated through two stages of library sorting. In the first stage of sorting, a single-chain variable fragment (scFv) yeast surface display library was generated by diversifying the heavy-chain complementarity-determining region 3 (HCDR3) of 4D5scFv (Julian et al., 2019; Stimple et al., 2019; Tiller et al., 2017). The scFv was genetically fused to the C-terminus of the yeast Aga2 protein via a flexible linker, enabling the antibody to be displayed on the cell surface. The yeast-displayed antibody library was sorted for binding to liraglutide fibrils (and soluble liraglutide as a control) immobilized on magnetic beads (Dynabeads M-280 Tosylactivated, 14203, Invitrogen). To prepare the beads, 8x10 7 beads were first washed (2x) with 1 mL of sterile PBS. Soluble liraglutide (100 μg, from a 6 mg / mL stock solution in the drug composition buffer) was diluted into PBS containing the magnetic beads (final volume 800 μL) and coupled to the beads overnight (4 °C, without agitation). For beads coated with fibrillar liraglutide, 100 μg of liraglutide fibrils were coupled to the beads in 800 μL of PBS overnight at room temperature with mixing by inversion. The next day, the beads were washed (2x) with 1 mL of PBS supplemented with 10 mM glycine to quench unreacted tosyl groups on the beads, then washed (2x) with 1 mL of PBS supplemented with 1 g / L BSA (PBS-B), and then incubated with yeast. The liraglutide fibril-coated beads were subjected to eight rounds of positive selection in PBS-B supplemented with 1% milk. To isolate yeast carrying conformation-specific antibodies against liraglutide

[0229] fibrils, the last three rounds of sorting combined negative selection against beads coated with soluble liraglutide in PBS-B, followed by positive selection against liraglutide fibrils.

[0230] In the second stage of library sorting (affinity maturation), a sub-library was designed for one of the best clones from the first stage of sorting. The second-generation library diversified the sites in LCDR1, LCDR3, and HCDR2. This library underwent four rounds of selection against liraglutide fibrils. The first two rounds of sorting combined two consecutive negative selections against soluble liraglutide (immobilized on magnetic beads), followed by positive selection against immobilized liraglutide fibrils. Negative selection was performed in PBS-B, while positive selection was performed in PBS-B supplemented with 1% milk. We also performed three consecutive negative selections against beads coated with proglucagon fibrils in rounds 3 and 4. Beads coated with proglucagon fibrils were prepared as previously described (Stimple et al., 2019).

[0231] As previously described (Stimple et al., 2019), selected antibodies were cloned into the mammalian expression vector anti-Notch1_E6-pBIOCAM5. Briefly, the insert and backbone plasmid were digested with NcoI and NotI, purified, and ligated. Insertion of the scFv-encoding fragment was confirmed by Sanger sequencing. These plasmids express a bivalent scFv-human Fc fusion protein with 6xHis and 3xFLAG tags at the C-terminus of the antibody.

[0232] Antibody Expression and Purification

[0233] Proteins were expressed using the Expi293F expression system (Catalog number A14635). Expi293F cells were passaged and expanded until the cells reached a density of approximately 3 - 5 million viable cells per mL. The plasmid (30 μg) was transfected into 25 mL of Expi293 cells. The complex of ExpiFectamine 293 and plasmid DNA was prepared as described in the manufacturer's guidelines. Briefly, the plasmid DNA and ExpiFectamine reagent were diluted with Opti-MEM medium and mixed by gentle pipetting. After incubation for 5 minutes, the diluted transfection reagent was mixed with the diluted DNA. The complex of transfection reagent and DNA was incubated at room temperature for 20 minutes and then added to the Expi293 cells. The cells were incubated with shaking at 37 °C and 5% CO 2 2. The enhancer 1 and 2 solutions were added to the cells 20 hours (post-transfection) according to the manufacturer's instructions. Three days later, the medium containing the secreted antibody was collected and centrifuged at 3400 x g for 45 minutes to remove cells and associated debris.

[0234] Purify the antibody using Protein A chromatography. Wash the Protein A beads (20334, Thermo Fisher Scientific) with PBS and incubate them with glycine buffer (pH 2.5) for 20 minutes. Next, wash the beads with PBS, then add 0.5 mL of the beads to 30 mL of clarified medium and incubate overnight at 4°C. The next day, add the medium containing the Protein A beads to a 10 mL purification column (89898, Thermo Fisher Scientific). Collect the beads by vacuum filtration and wash them thoroughly with PBS (100 mL). Then incubate the Protein A beads with 2 mL of 0.1 M glycine buffer (pH 3.0) for 15 minutes and collect the buffer (with the eluted protein) by centrifugation. Then use Zeba Spin Desalting Columns (89891, Thermo Fisher Scientific) to perform buffer exchange on the eluted antibody into PBS. Measure the protein concentration by absorbance measurement at 280 nm (extinction coefficient is 168,460 - 205,360 M -1 cm -1 ).

[0235] Size Exclusion Chromatography

[0236] Perform analytical and preparative size exclusion chromatography (SEC) experiments using a Shimadzu Prominence HPLC system. The running buffer is 137 mM sodium chloride, 2.7 mM sodium-potassium, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, and 200 mM arginine. The column flow rate is 0.75 mL / min. Inject the antibody sample (0.1 mg / mL) (100 μL) into the column (GE 28990944, Superdex 200 Increase 10 / 300GL column, inner diameter 10 mm, length 300 mm) and monitor the absorbance signals at 220 and 280 nm. For preparative SEC, use an FRC-10A fraction collector to separate the monomer fraction.

[0237] Experiment (I): Preparation of Liraglutide Amyloid Fibrils

[0238] Prepare a test solution of liraglutide at 6 mg / mL in a pharmaceutical composition buffer (14.0 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium hydrogen phosphate dihydrate), and adjust the final pH to 8.15 using NaOH and / or HCl as needed, followed by syringe filtration (0.22 μm PES filter). Aliquot 1 mL of the liraglutide solution into microcentrifuge tubes, add a single 3 mm glass bead (Sigma Z265926) to each tube, and incubate the tubes in a thermal mixer at 37 °C with orbital shaking at 300 rpm for 15 - 20 days.

[0239] Analyze the fibril formation by taking a small sample (about 75 μL) of the test solution from the tube and analyzing it according to Test (V) (ThT assay) described herein, using the positive ThT signal to monitor fibril formation. When the sample shows at least 5 - fold fluorescence compared to the freshly prepared test solution in Test (V) (ThT assay) herein, sediment the fibrils at 221,000 x g (1 hour, 4 °C). Fibrils are observed at the bottom of the tube, e.g., gel - like fibrils. Remove the supernatant from the tube (retain the supernatant for analysis by Test (VII) (BCA assay) herein). Wash the pellet gently once with the pharmaceutical composition buffer at pH 8.15 (without disturbing the pellet), then resuspend it in the original volume of the pharmaceutical composition buffer at pH 8.15 (taking into account any volume removed for ThT analysis), and store at 4 °C. The concentration of the fibrils is determined according to Test (VII) herein; it is important to resuspend the fibril pellet in exactly the same total volume after centrifugation for accurate calculation.

[0240] Experiment (II): Preparation of Semaglutide Amyloid Fibrils

[0241] Aliquot 1 mL of a test solution of semaglutide at 6 mg / mL (optionally containing 50 mM NaCl) and a pharmaceutical composition buffer (14.0 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium hydrogen phosphate dihydrate) adjusted to pH 6.9 using NaOH and / or HCl as needed into microcentrifuge tubes, add a single 3 mm glass bead (Sigma Z265926) to each tube, and incubate the tubes in a thermal mixer at 37 °C with orbital shaking at 300 rpm for 15 - 20 days.

[0242] A small test solution sample (about 75 μL) is taken out from the tube and analyzed according to Test (V) (ThT assay) described herein, and the positive ThT signal is used to monitor fibril formation. When the sample shows at least 5 times the fluorescence of freshly prepared semaglutide in the pH 6.9 pharmaceutical composition buffer in Test (V) (ThT assay), the fibrils are sedimented at 221,000 x g (1 hour, 4 °C). Fibrils are observed at the bottom of the tube, such as gel-like fibrils. The supernatant is taken out from the tube (the supernatant is reserved for analysis by Test (VII) (BCA assay)). The precipitate is gently washed once with the pH 6.9 pharmaceutical composition buffer (without disturbing the precipitate), then resuspended in the original volume of the pH 6.9 pharmaceutical composition buffer (taking into account any volume taken out for ThT analysis), and stored at 4 °C. The concentration of the fibrils is determined according to Test (VII) herein; to make this calculation accurate, it is important to resuspend the fibril precipitate in exactly the same total volume after centrifugation.

[0243] Experiment (III): Antibody Specificity Ratio (Method 1)

[0244] The antibody specificity ratio is determined as follows:

[0245] 1. ELISA plate preparation (3 plates):

[0246] a. For fibril-coated ELISA plates: Resuspend the liraglutide fibrils prepared according to Test (I) herein in the pharmaceutical composition buffer (the fibril concentration is determined by Test (VII) (BCA assay) herein). Approximately 300 μL of the sample is sonicated in a microcentrifuge tube on ice (3 cycles, 10 seconds on / 30 seconds off, 100% amplitude; FB-120 Sonic Dismembrator, Thermo Fisher Scientific). Dilute the solution to 25 μM liraglutide fibrils in PBS and dispense 100 μL of the sample into each well of a 96-well Nunc MaxiSorp ELISA plate (product number: 439454).

[0247] b. For soluble liraglutide-coated plates: Dissolve 6 mg / mL liraglutide in the pharmaceutical composition buffer (14 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium phosphate dihydrate) whose pH is adjusted to 8.15 with NaOH and / or HCl if necessary, and filter the solution through a 0.22 μm PES filter. Dilute the solution to 25 μM liraglutide in PBS and dispense 100 μL of the sample into each well of a 96-well NuncMaxiSorp ELISA plate (product number: 439454).

[0248] i. For the "background" plate: Dispense 100 μL of PBS into each well of a 96-well Nunc MaxiSorp ELISA plate (product number: 439454).

[0249] 2. Cover the plate with parafilm, wrap it in aluminum foil, and incubate overnight at 4 °C.

[0250] 3. The next day, wash the plate 3 times by adding 300 μL of PBS to each well.

[0251] 4. Block the plate by adding 300 μL of PBS supplemented with 0.1% Tween 20 and 10 g / L BSA to each well. Then cover the plate with parafilm, wrap it in aluminum foil, and incubate for 3 hours at room temperature.

[0252] 5. While blocking the plate, centrifuge the antibody samples at 21,000 x g for 5 minutes in a centrifuge and measure the concentration of the supernatant according to the absorbance at 280 nm. Each antibody is serially diluted to 5 nM in PBS supplemented with 0.1% Tween 20 and 1 g / L BSA.

[0253] 6. Wash the plate 3 times by adding 300 μL of PBS to each well

[0254] 7. Dispense 100 μL of the antibody solution into each well. Each antibody is tested in duplicate (i.e., 2 wells per antibody per plate). Then cover the plate with parafilm, wrap it in aluminum foil, and incubate for 1 hour at room temperature.

[0255] 8. The secondary antibody solution is prepared by diluting the secondary antibody (goat anti-human IgG-Fc HRP conjugate, Invitrogen A18817, stock concentration: 0.5 mg / mL in 50% glycerol) 1:1000 into PBS supplemented with 0.1% Tween 20 and 10 g / L BSA.

[0256] 9. Wash the plate 3 times by adding 300 μL of PBS to each well.

[0257] 10. Dispense 100 μL of the secondary antibody solution into each well. Then cover the plate with parafilm, wrap it in aluminum foil, and incubate for 1 hour at room temperature. During the secondary antibody incubation, take out the 1-Step Ultra-TMB ELISA substrate (Thermo Fisher Scientific, 34208) from the refrigerator to equilibrate the solution to room temperature and prepare 2 M (4 N) H 2 SO 4 。

[0258] 11. Wash the plate 3 times by adding 300 μL of PBS to each well.

[0259] 12. Add 100 μL of Ultra-TMB to each well and incubate until a yellow product is formed (5 - 10 minutes).

[0260] 13. Quench the reaction by adding 100 μL of 2M H 2 SO 4 .

[0261] 14. Read the absorbance of each well at 450 nm using a microplate reader (BioTek Synergy Neo).

[0262] Calculation: Calculate the ratio of the ELISA signal (absorbance at 450 nm) of each antibody against the fibril-coated plate to its signal against the soluble liraglutide-coated plate and to the signal of the background plate. These ratios are the fibril / soluble ratio and the fibril / background ratio reported herein. For example: If the signal given by the antibody for liraglutide fibrils is 1.5, for soluble liraglutide is 0.05, and for the background plate is 0.1, then the fibril / soluble ratio will be 1.5 / 0.05 = 30, and the fibril / background ratio will be: 1.5 / 0.1 = 15.

[0263] Experiment (II-B): Antibody Specificity Ratio (Method 1B)

[0264] 1. The night before the assay: Dissolve BSA in PBS at 1 mg / mL, then filter-sterilize through a 0.22 μm PES filter with a 30 cc luer-lock syringe and dispense 150 μL of the solution into each well of a Nunc MaxiSorp (product number: 439454) 96-well ELISA plate. Cover the plate with parafilm, wrap with aluminum foil, and incubate overnight at 4°C.

[0265] 2. Remove the ELISA plate (coated with BSA) from the refrigerator and wash the wells 3 times with 300 μL of PBS.

[0266] a. For the fibril-coated ELISA plate: Resuspend the liraglutide fibrils prepared according to Test (I) herein in the drug composition buffer (determine the fibril concentration by Test (VII) herein (BCA assay)), sonicate approximately 300 μL of the sample in a microcentrifuge tube on ice (3 cycles, 10 seconds on / 30 seconds off, 100% amplitude; FB-120 Sonic Dismembrator, Thermo Fisher Scientific). Dilute the solution to 10 μM liraglutide fibrils in PBS and dispense 100 μL of the sample into each well.

[0267] b. For the liraglutide-coated plate: Dissolve 6 mg / mL liraglutide in a pharmaceutical composition buffer (14 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium phosphate dihydrate) with the pH adjusted to 8.15 using NaOH and / or HCl as needed, and filter the solution through a 0.22 μm PES filter. Dilute the solution to 10 μM liraglutide in PBS and dispense 100 μL of the sample into each well.

[0268] i. For the "background" plate: Dispense 100 μL of PBS into each well of a 96-well Nunc MaxiSorp ELISA plate (product number: 439454).

[0269] 3. Cover the plate with a plastic film, wrap it with aluminum foil, and incubate it at room temperature for 3 hours without agitation.

[0270] 4. During these 3 hours, centrifuge the antibody samples at 21,000 x g for 5 minutes in a centrifuge and measure the concentration of the supernatant based on the absorbance at 280 nm. Each antibody is serially diluted to 5 nM in PBS supplemented with 0.1% Tween 20 and 1 g / L BSA (unless the concentration is specified otherwise).

[0271] 5. Wash the plate 3 times by adding 300 μL of PBS to each well.

[0272] 6. Dispense 100 μL of the antibody solution into each well. Then cover the plate with a plastic film, wrap it with aluminum foil, and incubate it at room temperature for 1 hour.

[0273] 7. The secondary antibody solution is prepared by diluting the secondary antibody (goat anti-human IgG-Fc HRP conjugate, Invitrogen A18817, stock concentration: 0.5 mg / mL in 50% glycerol) 1:1000 into PBS supplemented with 0.1% Tween 20 and 10 g / L BSA.

[0274] 8. Wash the plate 3 times by adding 300 μL of PBST (PBS supplemented with 0.1% Tween 20) to each well.

[0275] 9. Dispense 100 μL of the secondary antibody solution into each well. Then cover the plate with a plastic film, wrap it with aluminum foil, and incubate it at room temperature for 1 hour. During the incubation of the secondary antibody, take out the 1-Step Ultra-TMB ELISA substrate (Thermo Fisher Scientific, 34208) from the refrigerator to equilibrate the solution to room temperature and prepare 2 M (4 N) H 2 SO 4 .

[0276] 10. Wash the plate three times by adding 300 μL of PBST to each well.

[0277] 11. Add 100 μL of Ultra-TMB to each well and incubate until a yellow product forms (5 - 10 minutes).

[0278] 12. Quench the reaction by adding 100 μL of 2M H 2 SO 4 to it.

[0279] 13. Read the absorbance of each well at 450 nm using a microplate reader (BioTek Synergy Neo).

[0280] Calculation: Calculate the ratio of the ELISA signal (absorbance at 450 nm) of each antibody against the fibril-coated plate to its signal against the soluble liraglutide-coated plate and to the signal of the background plate. These ratios are the fibril / soluble ratio and the fibril / background ratio reported herein. For example: If the signal given by an antibody for liraglutide fibrils is 1.5, for soluble liraglutide is 0.05, and for the background plate is 0.1, then the fibril / soluble ratio will be 1.5 / 0.05 = 30, and the fibril / background ratio will be: 1.5 / 0.1 = 15.

[0281] Experiment (IV): Antibody Specificity Ratio (Method 2)

[0282] Determine the antibody specificity ratio as follows:

[0283] The night before the assay: Dissolve BSA at 1 mg / mL in PBS, then filter-sterilize it through a 0.22 μm PES filter with a 30 cc luer-lock syringe and dispense 150 μL of the solution into each well of a Nunc MaxiSorp (product number: 439454) 96-well ELISA plate. Cover the plate with a plastic film, wrap it with aluminum foil, and incubate overnight at 4 °C.

[0284] On the day of the assay:

[0285] 1. Dissolve liraglutide at 60 mg / mL in a pharmaceutical composition buffer (14 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium phosphate dihydrate) whose pH is adjusted to 8.15 with NaOH and / or HCl as needed, and filter it through a 0.22 μm PES filter. Dilute the solution to 6 mg / mL (1600 μM) liraglutide in PBS. This solution is referred to as "the solution of soluble liraglutide".

[0286] 2. Resuspend the liraglutide fibrils (hereinafter: fibrils) obtained according to Test (I) herein in about 300 μL of the drug composition buffer at pH 8.15 (the concentration of the fibrils was determined by Test (VII) herein (BCA assay)), and perform sonication on ice in a microcentrifuge tube (3 cycles, 10 seconds on / 30 seconds off, 100% amplitude; FB-120 Sonic Dismembrator, Thermo Fisher Scientific). This solution is referred to as the "fibril solution".

[0287] 3. Dilute the sonicated fibril solution into a solution of soluble liraglutide such that the final concentration of the fibrils is 100 μM. Then further serially dilute this solution into a solution of soluble liraglutide to obtain samples of 0.1 μM fibrils. Use two controls: i) PBS (peptide-free), and ii) fibril-free (solution of only soluble liraglutide).

[0288] 4. Remove the ELISA plate (coated with BSA) from the refrigerator and wash the wells 3 times with 300 μL of PBS.

[0289] 5. Dispense 100 μL of each sample or control from above (3) into the wells of the freshly washed plate, cover the plate with a plastic film, wrap it with aluminum foil, and incubate it at room temperature without stirring for 3 hours.

[0290] 6. During the above 3-hour incubation, centrifuge approximately 75 μL of the antibody to be tested (e.g., scFv-Fc fusion protein) at 21,000 x g for 5 minutes to sediment any particles. Take out the supernatant and measure the A280nm of this supernatant to calculate the antibody concentration. Serially dilute the antibody in PBS + 0.1% Tween 20 + 1 g / L BSA to 5 nM and keep it on ice until use.

[0291] 7. At the end of the 3-hour incubation, wash the wells of the plate 3 times with 300 μL of PBS.

[0292] 8. Add 100 μL of 5 nM antibody to each well. Cover the plate with a plastic film, wrap it with aluminum foil, and incubate it at room temperature for 1 hour.

[0293] 9. During the above 1-hour incubation, dilute the secondary antibody (goat anti-human IgG-Fc HRP conjugate, Invitrogen A18817, stock concentration: 0.5 mg / mL in 50% glycerol) 1:1000 into PBS + 0.1% Tween 20 + 10 g / L BSA.

[0294] 10. At the end of the 1-hour incubation, wash the wells 3 times with 300 μL of PBS.

[0295] 11. Add 100 μL of the secondary antibody solution to each well. Cover the plate with a plastic film, wrap it with aluminum foil, and incubate at room temperature for 1 hour.

[0296] 12. During the incubation of the secondary antibody, take out the 1-Step Ultra-TMB ELISA substrate (ThermoFisher Scientific, 34208) from the refrigerator to equilibrate the solution to room temperature. Prepare 2 M (4 N) H 2 SO 4 .

[0297] 13. At the end of the 1-hour incubation, wash the wells three times with 300 μL of PBS.

[0298] 14. Add 100 μL of Ultra-TMB to each well and incubate for 10 minutes.

[0299] 15. Quench the reaction by adding 100 μL of 2 M H 2 SO 4 .

[0300] 16. Read the absorbance at 450 nm in a microplate reader (BioTek Synergy Neo).

[0301] Calculation: Divide the ELISA signal of each antibody in the wells containing fibrils by the ELISA signal of the same antibody in the control wells with soluble liraglutide and no fibrils. This ratio is the mixture / soluble specific ratio. For example: If a given antibody gives a signal of 1.5 for 0.1 μM liraglutide fibrils mixed with soluble liraglutide and a signal of 0.05 for soluble liraglutide (lacking fibrils): the mixture / soluble specific ratio will be 1.5 / 0.05 = 30.

[0302] Experiment (V): ThT Assay

[0303] The presence or absence of fibrils in the test solution of the peptide is analyzed immediately after fibril formation. The peptide concentration before fibril formation is 6 mg / ml. The peptide can be liraglutide or semaglutide. Liraglutide fibrils can be prepared according to Test (I) herein. Semaglutide fibrils can be prepared according to Test (II) herein. 75 μL of the test solution sample is mixed with 1.36 μL of the ThT stock solution (stock concentration: 2200 μM ThT) to achieve a final ThT concentration of 40 μM in the peptide / ThT mixture. For liraglutide, the final concentration in this mixture is 1571 μM liraglutide (calculated before fibrillization). 50 μL of the peptide / ThT mixture sample is added to the wells of a black 384-well plate (Fisherbrand 384-well polystyrene plate, 12566624, ThermoFisher Scientific). After 5 - 10 minutes, the ThT fluorescence (λex = 444 nm, λem = 482 nm) value is measured using a Biotek Synergy Neo microplate reader.

[0304] Experiment (VI): Comparison of ThT Assay and Antibody Assay

[0305] The detection of liraglutide fibrils in a mixture with soluble liraglutide is determined using the ThT detection method as compared to an antibody assay.

[0306] 1. Dissolve liraglutide at 60 mg / mL in a pharmaceutical composition buffer (14 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium phosphate dihydrate) with the pH adjusted to 8.15 using NaOH and / or HCl as needed, and filter through a 0.22 μm PES filter. Dilute the solution to 6 mg / mL (1600 μM) liraglutide in PBS. This solution is referred to as the "solution of soluble liraglutide".

[0307] 2. Resuspend the liraglutide fibrils (hereinafter: fibrils) obtained according to Test (I) herein in a pharmaceutical composition buffer at pH 8.15 (the concentration of the fibrils is determined by Test (VII) (BCA assay) herein) in a microcentrifuge tube and sonicate on ice (3 cycles, 10 seconds on / 30 seconds off, 100% amplitude; FB-120 Sonic Dismembrator, Thermo Fisher Scientific). This solution is referred to as the "fibril solution".

[0308] 3. Dilute the ultrasonically treated fibril solution into a solution of soluble liraglutide such that the final concentration of fibrils is 100 μM. Then further serially dilute this solution into a solution of soluble liraglutide to obtain samples of 0.001, 0.0025, 0.01, 0.025, 0.1, 0.25, 1, 2.5, 10, and 25 μM fibrils. Use two controls: i) PBS (peptide-free), and ii) fibril-free (solution of soluble liraglutide only).

[0309] a. At this time, add (100 μL) the solution obtained by diluting the fibril solution into a solution of soluble liraglutide (from step 3) to the wells of an ovalbumin-coated ELISA plate and incubate at room temperature for 2 hours, after which ELISA detection is performed using the antibody of the present invention. The remaining ELISA protocol is carried out as described in experiment (IV) (after step 7).

[0310] 4. Incubate the residual samples (the mixture of fibrils and soluble liraglutide from step 3, and the PBS control and soluble liraglutide control) in microcentrifuge tubes at room temperature for 2.5 hours.

[0311] 5. Prepare a stock solution of thioflavin T (ThT) at a concentration of 2200 μM. Add ThT to the samples (initial total peptide concentration of 1600 μM) to a final concentration of 40 μM, and add 50 μM samples of the peptide / ThT mixture to the wells of a black 384-well plate (Fisherbrand 384-well polystyrene plate, 12566624, Thermo Fisher Scientific). Measure the ThT fluorescence (λex = 444 nm, λex = 482 nm) values of each sample using a Biotek Synergy Neo microplate reader. The final (total) peptide concentration in the peptide / ThT mixture is 1571 μM (calculated before fibrillation).

[0312] 6. Divide the fluorescence measurement value of the solution containing fibrils by the fluorescence measurement value of the solution of soluble liraglutide (fibril-free control), and report this ratio as the mixture / soluble ratio.

[0313] For the detection of liraglutide fibrils in a mixture with soluble liraglutide, use the ThT detection method as compared to the antibody assay.

[0314] Experiment (VI-B): Comparison of ThT Assay and Antibody Assay

[0315]

[0316] ​1. The night before the assay: Dissolve BSA at 1 mg / mL in PBS, then filter-sterilize through a 0.22 μm PES filter with a 30 cc luer-lock syringe, and dispense 150 μL of the solution into each well of a Nunc MaxiSorp (product number: 439454) 96-well ELISA plate. Cover the plate with a plastic film, wrap it with aluminum foil, and incubate overnight at 4 °C.

[0317] 2. On the day of the assay, fix the liraglutide mixture on the BSA-coated plate.

[0318] a. Dissolve liraglutide at 60 mg / mL in a pharmaceutical composition buffer (14 mg / mL propylene glycol, 5.5 mg / mL phenol, 1.42 mg / mL disodium phosphate dihydrate) with the pH adjusted to 8.15 using NaOH and / or HCl as needed, and filter through a 0.22 μm PES filter. Dilute the solution to 6 mg / mL (1600 μM) liraglutide in PBS. This solution is referred to as "solution of soluble liraglutide".

[0319] b. Resuspend the liraglutide fibrils (hereinafter: fibrils) obtained according to test (I) herein in approximately 300 μL of the pharmaceutical composition buffer at pH 8.15 (determine the concentration of fibrils by test (VII) herein (BCA assay)) in a microcentrifuge tube and sonicate on ice (3 cycles, 10 s on / 30 s off, 100% amplitude; FB-120 Sonic Dismembrator, Thermo Fisher Scientific). This solution is referred to as "fibril solution".

[0320] c. Dilute the sonicated fibril solution into the solution of soluble liraglutide such that the final concentration of fibrils is 100 μM. Then further serially dilute this solution into the solution of soluble liraglutide to obtain samples of 0.001, 0.0025, 0.01, 0.025, 0.1, 0.25, 1, 2.5, 10, and 25 μM fibrils. Use two controls: i) PBS (peptide-free), and ii) fibril-free (only solution of soluble liraglutide).

[0321] d. Add (100 μL) the solution obtained by diluting the fibril solution into the solution of soluble liraglutide to the wells of the BSA-coated ELISA plate and incubate for 3 hours at room temperature.

[0322] 3. The remaining ELISA protocol is carried out as described in test (IV) with some modifications.

[0323] a. During this 3-hour process, the antibody samples were centrifuged at 21,000 x g for 5 minutes in a centrifuge, and the concentration of the supernatant was measured based on the absorbance at 280 nm. Each antibody was serially diluted to 5 nM in PBS (PBST) supplemented with 0.1% Tween 20.

[0324] b. The plate was washed 3 times by adding 300 μL of PBS to each well.

[0325] c. 100 μL of the antibody solution was dispensed into each well. Then the plate was covered with a plastic film, wrapped with aluminum foil, and incubated at room temperature for 1 hour.

[0326] d. The secondary antibody solution was prepared by diluting the secondary antibody (goat anti-human IgG-Fc HRP conjugate, Invitrogen A18817, stock concentration: 0.5 mg / mL in 50% glycerol) 1:1000 into PBS supplemented with 0.1% Tween 20 and 10 g / L BSA.

[0327] e. The plate was washed 3 times by adding 300 μL of PBST to each well.

[0328] f. 100 μL of the secondary antibody solution was dispensed into each well. Then the plate was covered with a plastic film, wrapped with aluminum foil, and incubated at room temperature for 1 hour. During the secondary antibody incubation, 1-Step Ultra-TMB ELISA substrate (Thermo Fisher Scientific, 34208) was taken out of the refrigerator to equilibrate the solution to room temperature and prepare 2 M (4 N) H 2 SO 4 。

[0329] g. The plate was washed 3 times by adding 300 μL of PBST to each well.

[0330] h. 100 μL of Ultra-TMB was added to each well and incubated until a yellow product formed (5 - 10 minutes).

[0331] i. The reaction was quenched by adding 100 μL of 2 M H 2 SO 4 。

[0332] j. The absorbance of each well was read at 450 nm in a microplate reader (BioTek Synergy Neo).

[0333] 4. The residual samples (the mixture of fibrils and soluble liraglutide from step 3, as well as the PBS control and the soluble liraglutide control) were incubated at room temperature in microcentrifuge tubes for 2.5 hours.

[0334] 5. Prepare a stock solution of Thioflavin T (ThT) at a concentration of 2200 μM. Add ThT to the sample (initial total peptide concentration of 1600 μM) to a final concentration of 40 μM, and add 50 μM of the peptide / ThT mixture sample to the wells of a black 384-well plate (Fisherbrand 384-well polystyrene plate, 12566624, Thermo Fisher Scientific). Measure the ThT fluorescence (λem = 444 nm, λem = 482 nm) values of each sample using a Biotek Synergy Neo microplate reader. The final (total) peptide concentration in the peptide / ThT mixture is 1571 μM (calculated before fibrillization).

[0335] 6. Divide the fluorescence measurement value of the solution containing fibrils by the fluorescence measurement value of the solution of soluble liraglutide (no fibril control), and report this ratio as the mixture / soluble ratio.

[0336] Experiment (VII): BCA Assay

[0337] The concentration of fibrils (such as liraglutide fibrils) is determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, 23225), with liraglutide as the standard for liraglutide fibrils and semaglutide as the standard for semaglutide fibrils (instead of BSA). Since the degree of reaction of phenol from the drug composition buffer with the BCA reagent varies according to the sample dilution, controls are run for quantification to account for the background signal generated by phenol. The concentration of fibrils in the resuspended fibril solution from Experiment (I) or Experiment (II) herein is determined by subtracting the peptide concentration in the supernatant (after ultracentrifugation) from the initial concentration (6 mg / mL) during fibril assembly. The analysis of liraglutide fibrils is performed as follows:

[0338] 1. Dissolve liraglutide at 6 mg / mL (6000 μg / mL) in the drug composition buffer at pH 8.15. This is referred to as the "solution of soluble liraglutide".

[0339] 2. For the standards, dilute the solution of soluble liraglutide from (1) into PBS at concentrations of 2000, 1500, 1000, 750, 500, 250, 125, 25, and 0 μg / mL, and prepare "blanks" that contain the same amount of the drug composition buffer (pH 8.15) diluted in PBS (but lacking the peptide). For example, to prepare 600 μL of the 2000 μg / mL standard, 200 μL of the solution from (1) and 400 μL of PBS are required. To prepare the blank, mix 200 μL of the drug composition buffer (without peptide) and 400 μL of PBS.

[0340] 3. Dilute the supernatant of the ultracentrifuged fibrils obtained from the present experiment (I) (in the drug composition buffer (pH 8.15)) into PBS at the following dilution factors: 1:2, 1:4, 1:8, 1:16, 1:32. "Blanks" are also prepared for these samples, which contain the drug composition buffer (pH 8.15) diluted into PBS at the same dilution factors.

[0341] 4. Add 10 μL of the standards (and separate wells with the corresponding blanks) and 10 μL of the diluted samples (and separate wells with the corresponding blanks) to each well of a transparent (non-binding or low-binding) flat-bottom 96-well plate.

[0342] 5. Prepare the BCA working reagent, add 225 μL to each well, and cover the plate with a plastic film.

[0343] 6. Incubate the plate at 37 °C until sufficient purple color has formed (this usually occurs relatively quickly, and in some samples, some colors are usually almost immediately visible).

[0344] 7. Read the absorbance at 562 nm in a microplate reader.

[0345] 8. Subtract the "blank" absorbance value of the standards from the values of the standards. Fit a standard curve to the (background-subtracted) absorbance obtained by plotting the peptide concentration versus the absorbance and fitting a second-order polynomial.

[0346] 9. Subtract the "blank" absorbance value of the supernatant from the values of the supernatant dilutions. Using the standard curve from (8), determine the peptide concentration of the supernatant samples and multiply them by their dilution factors to calculate the liraglutide concentration in the undiluted supernatant. Samples with concentrations in the range of approximately 250 - 1000 μg / mL are preferably calculated (this is the midpoint of the accurate range of the BCA assay).

[0347] 10. Since the fibrils were assembled at a concentration of 6 mg / mL (6000 μg / mL), the peptide concentration of the supernatant was subtracted therefrom to obtain the concentration of the fibrils in the resuspended sample. To make this calculation accurate, it was important to resuspend the fibril precipitate in Experiment (I) at exactly the same total volume after centrifugation.

[0348] A similar procedure can be used for semaglutide fibrils, except that Experiment (I) mentioned herein should be replaced with Experiment (II) herein.

[0349] Results

[0350] Example 1: Antibody

[0351] An antibody (scFv-Fc) having the amino acid sequence listed in Table 1 was prepared and purified by recombinant expression.

[0352] Table 1 lists the complete sequence of each antibody; the bold text shows the CDR positions (shown in the order of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, and H-CDR3); the CDRs are defined according to the Kabat antibody numbering scheme. Table 2 lists the variable light chain (V L sequence), variable heavy chain (V H sequence) amino acid sequences of the antibodies in Table 1. Table 3 lists the CDRs of the antibodies in Table 1.

[0353] Table 1. Antibody Complete Sequence Information

[0354]

[0355]

[0356]

[0357]

[0358] Table 2. Antibody Variable Light Chain (V L ) and Variable Heavy Chain (V H ) Amino Acid Sequences

[0359]

[0360]

[0361] Table 3. Antibody CDR Sequences

[0362]

[0363]

[0364] Example 2: Liraglutide Amyloid Fibril Specificity of Antibody

[0365] The ability of the antibody of Example 1 to bind liraglutide fibrils was tested alone, compared to the background and / or soluble liraglutide. The testing was performed according to Assay (III) or Assay (IV) as defined herein. The results are shown in Tables 4 and 5.

[0366] Table 4. Liraglutide fibril specificity of antibodies determined according to Assay (III) herein using a 25 μM liraglutide fibril concentration

[0367]

[0368]

[0369] Table 5. Liraglutide fibril specificity of antibodies determined in a mixture with soluble liraglutide according to Assay (IV) herein using a 0.1 μM liraglutide fibril concentration

[0370]

[0371] The results in Tables 4 and 5 show that the antibodies tested bind significantly more to liraglutide fibrils than to soluble liraglutide. The results in Table 4 also show that the antibodies tested bind significantly more to liraglutide fibrils than to the background. The results in Table 5 show that the antibodies tested also bind significantly more to liraglutide fibrils when tested in a mixture with a high concentration of soluble liraglutide.

[0372] Example 3: Detection Sensitivity of Liraglutide Amyloid Fibrils - Comparison between Antibody and ThT Assay

[0373] The ability of the ThT assay to bind liraglutide fibrils in a mixture containing excess soluble liraglutide was tested to allow for sensitivity comparison with the antibodies of the present invention. This experiment was performed according to Assay (VI) herein. The results are shown in Table 6.

[0374] Table 6. Detection sensitivity of liraglutide fibrils; results are reported as the specificity ratio (liraglutide fibrils in mixture / soluble liraglutide).

[0375]

[0376] Example 4: Concentration-Dependent Binding Analysis of Liraglutide Antibody

[0377] Antibodies were prepared as described in the "Antibody Expression and Purification" section. Antibodies E, M, and N were purified in two steps with a yield > 20 mg / L. The purified antibodies were mainly monomers, as demonstrated by analytical size exclusion chromatography (for E, M, and N, > 95% monomers; Figure 1 ). The sensitivity of assays using the two-step purified antibodies (> 95% monomers) was also enhanced by removing BSA during the first antibody incubation. These changes led to an improved assay (III-B).

[0378] Antibodies E, M, and N of Example 1 were purified in two steps (> 95% monomers) and tested for their ability to bind liraglutide fibrils alone, compared to background and / or soluble liraglutide. Testing was performed according to Assay (III-B) herein. The results are shown in Table 7 (for aggregated and soluble liraglutide, raw (minus non-background) antibody binding signal) and Table 8 (liraglutide fibril specificity of the antibody (liraglutide fibrils / monomeric liraglutide)). Three independent experiments were performed and the reported values are the averages.

[0379] Table 7: Antibody binding signals to aggregated and soluble liraglutide for the two-step purified antibodies.

[0380]

[0381] Table 8: Liraglutide fibril specificity of the antibody (liraglutide fibrils / liraglutide monomer) for the two-step purified antibodies.

[0382]

[0383]

[0384] The results in Tables 7 and 8 show that the tested antibodies bound significantly more to liraglutide fibrils than to soluble liraglutide.

[0385] It was also observed that using highly purified antibodies (> 95% monomers) in the assay had the advantage of making Assay (III-B) more reproducible than Assay (III) previously using one-step purified antibodies (> 5% antibody aggregates), because it was easier to control the amount of antibody aggregates in different batches of two-step purified antibodies (> 95% monomers). Removing antibody aggregates using size exclusion chromatography decreased the antibody sensitivity at low antibody concentrations because antibody aggregates contributed to the binding to liraglutide fibrils. However, the increased antibody purity allowed the use of higher antibody concentrations due to lower background signals, which made it possible to increase the detection sensitivity.

[0386] Example 5: Detection Sensitivity of Liraglutide Amyloid Fibrils - Comparison between Antibody and ThT Assay

[0387] Antibodies were prepared as described in the "Antibody Expression and Purification" section. Antibodies E, M, and N were purified in two steps with a yield > 20 mg / L. The purified antibodies were mainly monomers, as demonstrated by analytical size exclusion chromatography (for E, M, and N, > 95% monomer; Figure 1 ). The sensitivity of the assay using the two-step purified antibodies (> 95% monomer) was also enhanced by removing BSA and increasing the antibody concentration (from 5 nM to 50 nM) during the first antibody incubation, resulting in an improved assay (VI-B).

[0388] The ability of the ThT assay to bind to liraglutide fibrils in a mixture containing excess soluble liraglutide was tested to allow for a sensitivity comparison with antibody M of the present invention. This experiment was conducted according to assay (VI-B) herein. The results are shown in Table 9.

[0389] Table 9. Sensitivity of detecting liraglutide fibrils using assay (VI-B) for two-step purified antibody M (> 95% monomer); results are reported as the specificity ratio (liraglutide fibrils in the mixture / soluble liraglutide), ThT concentration = 0.4 μM.

[0390]

[0391] The results in Table 9 show that the antibodies of the present invention detect liraglutide fibrils with a sensitivity several orders of magnitude higher than the ThT assay. Additionally, it was also found that the antibodies of the present invention detect liraglutide fibrils at fibril concentrations at which no signal was recorded in the ThT assay.

[0392] It was also observed that using highly purified antibodies (> 95% monomer) in the assay has the advantage of making assay (VI-B) more reproducible than assay (VI) previously using one-step purified antibodies (> 5% antibody aggregates), because the amount of antibody aggregates is more easily controlled in different batches of two-step purified antibodies (> 95% monomer). Removing antibody aggregates using size exclusion chromatography reduces the antibody sensitivity at low antibody concentrations (e.g., 5 nM) because antibody aggregates contribute to the binding to liraglutide fibrils. However, the increased antibody purity allows for the use of higher antibody concentrations (50 nM instead of 5 nM) due to the lower background signal, which enables an improvement in detection sensitivity.

[0393] Example 6: Reproducibility of Antibody with Purity > 95% Monomer

[0394] The antibody M of Example 1 was purified in two steps (>95% monomer), and its ability to bind liraglutide fibrils in the presence of excess soluble liraglutide was tested compared to soluble liraglutide. The test was conducted according to the assay (VI-B) herein. Two different batches of the antibody were tested, and a total of four independent experiments were performed. The results are shown in Table 10.

[0395] Table 10. Sensitivity of detecting liraglutide fibrils for the dual-sorted antibody M using assay (VI-B); results are reported as specific ratio (liraglutide fibrils in mixture / soluble liraglutide), ThT concentration = 0.4 μM.

[0396]

[0397] The results in Table 10 show that using a highly purified antibody (>95% monomer) in the assay led to very reproducible results.

[0398] Although certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. Accordingly, it is to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true scope of the invention.

Claims

1. An antibody or an antigen-binding fragment thereof that binds to liraglutide fibrils, wherein the antibody or the antigen-binding fragment thereof comprises the H-CDR1, H-CDR2, and H-CDR3 sequences of the heavy-chain variable region and the L-CDR1, L-CDR2, and L-CDR3 sequences of the light-chain variable region, and wherein the H-CDR3, H-CDR2, and H-CDR1 sequences and the L-CDR3, L-CDR2, and L-CDR1 sequences correspond respectively to: a. SEQ ID NO: 37, 38, and 39, and SEQ ID NO: 40, 41, and 42; b. SEQ ID NO: 43, 44, and 45, and SEQ ID NO: 46, 47, and 48; c. SEQ ID NO: 49, 50, and 51, and SEQ ID NO: 52, 53, and 54; d. SEQ ID NO: 55, 56, and 57, and SEQ ID NO: 58, 59, and 60; e. SEQ ID NO: 61, 62, and 63, and SEQ ID NO: 64, 65, and 66; f. SEQ ID NO: 67, 68, and 69, and SEQ ID NO: 70, 71, and 72; g. SEQ ID NO: 73, 74, and 75, and SEQ ID NO: 76, 77, and 78; h. SEQ ID NO: 79, 80, and 81, and SEQ ID NO: 82, 83, and 84; i. SEQ ID NO: 85, 86, and 87, and SEQ ID NO: 88, 89, and 90; j. SEQ ID NO: 91, 92, and 93, and SEQ ID NO: 94, 95, and 96; k. SEQ ID NO: 97, 98, and 99, and SEQ ID NO: 100, 101, and 102; l. SEQ ID NO: 103, 104, and 105, and SEQ ID NO: 106, 107, and 108; m. SEQ ID NO: 115, 116, and 117, and SEQ ID NO: 118, 119, and 120; or n. SEQ ID NO: 121, 122, 123, and SEQ ID NO: 124, 125, and 126.

2. The antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody or the antigen-binding fragment thereof comprises a light-chain variable region (VL) sequence and a heavy-chain variable region (VH) sequence, and wherein the VL sequence and the VH sequence correspond respectively to: i. SEQ ID NO: 13 and 14; ii. SEQ ID NO: 15 and 16; iii. SEQ ID NO: 17 and 18; iv. SEQ ID NO: 19 and 20; v. SEQ ID NO: 21 and 22; vi. SEQ ID NO: 23 and 24; vii. SEQ ID NO: 25 and 26; viii. SEQ ID NO: 27 and 28; ix. SEQ ID NO: 29 and 30; x. SEQ ID NO: 31 and 32; xi. SEQ ID NO: 33 and 34; xi. SEQ ID NO: 35 and 36; xiii. SEQ ID NO: 111 and 112; or xiv. SEQ ID NO: 113 and 114。 3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 109, and 110.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof is a single-chain Fv fragment.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody or antigen-binding fragment thereof is a single-chain Fv fragment comprising an Fc domain.

6. An antibody or antigen-binding fragment thereof that binds to liraglutide fibrils, comprising: a heavy-chain variable region comprising H-CDR1, H-CDR2, and H-CDR3 and a light-chain variable region comprising L-CDR1, L-CDR2, and L-CDR3, wherein the H-CDR1, H-CDR2, and H-CDR3 correspond to SEQ ID NO: 63, 62, and 61, respectively, and the L-CDR1, L-CDR2, and L-CDR3 correspond to SEQ ID NO: 66, 65, and 64, respectively.

7. An antibody or antigen-binding fragment thereof comprising a light-chain variable region (VL) sequence and a heavy-chain variable region (VH) sequence, wherein the VL sequence and VH sequence correspond to SEQ ID NO: 21 and 22, respectively.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the antibody or antigen-binding fragment thereof comprises the sequence of SEQ IDNO:

5.

9. An antibody or antigen-binding fragment thereof that binds to liraglutide fibrils, comprising: a heavy-chain variable region comprising H-CDR1, H-CDR2, and H-CDR3 and a light-chain variable region comprising L-CDR1, L-CDR2, and L-CDR3, wherein the H-CDR1, H-CDR2, and H-CDR3 correspond to SEQ ID NO: 117, 116, and 115, respectively, and the L-CDR1, L-CDR2, and L-CDR3 correspond to SEQ ID NO: 120, 119, and 118, respectively.

10. An antibody or antigen-binding fragment thereof comprising a light-chain variable region (VL) sequence and a heavy-chain variable region (VH) sequence, wherein the VL sequence and VH sequence correspond to SEQ ID NO: 111 and 112, respectively.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the antibody or antigen-binding fragment thereof comprises the sequence of SEQ ID NO:

109.

12. An antibody or antigen-binding fragment thereof that binds to liraglutide fibrils, comprising: a heavy chain variable region comprising H-CDR1, H-CDR2, and H-CDR3 and a light chain variable region comprising L-CDR1, L-CDR2, and L-CDR3, wherein the H-CDR1, H-CDR2, and H-CDR3 correspond to SEQ ID NOs: 123, 122, and 121, respectively, and the L-CDR1, L-CDR2, and L-CDR3 correspond to SEQ ID NOs: 126, 125, and 124, respectively.

13. An antibody or antigen-binding fragment thereof, comprising a light chain variable region (VL) sequence and a heavy chain variable region (VH) sequence, wherein the VL sequence and VH sequence correspond to SEQ ID NOs: 113 and 114, respectively.

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein the antibody or antigen-binding fragment thereof comprises the sequence of SEQ ID NO:

110.

15. The antibody or antigen-binding fragment thereof according to any one of claims 7-8, 10-11, and 13-14, wherein the antibody binds to liraglutide fibrils.

16. Use of the antibody or antigen-binding fragment thereof as defined in any one of claims 1-15 for identifying liraglutide fibrils and / or for removing fibrils from a mixture comprising liraglutide fibrils and soluble liraglutide as an affinity ligand.

17. A method for identifying and / or quantifying liraglutide fibrils, the method comprising the following steps: contacting the antibody or antigen-binding fragment thereof as defined in any one of claims 1-15 with liraglutide.

18. The method according to claim 17, wherein the method further comprises the following steps: detecting the antibody that binds to liraglutide fibrils.

19. The method according to claim 18, wherein the method further comprises the following steps: quantifying the antibody that binds to liraglutide fibrils.

20. The method according to claim 19, wherein the method comprises quantifying the antibody that binds to liraglutide fibrils by using a standard of the fibrils.

21. An assay method for detecting liraglutide fibrils relative to soluble and / or monomeric liraglutide, comprising using the antibody or antigen-binding fragment thereof as defined in any one of claims 1-15, wherein the antibody or antigen-binding fragment thereof is capable of detecting liraglutide fibrils at a fibril concentration of 1-1000 ppm in solution.

22. The assay method according to claim 21, wherein the antibody or antigen-binding fragment thereof is capable of detecting liraglutide fibrils at a fibril concentration of 1-10 ppm in solution.

23. The assay method according to claim 21, wherein the antibody or antigen-binding fragment thereof is capable of detecting liraglutide fibrils at a fibril concentration of 10-100 ppm in solution.

24. The assay method according to claim 21, wherein the antibody or antigen-binding fragment thereof is capable of detecting liraglutide fibrils at a concentration of 100-1000 ppm in solution.

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