Novel BSSL Antibody

Abstract

The present invention relates to novel isolated antibodies and antigen-binding fragments thereof that bind to human bile salt-stimulated lipase (hBSSL). The antibodies and antigen-binding fragments thereof bind to a previously uncharacterized epitope located at the N-terminal portion of hBSSL and identified as comprising amino acid residues 7-12 and amino acid residues 42-55. The present invention also relates to the medical use of the antibodies and / or antigen-binding fragments thereof, in particular in the treatment of inflammatory conditions, and to related pharmaceutical compositions.

Description

Technical Field

[0001] The present invention relates to novel isolated antibodies and antigen-binding fragments thereof that bind to a previously uncharacterized epitope of bile salt-stimulated lipase (BSSL) located in the N-terminal portion of the BSSL protein. The present invention also relates to the medical uses of the antibodies and antigen-binding fragments thereof, particularly in the treatment of inflammatory conditions, and to related pharmaceutical compositions. Also disclosed herein is the use of the antibodies or antigen-binding fragments thereof as molecular tools for detecting BSSL and / or diagnosing BSSL-related diseases. Background Art

[0002] Inflammatory disorders, including autoimmune and autoinflammatory diseases, remain a significant threat to human health. Despite advances in the treatment of inflammatory disorders, improved therapies are still sought.

[0003] Inflammatory conditions include a large number of illnesses and diseases characterized by inflammation, including autoimmune diseases and autoinflammatory diseases. Inflammation can occur, for example, as a response to infection, injury, allergens and / or toxins, or as a response to the body itself, such as an autoimmune process. Autoimmune diseases occur when the body's immune system mistakenly attacks and destroys healthy body tissues. It is reported that there are more than about 80 known autoimmune diseases.

[0004] Some inflammatory conditions are chronic. Chronic inflammation occurs when the inflammatory response persists, keeping the body in a constant state of alert. Examples of inflammatory diseases and conditions that include chronic inflammation are rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA), and inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD).

[0005] RA is a chronic, inflammatory, systemic autoimmune disease. Current therapies for RA include nonsteroidal anti-inflammatory drugs (NSAIDs) for pain management, disease-modifying antirheumatic drugs (DMARDs), and biologic agents that target specific proinflammatory cytokines or cell surface receptors on various cell types.

[0006] JIA, also known as juvenile rheumatoid arthritis (JRA), is the most common form of arthritis in children and adolescents. JIA develops before age 16, and the cause of JIA is largely unknown. The main focus of treating JIA is helping children resume their normal levels of physical and social activity. Most children are treated with NSAIDs and intra-articular corticosteroid injections. Methotrexate, a powerful DMARD, helps suppress joint inflammation in most JIA patients with polyarthritis, although it is reportedly less useful in systemic arthritis, and many children receive TNFα inhibitor drugs, such as etanercept.

[0007] IBD is a term used to describe conditions involving chronic inflammation in the digestive tract. IBD includes UC and CD.

[0008] The goal of IBD treatment is to reduce the inflammation that causes signs and symptoms. In the best case, this can lead not only to symptom relief, but also to long-term remission and a reduced risk of complications. IBD treatment typically involves medications such as anti-inflammatory drugs (NSAIDs), immune system suppressants and / or biologic agents, as well as surgery.

[0009] Bile salt-stimulated lipase (BSSL), also known as bile salt-dependent lipase (BSDL), carboxyl ester lipase (CEL), or bile salt-activated lipase (BAL), is a lipolytic enzyme encoded by the CEL gene and is expressed in the exocrine pancreas and secreted into the intestinal lumen of all species studied to date, where it contributes to the digestion of lipids.

[0010] In some species, including humans, primates, dogs, cats, and mice, BSSL is also expressed in the lactating mammary gland and secreted into milk. Furthermore, BSSL has been found at low but significant levels in the serum of healthy individuals and is involved in the regulation of lipoprotein metabolism and atherosclerosis. BSSL has also been found to play a role in inflammatory processes.

[0011] BSSL can be isolated from a suitable tissue such as human milk. Alternatively, recombinant BSSL can be produced by isolating DNA encoding BSSL using standard methods.

[0012] DNA encoding BSSL can be conveniently isolated from commercially available RNA, cDNA libraries, genomic DNA, or genomic DNA libraries using conventional molecular biology techniques such as library screening and / or polymerase chain reaction (PCR).

[0013] Methods for purifying BSSL from different tissues and transfected cell lines are known in the art [1].

[0014] Reference [2] describes antigen-binding compounds that bind to BSSL or fetal acinar pancreatic protein (FAPP). Compounds that recognize the C-terminal peptide (J28 epitope) of BSSL are disclosed. FAPP is an oncofetal form of BSSL characterized by the J28 carbohydrate-dependent epitope. The antigen-binding compounds are said to induce apoptosis and / or slow the proliferation of tumor cells expressing the BSSL or FAPP polypeptide. Reference [2] describes compounds that are capable of directly targeting tumor cells, particularly pancreatic tumor cells expressing BSSL or FAPP, and causing their death by apoptosis and / or halting their proliferation.

[0015] References [3, 4] describe the discovery that BSSL plays a role in inflammatory processes and that inhibiting or eliminating BSSL prevents the development of chronic arthritis in animal models. References [3, 4] disclose that BSSL protein is present in inflammatory cells and inflamed tissues and protects BSSL-deficient mice from developing inflammatory diseases, such as collagen-induced arthritis (CIA).

[0016] Although the treatment of inflammatory conditions such as autoinflammatory and autoimmune diseases has improved significantly over the years with the introduction of new drugs and drug classes (e.g., antibodies), most regimens and drugs still have in common that they aim to suppress the immune system, as is the case with all TNFα inhibitor drugs and corticosteroids. This in turn increases the risk of secondary infections and complications.

[0017] Therefore, there remains a significant clinical need for new selective drugs, preferably biologic drugs, for the treatment, prevention and protection of inflammatory diseases, which are directed against different and / or novel targets involved in inflammatory signaling and processes and which do not act primarily by suppressing the immune system and which are therefore expected to have fewer and / or less severe side effects. Summary of the Invention

[0018] A general objective is to provide antibodies or antigen-binding fragments thereof that specifically bind bile salt-stimulated lipase (BSSL), such as human BSSL (hBSSL).

[0019] The embodiments as disclosed herein meet this and other objectives.

[0020] The invention is defined by the independent claim. Further embodiments of the invention are defined in the dependent claims.

[0021] One aspect of the present invention relates to an isolated antibody or antigen-binding fragment thereof comprising three complementarity determining regions (CDRs) of a heavy chain variable region (HCVR), denoted HCDRs, and three CDRs of a light chain variable region (LCVR), denoted LCDRs. In this aspect, the first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 8, or an amino acid sequence that is at least 75% identical to SEQ ID NO: 8, and the third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence that is at least 83% identical to SEQ ID NO: 9. Furthermore, the first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 10 or an amino acid sequence that is at least 80% identical to SEQ ID NO: 10, the second LCDR comprises, preferably consists of, the amino acid sequence ATS or an amino acid sequence that is at least 66% identical to the amino acid sequence ATS, preferably AAS, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11 or an amino acid sequence that is at least 87% identical to SEQ ID NO: 11.

[0022] Another aspect of the present invention relates to an isolated antibody or antigen-binding fragment thereof comprising a polypeptide selected from the group consisting of ZH1-[GYTFTSYN]-ZH2-[X 53 GVIX 57 PGDGX 64 TSYX 68 QKFX 72 ]-ZH3-[ARDYYGSSPLGY]-ZH4 and a heavy chain variable region (HCVR) consisting of an amino acid sequence selected from ZL1-[X 24 ASX 27 SISYX 39 N]-ZL2-[AX 57 SX 66 LX 68 ]-ZL3-[HQRSSX 115 In this regard, ZH1, ZH2, ZH3 and ZH4 each independently represent zero, one or several independently selected amino acid residues, and X 53 Selected from I and M, X 57 Selected from N and Y, X 64 Selected from A and S, X 68 is selected from A and N, and X 72is selected from K and Q. In addition, ZL1, ZL2, ZL3 and ZL4 each independently represent zero, one or several independently selected amino acid residues, X 24 Selected from S and R, X 27 Selected from S and P, X 39 Choose from M, L, X 57 Selected from A and T, X 66 Selected from K and S, X 68 Selected from A and P, and X 115 Selected from S, T and Y.

[0023] Another aspect of the present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of BSSL, preferably hBSSL, comprising a first surface comprising the amino acid sequence according to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, preferably at least 83% identical to SEQ ID NO: 1, and a second surface comprising the amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence that is at least 80%, preferably at least 85% or at least 92% identical to SEQ ID NO: 2.

[0024] Yet another aspect of the present invention relates to a pharmaceutical composition comprising the isolated antibody and / or antigen-binding fragment thereof according to the above and a pharmaceutically acceptable carrier or excipient.

[0025] Other aspects of the present invention relate to an isolated antibody or antigen-binding fragment thereof according to the above, or a pharmaceutical composition according to the above, for use as a medicament and for the treatment and / or prevention of inflammatory diseases.

[0026] A related aspect of the present invention defines the use of an isolated antibody or antigen-binding fragment thereof according to above or a pharmaceutical composition according to above for the manufacture of a medicament for the treatment and / or prevention of an inflammatory disease.

[0027] Another related aspect of the present invention provides a method for treating and / or ameliorating and / or preventing and / or prophylactic inflammatory diseases, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof, or a pharmaceutical composition, as described above.

[0028] Other aspects of the present invention relate to polynucleotides encoding the antibodies or antigen-binding fragments thereof according to the above, expression vectors comprising the polynucleotides and cells comprising the antibodies or antigen-binding fragments thereof according to the above, the polynucleotides according to the above and / or the expression vectors according to the above.

[0029] Another aspect of the present invention relates to a method for detecting the presence of BSSL and / or quantifying the amount of BSSL in a sample. The method comprises contacting the sample with an isolated antibody or antigen-binding fragment thereof as described above, and detecting the presence of BSSL in the sample and / or quantifying the amount of BSSL in the sample based on the amount of the isolated antibody or antigen-binding fragment thereof that binds to BSSL.

[0030] Another aspect of the present invention relates to a method for diagnosing a BSSL-related disorder. The method comprises contacting a sample from a subject with an isolated antibody or antigen-binding fragment thereof as described above, and detecting the presence of BSSL in the sample and / or quantifying the amount of BSSL in the sample based on the amount of the isolated antibody or antigen-binding fragment thereof that binds to BSSL. The method further comprises determining whether the subject has a BSSL-related disorder based on the results of the detection and / or quantification.

[0031] Yet another aspect of the invention relates to a BSSL epitope comprising a first surface comprising the amino acid sequence according to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, preferably at least 83% identical to SEQ ID NO: 1, and a second surface comprising the amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence that is at least 80%, preferably at least 85% or at least 92% identical to SEQ ID NO: 2.

[0032] The antibodies and antigen-binding fragments of the present invention bind to a previously uncharacterized epitope in hBSSL that is distinct from the enzymatic active site. Thus, the antibodies and antigen-binding fragments can bind to hBSSL without competing with the enzymatic activity of hBSSL. The antibodies and antigen-binding fragments of the present invention can be used to treat and / or prevent inflammatory conditions while alleviating the aforementioned and other shortcomings of current therapies. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in which:

[0034] Figure 1 Shown is the interaction between hBSSL and immobilized AS20 mIgG1. The sensorgram was fitted to a 1:1 binding model. Due to the good fit, the sensorgram and the fitted line are indistinguishable.

[0035] Figure 2 Steady-state analysis of the interaction between mBSSL and immobilized AS20 mIgG1 is shown. D Taken from half of the maximal response, where max has been automatically corrected for high volume effects, with an offset of -173 RU (from top). Volume effects have not been removed from the graph.

[0036] Figure 3a Shown is a graph illustrating AS20 scFv ELISA binding to non-biotinylated and biotinylated human BSSL. The absorbance values ​​shown (y-axis) are the average of replicates. Figure 3b Shown is a graph illustrating AS20 scFv ELISA binding to mouse and human BSSL and an unrelated protein. The absorbance values ​​shown (y-axis) are the average of replicates.

[0037] Figure 4 Multiplex bead assay showing the ability of AS20 scFv (left bar) to bind to human BSSL and 30 different unrelated proteins A positive control scFv expected to bind b non-related protein_1, non-related scFv_1 (right bar) was also included in the assay.

[0038] Figure 5 Shown are the results of an HTRF-based competition assay analyzing AS20 scFv binding to mouse and human BSSL.

[0039] Figure 6 Sequence comparison between AS20, AS20 CDR grafts, and the AS20 humanized library scaffold. * indicates a stop in the reading frame, introduced into LCDR3 to ensure that only clones mutagenized in this region are displayed on phage. X indicates the position of mutagenesis in the AS20 humanized library. The boundaries of the CDRs are defined by Kabat, and the residue numbering is defined by the IMGT nomenclature [5]. According to IMGT, eHCDR2, eLCDR1, and eLCDR2 represent extended HCDR2, LCDR1, and LCDR2 regions that include amino acid positions outside the corresponding CDR regions.

[0040] 7 is a graph showing trends in size exclusion chromatography data at A) +40°C and B) +4°C as described in Example 12.

[0041] Figure 8 is a graph showing the mean Tm1 (circles) and Tm2 (squares) values ​​plotted for each candidate. Bars represent standard deviation; a) +4°C b) +40°C.

[0042] Figure 9 Results from DLS analysis are shown, showing the intensity and size of the candidates. Samples were analyzed after 30 days of storage at -80°C (dashed arrow), +4°C (dotted arrow) and +40°C (solid arrow).

[0043] Figure 10Schematic representation of the BSSL structures with the designated epitope regions of the pre-named antibody candidates (aa 7-12 of S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116, S-SL048-118; aa 42-55 of S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116, S-SL048-118; aa 84-101 of S-SL048-46; aa 174-180 of S-SL048-116; aa 283-295 of S-SL048-11).

[0044] Figure 11 is a representation of the S-SL048-106 svFv with potential post-translational liabilities highlighted.

[0045] Figure 12 is a cartoon representation of t-hBSSL showing the "oven glove" view and the epitope enclosed in a dashed circle and light grey around the back of the glove. Chains are shown as arrows and helices are shown as spirals.

[0046] Figure 13 The left panel shows t-hBSSL in dark grey surface representation, with the AS20-Fab interacting sequences in light grey. The variable regions of the heavy and light chains are shown in ribbon representation (light chain in grey, heavy chain in black). The right panel shows the same view, but with t-hBSSL in stick representation, with the active site triad highlighted in black. In the left panel, the active site is hidden beneath the surface.

[0047] Figure 14 is a table showing the amino acid sequence differences between the 38 candidate scFvs.

[0048] Figure 15A and 15B This is an overview of the design of a combinatorial scFv library of heavy chain variable regions as described in Example 5.

[0049] Figure 16A and 16B This is an overview of the design of the combinatorial scFv library of light chain variable regions as described in Example 5.

[0050] Figure 17 Figures show BSSL activity assays according to Example 20. A) and B) show the results of triglyceride hydrolysis assays, and C) and D) show the results of cholesterol ester hydrolysis assays. Note that chimeric AS20 is indicated as AS20 in the figure.

[0051] Figure 18Arthritis severity is shown. CAIA developed in mice after CIA-MAB-50 injection, treatment modality: (A) isotype control anti-NP hIgG1 LALA-PG (90 mg / kg), AS20 hIgG1 LALA-PG (90, 30, and 10 mg / kg) every 4 days from day -1 to day 15. (B) isotype control and AS20 hIgG1 LALA-PG 90 mg / kg. (C) isotype control and AS20 IgG1 LALA-PG 30 mg / kg. (D) isotype control and AS20hIgG1 LALA-PG 10 mg / kg. For ethical reasons (high score), two animals were removed before termination (day 12), one in the AS20 hIgG1 LALA-PG 10 mg / kg group and one in the isotype control group. These animals are included in the results until the day of removal. Data are expressed as mean ± SEM. *p<0.05; **p<0.01.

[0052] Figure 19 Disease parameter graphs are shown. CAIA disease parameters include animals treated ip with isotype control anti-NP hIgG1 LALA-PG (90 mg / kg), AS20hIgG1 LALA-PG (90, 30, and 10 mg / kg) every 4 days from day -1 to day 15. (A) Mean CAIA score (sum of scores during the experiment divided by the number of scoring days). (B) Maximum CAIA score. (C) Total disease burden (AUC). (D) Percent inhibition. For ethical reasons (high score), two animals were removed before termination (day 12), one in the AS20 hIgG1 LALA-PG 10 mg / kg group and one in the isotype control group. These animals were included only in the maximum score. Data are expressed as mean ± SEM. *p<0.05; **p<0.01.

[0053] Figure 20 Graphs showing the total number of cell subsets are shown. Data are presented as mean ± SD.

[0054] Figure 21 Graphs showing percentages of cell subsets are shown. Data are presented as mean ± SD.

[0055] Figure 22 The structure of the Fab-BSSL complex, drawn as a cartoon, is shown. S-SL048-116 Fab in dimeric complex with light and heavy chains and BSSL. The same complex is rotated 180° to the right.

[0056] Figure 23Interaction between BSSL and S-SL048-116 Fab. Variable Ig domain of Fab with light and heavy chains interacting with BSSL. Two important amino acids of the epitope, Arg 176 and Gln 52, are depicted as balls and sticks.

[0057] Figure 24 Arthritis severity is shown. CAIA developed in mice after CIA-MAB-50 injection, treatment modalities: (A) Vehicle and S-SL048-116 (SOL-116) (90, 30, and 10 mg / kg) every 4 days from day -1 to day 15. (B) Vehicle and S-SL048-116, 90 mg / kg. (C) Vehicle and S-SL048-116, 30 mg / kg. (D) Vehicle and S-SL048-116, 10 mg / kg. For ethical reasons, three animals were removed before termination, 2 in the vehicle group (days 7 and 15) and 1 in the 90 mg / kg group (day 12). These animals were included in the results until the day of removal, and the animals removed on day 7 were completely excluded from the data. Data are expressed as mean ± SEM.

[0058] Figure 25 CAIA disease parameters are shown, including animals treated with vehicle and S-SL048-116 (SOL-116) ip at different doses (90, 30, and 10 mg / kg). (A) Mean CAIA score (sum of scores during the experiment divided by the number of scoring days). (B) Maximum CAIA score. (C) Total disease burden (AUC). (D) Percent inhibition. For ethical reasons, three animals were removed before termination, two in the vehicle group (days 7 and 15) and one in the 90 mg / kg group (day 12). These animals are included only in the maximum score. Data are expressed as mean ± SEM.

[0059] Figure 26 Total leukocyte counts in (A) spleen and (B) mesenteric lymph nodes are shown. Data are presented as mean ± SEM. **p < 0.01.

[0060] Figure 27 The proportion of NK cells in CD45+ cells in (A) spleen, (B) blood, and (C) mesenteric lymph nodes is shown. Data are expressed as mean ± SEM. ****p < 0.001. DETAILED DESCRIPTION

[0061] definition

[0062] The term "isolated," when used in connection with an antibody, for example, in the expression "isolated antibody," means that the antibody has been removed from its original environment. As used herein, an isolated antibody is one that is substantially free of other antibodies with different antigenic specificities. For example, an isolated antibody that specifically binds to BSSL, particularly human BSSL (hBSSL), is substantially free of antibodies that specifically bind to antigens other than BSSL. However, an isolated antibody that specifically binds to hBSSL may have cross-reactivity with other antigens, such as BSSL molecules from other species, such as mouse or murine BSSL (mBSSL). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. For example, an isolated antibody or antigen-binding fragment thereof may be purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis, e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), capillary electrophoresis, or chromatography, e.g., ion exchange or reversed-phase high performance liquid chromatography (HPLC). The skilled person will understand that although the word "isolated" is not explicitly mentioned each time the term "antibody or antigen-binding fragment thereof" is used, the present invention relates to isolated antibodies or antigen-binding fragments thereof.

[0063] The term "isolated humanized antibody" and the like as used herein refers to an isolated antibody that has been humanized.

[0064] In the context of this article, the term "antigen binding fragment" means a fragment or portion of an antibody that substantially retains antigen binding properties. An antigen binding fragment is a portion or region of an antibody molecule or derivative thereof that retains all or a significant portion of the antigen binding of the corresponding full-length antibody. An antigen binding fragment may comprise one or more complementary determining region (CDR) sequences of an antibody or a portion of these CDR sequences, a portion or all of a heavy chain variable region (HCVR), a portion or all of a light chain variable region (LCVR), or a combination thereof. In one embodiment, the antigen binding fragment of an antibody may consist of the continuous amino acid sequence of the antibody from which it is obtained, or may consist of different portions of the amino acid sequence of an antibody, connected together with or without a linker. Examples of antigen binding fragments are single-chain variable fragments (scFv), Fab fragments, F(ab')2 fragments, F(ab')3 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, isolated complementary determining regions (CDRs) and nanobodies.

[0065] A "single chain variable fragment" or "single-chain variable fragment" ("scFv") is a fusion protein of the variable regions of the immunoglobulin heavy and light chains, connected to a short linker peptide of typically about 10 to 25 amino acids. ScFvs of the same or different types (having affinity for the same or different epitopes) can be combined in various ways known to those skilled in the art. Non-limiting examples of such combinations are tandem bi-scFvs, diabodies, tandem tri-scFvs, or triabodies.

[0066] The term "epitope" refers to the portion of an antigen that is recognized by the immune system, such as an antibody. An epitope is also called an antigenic determinant.

[0067] The term "paratope" refers to the portion of an antibody that binds an epitope.

[0068] As used herein, the terms "bind to," "have affinity for," "affinity," and the like refer to the property of an antibody or antigen-binding fragment thereof to bind to a target molecule. Standard assays for evaluating the ability of an antibody or antigen-binding fragment to bind to a target molecule include, for example, enzyme immunoassays (EIA), such as enzyme-linked immunosorbent assay (ELISA), Western blot, radioimmunoassay (RIA), surface plasmon resonance (SPR), Multiplex assays and flow cytometry analysis. As exemplified in the experimental section, the binding kinetics of the antibodies, such as binding affinity, can also be determined by standard methods known in the art, such as by System analysis to evaluate.

[0069] "Specifically binds to," "specifically binds to," and the like refer to molecules, such as antibodies or antigen-binding fragments thereof, that specifically bind to a target antigen without significantly binding to other molecules. The specificity of an antibody or antigen-binding fragment thereof can be determined based on affinity and / or avidity. Avidity (K) is the equilibrium constant for dissociation of the antigen from the antibody or antigen-binding fragment thereof. D ) is a measure of the strength of binding between an antigenic determinant (i.e., epitope) and the antigen binding site on an antibody or its antigen binding fragment. D The lower the value, the stronger the binding strength between the antigenic determinant and the antibody or its antigen-binding fragment. Alternatively, affinity can also be expressed as an affinity constant (K A ), which is 1 / K D As is clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest.

[0070] Typically, the antibody or antigen-binding fragment thereof will be expressed as 10 -5 to 10 -12 mol / L (M) or less, preferably 10 -7 to 10 - 12 M or less, more preferably 10 -8 to 10 -12 The equilibrium dissociation constant (K D ), that is, 10 5 to 10 12 M -1 or greater, preferably 10 7 to 10 12 M -1 or greater, more preferably 10 8 to 10 12 M -1 The affinity constant (K A ) binds to its antigen. Usually, more than 10 -4 Any K of M D Value (or less than 10 4 M -1 Any K A Preferably, the antibodies or antigen-binding fragments thereof of the embodiments will bind to BSSL with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 5 nM.

[0071] The terms "detection," "detecting," and the like include any means of detection, including direct and indirect detection.

[0072] Therefore, herein, all amino acids in the variable regions, including the CDRs described herein, are numbered according to the IMGT unique numbering defined by Marie-Paule Lefranc [5].

[0073] The term "Kabat numbering" and the like refer to a scheme for numbering amino acid residues in antibodies based on the variable region.

[0074] As used herein, the terms "monoclonal antibody," "monoclonal antibodies," and the like refer to one or more antibodies with monovalent affinity, meaning that each antibody molecule in a monoclonal antibody sample binds to the same epitope on an antigen. Monoclonal antibodies are produced by identical immune cells that are clones of a unique parent cell, such as a hybridoma cell line.

[0075] The term "polyclonal antibody" as used herein refers to a collection of antibodies that react against a specific antigen, but different antibody molecules that recognize different epitopes on the antigen may exist in the collection. Polyclonal antibodies are usually produced by inoculating a suitable mammal and purifying them from the serum of the mammal.

[0076] The term "human antibody derivative" refers to any modified form of a human antibody, such as a conjugate of an antibody and another agent or antibody.

[0077] As used herein, the term "full-length antibody" refers to an antibody of any class, such as immunoglobulin D (IgD), IgE, IgG, IgA, IgM or IgY, or any subclass thereof. The subunit structures and three-dimensional configurations of different classes of antibodies are well known.

[0078] As used herein, the term "chimeric antibody" refers to a recombinant or genetically engineered antibody, such as a mouse monoclonal antibody, that contains polypeptides or domains from a different species, such as human, introduced to reduce the immunogenicity of the antibody.

[0079] As used herein, the term "at least one" should be interpreted as one or more.

[0080] As will be understood by those skilled in the art, reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter itself. For example, descriptions referring to "about X" include descriptions of "X."

[0081] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length, and include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.

[0082] As used herein, "host cell" includes a single cell or cell culture that can be or has been a recipient of any vector of this document. Host cells include the progeny of a single host cell, and due to natural, accidental, or intentional mutations and / or changes, the progeny may not necessarily be completely identical in morphology or total DNA complement to the original parent cell. Host cells include cells transfected or infected with a vector comprising a nucleic acid herein. Host cells can be prokaryotic or eukaryotic.

[0083] The term "isolated" when used in connection with polynucleotides, polypeptides, etc., means that the molecule or polypeptide has been removed from its original environment.

[0084] As used herein, the terms "% identity" or "% identical" as used herein can be determined using methods well known in the art. For example, % identity is calculated as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm [6]. The comparison is performed over a window corresponding to the shortest aligned sequence. In some cases, the shortest aligned sequence may be the target sequence. In other cases, the query sequence may constitute the shortest aligned sequence. The amino acid residues or nucleotides at each position are compared, and the percentage of positions in the query sequence that have the same correspondence in the target sequence is reported as % identity.

[0085] As used herein, a "therapeutically effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0086] It should be understood that the aspects and embodiments described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments. As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise specified.

[0087] “hIgG1 LALA-PG” is as described in [7].

[0088] “hIgG4 S228P, hIgG4 S241P” as described in [8].

[0089] “G-SP140-8”, SP140 binding clone, negative control.

[0090] "expiHEK293 cells", human cells derived from the 293 cell line, and Expi293 TM Core components of the expression system.

[0091] The present invention provides antibodies and / or antigen-binding fragments thereof that can be used to treat and / or prevent inflammatory conditions while alleviating the aforementioned and other disadvantages of existing therapies. In addition, the present disclosure provides agents for diagnosing inflammatory conditions and for studying bile salt-stimulated lipase (BSSL) proteins.

[0092] More specifically, the present invention relates to novel isolated antibodies and antigen-binding fragments thereof that bind to a previously uncharacterized epitope of BSSL located in the N-terminal portion of the BSSL protein. The present invention also relates to the medical uses of the antibodies and antigen-binding fragments thereof, particularly in the treatment of inflammatory conditions, and to related pharmaceutical compositions. The present invention also discloses the use of the antibodies or antigen-binding fragments thereof as molecular tools for detecting BSSL and / or diagnosing BSSL-related diseases.

[0093] In one embodiment, whenever BSSL is mentioned, this also includes human BSSL (hBSSL), unless it is clear from the context that hBSSL is not intended to be included.

[0094] This disclosure describes a group of novel anti-BSSL antibodies, including antigen-binding fragments thereof, that bind to previously unidentified epitopes on hBSSL. The antibodies can be humanized or their CDR sequences can be grafted onto non-human scaffolds. The antibodies or antigen-binding fragments thereof can also bind to mouse or murine BSSL (mBSSL), although the affinity for hBSSL and mBSSL may differ due to amino acid differences in one of the epitopes bound by the antibodies and / or antigen-binding fragments thereof.

[0095] As is well known, antibodies are immunoglobulin molecules that are able to specifically bind to a target (antigen), such as a carbohydrate, polynucleotide, lipid, polypeptide, or other protein, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Antibodies are glycoproteins composed of at least two heavy (H) chains (HC) and two light (L) chains (LC) interconnected by disulfide bonds. The heavy chain variable region (HCVR) and the light chain variable region (LCVR) contain binding domains that interact with the antigen. The constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system, such as effector cells, and the first component of the classical complement system, complement component 1q (C1q).

[0096] The antibodies or antigen-binding fragments thereof disclosed herein can be used to inhibit or reduce at least some biological activities of the BSSL protein upon binding thereto. For example, binding can significantly or completely inhibit some biological activities of the BSSL protein. These effects of the antibodies or antigen-binding fragments thereof of the present invention are particularly surprising, given that the antibodies or antigen-binding fragments thereof of the present invention do not bind to the active site of BSSL. Therefore, the antibodies or antigen-binding fragments thereof of the present invention preferably do not significantly inhibit or reduce the enzymatic activity of BSSL, for example, the ability of BSSL to hydrolyze cholesterol esters (EC 3.1.1.13).

[0097] The antibodies or antigen-binding fragments thereof can be used to reduce the pro-inflammatory effects of BSSL in a subject in need thereof. Thus, the antibodies or antigen-binding fragments thereof can be used to treat and / or prevent various inflammatory diseases, as further described herein. These medical uses of the antibodies or antigen-binding fragments thereof of the present invention can be achieved without blocking the enzymatic activity of BSSL. Thus, the antibodies or antigen-binding fragments thereof of the present invention do not result in negative effects caused by inhibiting the enzymatic activity of BSSL, which may be associated with other anti-BSSL antibodies that bind or associate with the BSSL active site.

[0098] Furthermore, the antibodies or antigen-binding fragments thereof disclosed herein may be used for diagnostic purposes of BSSL-associated disorders, such as BSSL-associated inflammatory disorders.

[0099] As demonstrated in the experimental section, several approaches were used to attempt to develop humanized BSSL antibodies or antigen-binding fragments thereof, but despite a starting number of approximately 1,000,000 candidate scFvs, a surprisingly low number were found to exhibit sufficient binding affinity to the hBSSL protein. Of the 1,000,000 candidates, 68 initial candidates were identified, which were then reduced to 38 candidates and ultimately to 5 candidates selected for further evaluation and characterization. Thus, anti-BSSL antibodies or antigen-binding fragments thereof cannot be easily humanized using standard protocols and still have sufficient binding affinity to hBSSL. Therefore, humanization of anti-BSSL antibodies or antigen-binding fragments has been a major challenge, which has been overcome as described herein to obtain humanized antibodies or antigen-binding fragments thereof that still exhibit sufficient binding affinity to hBSSL. These antibodies or antigen-binding fragments thereof of the present invention have common structural and functional characteristics as described elsewhere herein.

[0100] Generation of antibodies and antigen-binding fragments

[0101] The antibodies and antigen-binding fragments thereof of the present invention were generated in a multi-step process with the goal of identifying antibodies and antigen-binding fragments thereof with sufficiently good binding affinity for hBSSL. Another object of the present invention is to provide humanized antibodies and antigen-binding fragments thereof. Some of the identified antibodies and antigen-binding fragments thereof were also found to bind to mBSSL with sufficient binding affinity. Although the present invention is not limited to humanized antibodies that bind to BSSL, humanized BSSL-binding antibodies and antigen-binding fragments thereof are disclosed herein.

[0102] Antibodies and antigen-binding fragments thereof are generated based on the sequence of the non-human monoclonal mBSSL antibody. DNA encoding the heavy and light chain immunoglobulins is obtained from a non-human hybridoma expressing this antibody and engineered to contain non-murine (eg, human) immunoglobulin sequences using standard molecular biology techniques.

[0103] However, it was surprisingly found that the CDR grafts constructed as disclosed in Example 6 did not have as high a binding affinity as expected based on the mBSSL antibody used to provide the CDR sequences. Therefore, in order to obtain antibodies and antigen-binding fragments thereof with sufficiently high binding affinity to hBSSL, the antibody framework (FW) had to be further modified by introducing mutations into the CDRs and adjacent FW regions, as disclosed in Example 5. This was achieved by constructing a humanized library for selecting and isolating scFv fragments that bind to mouse and human BSSL using phage display. Such phage display methods for isolating human antibodies are established in the art, see, for example, US 5,223,409; US 5,403,484; US 5,571,698; US 5,427,908; US 5,580,717; US 5,969,108; US 6,172,197; US 5,885,793; US 6,521,404; US 6,544,731; US ​​6,555,313; US 6,582,915 and US 6,593,081.

[0104] The resulting isolated antibodies and antigen-binding fragments thereof have minimal animal-derived CDR content, wherein only essential non-human germline residues are allowed. The remaining CDRs are converted to human v gene sequences, except for some new variations by introducing species-neutral essential de novo residues, such as in IMGT amino acid residues 62, 64, 68, 27, 66, 68, 115, and 116 (see Figures 15 and 16). These CDR sequences can be grafted onto human or non-human frameworks to prepare humanized or non-humanized antibodies and / or antigen-binding fragments thereof, depending on the intended use of the antibody.

[0105] In humanized antibodies, the constant regions and part of the variable regions, in addition to the CDR sequences, have a framework derived from human germline immunoglobulin sequences. However, in such humanized antibodies, the CDR sequences are derived from the germline of another mammalian species, such as a mouse, that has been grafted onto human framework sequences. Such humanized antibodies may also be referred to as CDR grafts in the context of the present invention. The advantage of using humanized antibodies is that they reduce the risk of immunogenic reactions that might occur if the antibodies were injected into human subjects when a framework from another species was used. This opens up their use in human medical applications. As disclosed in more detail elsewhere herein, the antibodies or antigen-binding fragments thereof disclosed herein can be used in diagnostic applications and for detecting BSSL proteins, such as hBSSL, in different types of samples.

[0106] Therefore, also disclosed herein is a method for producing an isolated antibody or antigen-binding fragment thereof according to the present invention. The method comprises culturing a host cell expressing the antibody or antigen-binding fragment thereof under conditions that allow expression of the antibody or antigen-binding fragment thereof from an expression vector comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof contained in the host cell. The method further comprises isolating the antibody or antigen-binding fragment thereof from the host cell or from the culture medium in which the host cell is cultured.

[0107] Epitope binding of antibodies or antigen-binding fragments thereof

[0108] The isolated antibody or antigen-binding fragment thereof has been found to bind to a previously unidentified epitope of the human BSSL protein located at the N-terminal portion of the BSSL protein. The epitope may form a conformational epitope of BSSL.

[0109] Surprisingly, the antibodies and antigen-binding fragments thereof produced according to the present invention have been found to bind to a previously unidentified epitope of the hBSSL protein. Even more surprisingly, the epitope was found not to be located near the lipid metabolism active site of BSSL, but rather to be located in the N-terminal portion of BSSL. This has several advantageous effects. One is that the antibodies or antigen-binding fragments thereof are less likely to cause negative side effects, as they do not significantly affect the enzymatic lipase activity of BSSL. Another advantage is that the antibodies or antigen-binding fragments thereof are suitable for studying the BSSL protein and its lipase activity, as this is not significantly affected by the antibodies or antigen-binding fragments thereof of the present invention.

[0110] The BSSL structure has been described as having a large core region consisting of a twisted 11-stranded β-sheet surrounded by α-helices and connecting loops ([9], Figure 12 ). There is a smaller 3-stranded beta sheet at the N-terminus. The structure has been likened to a left-hand oven-glove, with the palm containing the active site triad near the "thumb". Similarly, the small N-terminal beta sheet is located on the back of the hand near the "pinky finger", see Figure 12 The parts of the BSSL structure that interact with the Fab molecule are located in the small N-terminal β-sheet and the C-terminal part of αC, which is the third α-helix in the structure, see Figure 13 In other words, the binding region of the antibody is not close to the active site of the BBSL, but is on the opposite side of the BSSL.

[0111] The epitope region identified so far comprises residues 7-12 (chains 1 and 2) and 42-55 (the loop region of chain 3 that introduces the fold). The epitope is quite flat, with only a few characteristic residues protruding, namely Tyr7, Phe12, and Gln52 (the main interactions are listed in Table 25). The loop region 47-54 is well defined and forms a uniform surface. Proline 47 is important for stacking interactions with Tyr31 of the antibody, but overall, the surface here is flat. In a preferred embodiment, the epitope region also comprises residues 174-180 (the C-terminus of αC).

[0112] One aspect of the present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of BSSL, preferably hBSSL. The epitope comprises a first surface comprising, or defined by, an amino acid sequence according to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, such as at least 83%, identical to SEQ ID NO: 1. The epitope further comprises a second surface comprising, or defined by, an amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence that is at least 80%, such as at least 85% or at least 92% identical to SEQ ID NO: 2.

[0113] The first peptide comprising the amino acid sequence of SEQ ID NO: 1 defines a first surface of the epitope in BSSL, and the second peptide comprising the amino acid sequence of SEQ ID NO: 2 defines a second surface of the epitope in BSSL. Thus, the first surface comprises, or is defined by, the amino acid sequence of SEQ ID NO: 1, and the second surface comprises, or is defined by, the amino acid sequence of SEQ ID NO: 2.

[0114] In one embodiment, the first peptide comprises the amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence that is at least 80%, preferably at least 83%, more preferably at least 91% identical to SEQ ID NO: 3. SEQ ID NO: 3 is a longer amino acid sequence that comprises the amino acid sequence according to SEQ ID NO: 1 as a part thereof.

[0115] The isolated antibody or antigen-binding fragment thereof can further specifically bind to another peptide of BSSL, such as hBSSL, and a surface, i.e., a third peptide and a third surface. In one embodiment, the third peptide comprises the amino acid sequence according to SEQ ID NO:5, or an amino acid sequence that is at least 80%, preferably at least 85%, identical to SEQ ID NO:5. In another embodiment, the third peptide comprises the amino acid sequence according to SEQ ID NO:4, or an amino acid sequence that is at least 80%, preferably at least 83%, more preferably at least 88%, such as at least 94% identical to SEQ ID NO:4. In another embodiment, the third peptide comprises the amino acid sequence according to SEQ ID NO:6, or an amino acid sequence that is at least 80%, preferably at least 84%, more preferably at least 92% identical to SEQ ID NO:6.

[0116] In one embodiment, the isolated antibody or antigen-binding fragment thereof can specifically bind to a first peptide comprising, for example, consisting of, SEQ ID NO: 1, a second peptide comprising, for example, consisting of, SEQ ID NO: 2, and a third peptide comprising, for example, consisting of, SEQ ID NO: 4, or an amino acid sequence having respective identities as defined herein. Alternatively, such an antibody or antigen-binding fragment thereof can bind to the longer amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence having identities as specified herein, rather than the shorter amino acid sequence according to SEQ ID NO: 1.

[0117] In another embodiment, the isolated antibody or antigen-binding fragment thereof specifically binds to a first peptide comprising, for example, consisting of, SEQ ID NO: 1, a second peptide comprising, for example, consisting of, SEQ ID NO: 2, and a third peptide comprising, for example, consisting of, SEQ ID NO: 5, or an amino acid sequence having respective identities as defined herein. Alternatively, such an antibody or antigen-binding fragment thereof may specifically bind to the longer amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence having identities as specified herein, rather than to the shorter amino acid sequence according to SEQ ID NO: 1.

[0118] In another embodiment, the isolated antibody or antigen-binding fragment thereof specifically binds to a first peptide comprising, for example, consisting of, SEQ ID NO: 1, a second peptide comprising, for example, consisting of, SEQ ID NO: 2, and a third peptide comprising, for example, consisting of, SEQ ID NO: 6, or an amino acid sequence having respective identities as defined herein. Alternatively, such an antibody or antigen-binding fragment thereof may bind to the longer amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence having a specified identity as defined herein, rather than the shorter amino acid sequence according to SEQ ID NO: 1.

[0119] Another aspect of the invention relates to a BSSL epitope, e.g., a hBSSL epitope, comprising a first peptide comprising, e.g., consisting of, an amino acid sequence according to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, preferably at least 83% identical to SEQ ID NO: 1. The BSSL epitope further comprises, e.g., consisting of, a second peptide comprising, e.g., consisting of, an amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence that is at least 80%, preferably at least 85% or at least 92% identical to SEQ ID NO: 2.

[0120] In one embodiment, the first peptide comprises, eg consists of, an amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence having an identity thereto as defined herein.

[0121] The epitope may also comprise a third peptide comprising, e.g., consisting of, an amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence having an identity thereto as defined herein, an amino acid sequence according to SEQ ID NO: 5, or an amino acid sequence having an identity thereto as defined herein, or an amino acid sequence according to SEQ ID NO: 6, or an amino acid sequence having an identity thereto as defined herein.

[0122] These epitopes can be used to develop antibodies or antigen-binding fragments thereof that bind to BSSL proteins, such as hBSSL, for example, to treat and / or prevent BSSL-related conditions and / or as molecular tools for studying BSSL proteins. Therefore, the present invention also relates to the use of these epitopes for developing antibodies or antigen-binding fragments thereof. Because these epitopes are not located in the catalytic center of the BSSL protein lipase, the development of anti-BSSL antibodies or antigen-binding fragments is an attractive target.

[0123] The isolated antibody or antigen-binding fragment thereof according to the present invention may specifically bind to an epitope as defined herein.

[0124] Antigen-binding portion of antibodies and antigen-binding fragments thereof

[0125] A full-length antibody comprises two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (HVCR) and the first, second, and third constant regions (C H 1. C H 2 and C H 3). In this disclosure, the terms VH, VH, and HCVR are used interchangeably. Each light chain contains a light chain variable region (LVCR) and a light chain constant region (C L ). In this disclosure, the terms VL, VL and LCVR are used interchangeably.

[0126] The HCVR and LCVR regions can be further subdivided into hypervariable regions, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs or FWs). Each HCVR and LCVR consists of three CDRs and four FRs / FWs, arranged from N-terminus to C-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4.

[0127] As used herein, an extended CDR (eCDR) relates to an amino acid sequence comprising at least one additional amino acid residue beyond the amino acids of the CDR defined according to the IMGT nomenclature.

[0128] As is well known in the art, the paratope, also known as the antigen binding site, is the part of an antibody or its antigen binding fragment that recognizes and binds to an antigen. It is a small region of the antibody Fv region and contains part of the antibody heavy and light chains. Each arm tip of the Y-shaped antibody monomer is the paratope, which is a collection of 6 CDRs. The paratope consists of three light chain CDRs (LCDRs) and three heavy chain CDRs (HCDRs), which extend from the fold of antiparallel beta pleats.

[0129] In the following sections, the isolated antibodies or antigen-binding fragments thereof of the present invention are defined by the structural features of their CDRs, in other words, by the amino acid sequences of their HCDRs and / or LCDRs, or by the amino acid structure of the region comprising the HCDRs and / or LCDRs. A skilled person will appreciate that minor changes in the amino acid sequence, such as substitutions of one, two, three, four, or even more amino acid residues, may occur without affecting the functional properties of the isolated antibody or antigen-binding fragment thereof, such as its binding ability or binding affinity for BSSL, such as hBSSL. It should be understood that the first HCDR, second HCDR, third HCDR, first LCDR, second LCDR, and third LCDR may be independently selected from the amino acid sequences described.

[0130] Thus, one aspect of the present invention relates to an isolated antibody or antigen-binding fragment thereof comprising three CDRs of a HCVR (HCDR) and three CDRs of a LCHV (LCDR). In this aspect, the first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence that is at least 87%, such as at least 87.5%, identical to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 8, or an amino acid sequence that is at least 75% identical to SEQ ID NO: 8, and the third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence that is at least 83%, such as at least 91.6%, identical to SEQ ID NO: 9. Furthermore, in this aspect, the first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 10 or an amino acid sequence that is at least 80% identical to SEQ ID NO: 10, the second LCDR comprises, preferably consists of, the amino acid sequence ATS or an amino acid sequence that is at least 66% identical to the amino acid sequence ATS, such as AAS, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11 or an amino acid sequence that is at least 87% identical to SEQ ID NO: 11.

[0131] The experimental data presented in Example 22 suggest that the second LCDR may not be important for the interaction with BSSL. Accordingly, in an embodiment, the isolated antibody or antigen-binding fragment thereof comprises a first HCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence that is at least 87%, such as at least 87.5%, identical to SEQ ID NO: 7; a second HCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 8, or an amino acid sequence that is at least 75% identical to SEQ ID NO: 8; a third HCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence that is at least 83%, such as at least 91.6% identical to SEQ ID NO: 9; a first LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 10; and a third LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 11, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 11.

[0132] In one embodiment, the first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 18 and SEQ ID NO: 19, and the third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the first LCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 20, the second LCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of ATS and AAS, and the third LCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 21 and SEQ ID NO: 22.

[0133] In one embodiment, the first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 8, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 10, the second LCDR comprises, preferably consists of, the amino acid sequence ATS, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11.

[0134] In a specific embodiment, the isolated antibody or antigen-binding fragment thereof comprises an extended second HCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 12; an extended first LCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 14; and an extended second LCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 15.

[0135] In another specific embodiment, the isolated antibody or antigen-binding fragment thereof comprises an extended second HCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 12; an extended first LCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 16; and an extended second LCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 17.

[0136] In one embodiment, the first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 18, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 10, the second LCDR comprises, preferably consists of, the amino acid sequence ATS, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 21.

[0137] In a specific embodiment, the isolated antibody or antigen-binding fragment thereof comprises an extended second HCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 23; an extended first LCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 16; and an extended second LCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 15.

[0138] In one embodiment, the first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 8, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 20, the second LCDR comprises, preferably consists of, the amino acid sequence AAS, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11.

[0139] In a specific embodiment, the isolated antibody or antigen-binding fragment thereof comprises an extended second HCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 24; an extended first LCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 27; and an extended second LCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 29.

[0140] In one embodiment, the first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 19, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 20, the second LCDR comprises, preferably consists of, the amino acid sequence ATS, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 22.

[0141] In a specific embodiment, the isolated antibody or antigen-binding fragment thereof comprises an extended second HCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 25; an extended first LCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 26; and an extended second LCDR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 28.

[0142] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises a first HCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 7; an extended second HCDR comprising, preferably consisting of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 23, 24 and 25; and a third HCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises an extended first LCDR comprising, preferably consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 26 and SEQ ID NO: 27; an extended second LCDR comprising, preferably consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 28 and SEQ ID NO: 29; and a third LCDR comprising, preferably consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 21 and SEQ ID NO: 22.

[0143] In one embodiment, when the first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, it further comprises an amino acid sequence which is at least 87.5% identical to SEQ ID NO: 7.

[0144] In one embodiment, when the second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 8, it further comprises an amino acid sequence that is at least 75%, such as at least 87%, or at least 87.5% identical to SEQ ID NO: 8.

[0145] In one embodiment, when the third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9, it further comprises an amino acid sequence which is at least 83%, such as at least 91.6% identical to SEQ ID NO: 9.

[0146] In one embodiment, when the first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 10, it further comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 10.

[0147] In one embodiment, when the second LCDR comprises, preferably consists of, the amino acid sequence ATS or AAS, it further comprises an amino acid sequence that is at least 66% identical to either of the amino acid sequences ATS or AAS.

[0148] In one embodiment, when the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 11, it further comprises an amino acid sequence which is at least 87.5% identical to SEQ ID NO: 11.

[0149] In one embodiment, when the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 21, it further comprises an amino acid sequence which is at least 75%, such as at least 87.5% identical to SEQ ID NO: 21.

[0150] In one embodiment, when the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 22, it further comprises an amino acid sequence which is at least 75%, such as at least 87.5% identical to SEQ ID NO: 22.

[0151] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 12, it further comprises an amino acid sequence which is at least 77.8%, such as at least 83%, such as at least 83.3%, such as at least 88%, such as at least 88.9%, such as at least 94%, such as at least 94.4% identical to SEQ ID NO: 12.

[0152] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 18, it further comprises an amino acid sequence which is at least 75%, such as at least 87.5% identical to SEQ ID NO: 18.

[0153] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 19, it further comprises an amino acid sequence which is at least 75%, such as at least 87.5% identical to SEQ ID NO: 19.

[0154] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 23, it further comprises an amino acid sequence which is at least 77.8%, such as at least 83%, such as at least 83.3%, such as at least 88%, such as at least 88.9%, such as at least 94%, such as at least 94.4% identical to SEQ ID NO: 23.

[0155] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 24, it further comprises an amino acid sequence which is at least 77.8%, such as at least 83%, such as at least 83.3%, such as at least 88%, such as at least 88.9%, such as at least 94%, such as at least 94.4% identical to SEQ ID NO: 24.

[0156] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 25, it further comprises an amino acid sequence which is at least 77.8%, such as at least 83%, such as at least 83.3%, such as at least 88%, such as at least 88.9%, such as at least 94%, such as at least 94.4% identical to SEQ ID NO: 25.

[0157] In one embodiment, when the extended first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 14, it further comprises an amino acid sequence which is at least 80%, such as at least 90% identical to SEQ ID NO: 14.

[0158] In one embodiment, when the extended first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 16, it further comprises an amino acid sequence which is at least 80%, such as at least 90% identical to SEQ ID NO: 16.

[0159] In one embodiment, when the extended first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 20, it further comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 20.

[0160] In one embodiment, when the extended first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 26, it further comprises an amino acid sequence which is at least 80%, such as at least 90% identical to SEQ ID NO: 26.

[0161] In one embodiment, when the extended first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 27, it further comprises an amino acid sequence which is at least 80%, such as at least 90% identical to SEQ ID NO: 27.

[0162] In one embodiment, when the extended second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 15, it further comprises an amino acid sequence which is at least 66.7%, such as at least 83%, such as at least 83.3% identical to SEQ ID NO: 15.

[0163] In one embodiment, when the extended second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 17, it further comprises an amino acid sequence which is at least 66.7%, such as at least 83%, such as at least 83.3% identical to SEQ ID NO: 17.

[0164] In one embodiment, when the extended second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 28, it further comprises an amino acid sequence which is at least 66.7%, such as at least 83%, such as at least 83.3% identical to SEQ ID NO: 28.

[0165] In one embodiment, when the extended second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 29, it further comprises an amino acid sequence which is at least 66.7%, such as at least 83%, such as at least 83.3% identical to SEQ ID NO: 29.

[0166] The isolated antibodies or antigen-binding fragments thereof disclosed herein may also or alternatively be structurally described by the amino acid sequences of their HCVR and / or LCVR. The skilled artisan will appreciate that the HCVR and LCVR may be independently selected from the amino acid sequences described. As described above, the skilled artisan will appreciate that minor changes in the amino acid sequence, such as substitutions of one, two, three, four, or even more amino acid residues, may occur without affecting the functional properties of the isolated antibody or antigen-binding fragment thereof (e.g., its ability to bind hBSSL). The changes may be in the amino acid sequences of the CDRs, in the amino acid sequences outside of the CDR regions referred to herein as framework regions, or in the amino acid sequences of the CDRs and in the amino acid sequences outside of the CDR regions of the HCVR or LCVR.

[0167] Thus, in one embodiment, the antibody or antigen-binding fragment thereof comprises a HCVR comprising, and preferably consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 36, and an amino acid sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any one of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 36.

[0168] In a specific embodiment, the amino acid sequence of HVCR is selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 34 and SEQ ID NO: 36, and an amino acid sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any one of SEQ ID NO: 30, SEQ ID NO: 34 and SEQ ID NO: 36. In another specific embodiment, the amino acid sequence of HVCR is selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 36, and an amino acid sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any one of SEQ ID NO: 34 and SEQ ID NO: 36. For example, HCVR may comprise the amino acid sequence according to SEQ ID NO: 36, or an amino acid sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any one of SEQ ID NO: 36.

[0169] In one embodiment, the antibody or antigen-binding fragment thereof comprises a LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 38, and an amino acid sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any one of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 38.

[0170] In a specific embodiment, the amino acid sequence of LVCR is selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38, and an amino acid sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any one of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38. In another specific embodiment, the amino acid sequence of LVCR is selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38, and an amino acid sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any one of SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38; for example, selected from the group consisting of SEQ ID NO: 37 and SEQ ID NO: 38, and an amino acid sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any one of SEQ ID NO: 37 and SEQ ID NO: 38. For example, the HCVR may comprise an amino acid sequence according to SEQ ID NO: 37 and an amino acid sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any one of SEQ ID NO: 37.

[0171] In one embodiment, the antibody or antigen-binding fragment thereof comprises a HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36, and an amino acid sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any one of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36. The antibody or antigen-binding fragment thereof further comprises a LCVR comprising an amino acid sequence independently selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 38, and an amino acid sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any one of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 38.

[0172] In a specific embodiment, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 36 and a LCVR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 37.

[0173] In another specific embodiment, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 36 and a LCVR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 38.

[0174] In another embodiment, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 30 and a LCVR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 31.

[0175] In yet another embodiment, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 32 and a LCVR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 33.

[0176] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 34 and a LCVR comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 35.

[0177] One aspect of the present invention relates to an isolated antibody or antigen-binding fragment thereof comprising a HCVR and a LCVR, wherein the HCVR consists of an amino acid sequence selected from the group consisting of: i) ZH1-[GYTFTSYN]-ZH2-[X 53 GVIX 57 PGDGX 64 TSYX 68 QKFX 72 ]-ZH3-[ARDYYGSSPLGY]-ZH4, or an amino acid sequence having at least 92% identity to the sequence defined in i), such as 93% or higher, such as 94% or higher, such as 95% or higher, such as 96% or higher, such as 97% or higher, such as 98% or higher, such as 99% or higher identity to the sequence defined in i), and the LCVR consists of an amino acid sequence selected from the group consisting of: ii) ZL1-[X 24 ASX 27 SISYX 39 N]-ZL2-[AX 57 SX 66 LX 68 ]-ZL3-[HQRSSX 115 PT]-ZL4, or an amino acid sequence that is at least 87% identical to the sequence defined in ii), e.g. 88% or higher, for example 89% or higher, such as 90% or higher, such as 91% or higher, for example 92% or higher, such as 93% or higher, such as 94% or higher, such as 95% or higher, for example 96% or higher, such as 97% or higher, such as 98% or higher, for example 99% or higher, to the sequence defined in ii).

[0178] In this regard, ZH1, ZH2, ZH3 and ZH4 each independently represent zero, one or several independently selected amino acid residues, and ZL1, ZL2, ZL3 and ZL4 each independently represent zero, one or several independently selected amino acid residues. 53 Selected from I and M, X 57 Selected from N and Y, X 64 Selected from A and S, X 68 Selected from A and N, X 72 Selected from K and Q, X 24 Selected from S and R, X 27 Selected from S and P, X39 Choose from M, L, X 57 Selected from A and T, X 66 Selected from K and S, X 68 Selected from A and P, and X 115 is selected from the group consisting of S, T and Y. The numbering of amino acid residues is according to the IMTG numbering standard, i.e. for position "X" in the sequence i) or ii) as defined above n ”, where n is an integer and represents the position of amino acid residue X according to IMGT numbering.

[0179] GYTFTSYN is presented in SEQ ID NO: 7, X 53 GVIX 57 PGDGX 64 TSYX 68 QKFX 72 is presented in SEQ ID NO: 169, ARDYYGSSPLGY is presented in SEQ ID NO: 9, X 24 ASX 27 SISYX 39 N is presented in SEQ ID NO: 170, AX 57 SX 66 LX 68 Presented in SEQ ID NO: 171 and HQRSSX 115 PT is presented in SEQ ID NO:172.

[0180] Provided herein are antibodies or antigen-binding fragments thereof, wherein X in sequence i) n are independently selected from the group of possible residues listed in List A below. The skilled person will understand that X n can be selected from any of the listed possible groups of residues, and this selection is independent of X m The selection of amino acids in , where n≠m. Therefore, position X n Any of the listed possible residues in can be independently combined with any of the listed possible residues at any other variable position according to List A.

[0181] List A: List of possible amino acid residues in sequence i)

[0182] X 53 It can be I;

[0183] X 53 It can be M;

[0184] X 57 It can be N;

[0185] X 57 It can be Y;

[0186] X 59 It can be G;

[0187] X 59 It can be S;

[0188] X 64 It can be A;

[0189] X 64 It can be S;

[0190] X 68 Can be selected from A and T;

[0191] X 68 Can be selected from A and N;

[0192] X 68 Can be selected from T and N;

[0193] X 68 It can be A;

[0194] X 68 It can be N;

[0195] X 68 It can be T;

[0196] X 72 can be K; and

[0197] X 72 It can be Q.

[0198] In a similar manner, provided herein are antibodies or antigen-binding fragments thereof, wherein X in sequence ii) herein is k are independently selected from the group of possible residues listed in Table B below. The skilled person will understand that X k can be selected from any of the listed possible groups of residues, and this selection is independent of X l The selection of amino acids in , where k≠l. Therefore, position X in list A k Any of the listed possible residues in can be independently combined with any of the listed possible residues at any other variable position according to List B.

[0199] List B: List of possible amino acid residues in sequence ii)

[0200] X 24 It can be S;

[0201] X 24 It can be R;

[0202] X 27 It can be S;

[0203] X 27It can be P;

[0204] X 39 It can be M;

[0205] X 39 It can be L;

[0206] X 40 It can be H;

[0207] X 40 It can be N;

[0208] X 57 It can be A;

[0209] X 57 It can be T;

[0210] X 66 Can be selected from K and S;

[0211] X 66 Can be selected from R and S;

[0212] X 66 Can be selected from R and K;

[0213] X 66 It can be K;

[0214] X 66 It can be R;

[0215] X 66 It can be S;

[0216] X 68 Can be selected from A and P;

[0217] X 68 Can be selected from A and Q;

[0218] X 68 Can be selected from P and Q;

[0219] X 68 It can be A;

[0220] X 68 It can be P;

[0221] X 68 It can be Q;

[0222] X 105 It can be H;

[0223] X 105 It can be Q;

[0224] X 115 Can be selected from S and T;

[0225] X115 Can be selected from S and Y;

[0226] X 115 Can be selected from T and Y;

[0227] X 115 It can be S;

[0228] X 115 can be Y; and

[0229] X 115 It can be T.

[0230] For the sake of clarity, the selection of the amino acid residue at the amino acid position in sequence i) from List A is independent of the selection of the amino acid residue at the amino acid position in sequence ii) from List B. For the avoidance of doubt, List A and List B each disclose several specific and individualized examples according to the present disclosure, and the listed examples can be freely combined.

[0231] As defined above, ZH1, ZH2, ZH3 and ZH4 may each represent zero, one or several independently selected amino acid residues. The identity and number of amino acid residues in each of said ZH1, ZH2, ZH3 and ZH4 may be independently selected.

[0232] Similarly, ZL1, ZL2, ZL3, and ZL4 can each represent zero, one, or several independently selected amino acid residues. The identity and number of amino acid residues in each of ZL1, ZL2, ZL3, and ZL4 can be independently selected. Furthermore, ZL1 can be linked to ZH1 or ZH4 via an amino acid linker or other linker. Furthermore, ZL4 can be linked to ZH1 or ZH4 via an amino acid linker or other linker. Thus, the sequence defined in i) and the sequence defined in ii) can be part of an amino acid sequence, in other words, part of a polypeptide.

[0233] In one embodiment, ZH1 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 39, or an amino acid sequence which is at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to SEQ ID NO: 39.

[0234] In one embodiment, ZH2 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 40, or an amino acid sequence which is at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to SEQ ID NO: 40.

[0235] In one embodiment, ZH3 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 41, or an amino acid sequence which is at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to SEQ ID NO: 41.

[0236] In one embodiment, ZH4 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 42, or an amino acid sequence which is at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to SEQ ID NO: 42.

[0237] In one embodiment, ZL1 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 43, or an amino acid sequence that is at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to SEQ ID NO: 43.

[0238] In one embodiment, ZL2 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 44, or an amino acid sequence which is at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to SEQ ID NO: 44.

[0239] In one embodiment, ZL3 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 45, or an amino acid sequence which is at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to SEQ ID NO: 45.

[0240] In one embodiment, ZL4 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 46, or an amino acid sequence which is at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to SEQ ID NO: 46.

[0241] The skilled person will understand that the % identity of each of ZH1, ZH2, ZH3, ZH4, ZL1, ZL2, ZL3 and ZL4 to the amino acid sequence according to the respective SEQ ID NO: above is independent of the % identity of any other of ZH1, ZH2, ZH3, ZH4, ZL1, ZL2, ZL3 and ZL4 to their respective SEQ ID NO:. Thus, for example, ZH1 may exhibit 95% identity to SEQ ID NO: 39 and ZH2 may exhibit 99% identity to SEQ ID NO: 40.

[0242] In the humanization method for generating the novel anti-BSSL antibodies or antigen-binding fragments thereof of the present invention, several mutations are introduced in the CDRs and adjacent FW regions. For the purposes of this disclosure, each antibody HCVR and LCVR consists of three CDRs and four FR / FWs, one, two, or three of which may be extended CDRs (eCDRs), arranged from N-terminus to C-terminus in the following order as defined in Table 1.

[0243] Table 1-IMGT Number

[0244]

[0245] The three CDRs in HCRV are flanked by framework regions, which may be the ZH1, ZH2, ZH3 and ZH4 regions described above. The three CDRs in LCRV are flanked by framework regions, which may be the ZL1, ZL2, ZL3 and ZL4 regions described above.

[0246] The HVCR of the antibodies or antigen-binding fragments thereof constructed herein are listed in the amino acid sequences of the group consisting of SEQ ID NOs: 30, 32, 34, 36 and 47-84. Accordingly, the LVCR of the antibodies or antigen-binding fragments thereof constructed herein are listed in the amino acid sequences of the group consisting of SEQ ID NOs: 31, 33, 35, 37, 38 and 86-123. Specific examples of these antibodies or antigen-binding fragments thereof include, and preferably consist of, combinations of the following HVCR and LVCR: SEQ ID NOs: 30 and 31; SEQ ID NOs: 32 and 33; SEQ ID NOs: 34 and 35; SEQ ID NOs: 36 and 37; SEQ ID NOs: 36 and 38; SEQ ID NOs: 47 and 81; SEQ ID NOs: 48 and 82; SEQ ID NOs: 49 and 83; SEQ ID NOs: 50 and 84; SEQ ID NOs: 51 and 85; SEQ ID NOs: 52 and 86; SEQ ID NOs: 53 and 87; SEQ ID NOs: 54 and 88; SEQ ID NOs: 55 and 89; SEQ ID NOs: 56 and 90; SEQ ID NOs: 57 and 91; SEQ ID NOs: 58 and 92; SEQ ID NOs: 59 and 93; SEQ ID NOs: 60 and 94; SEQ ID NOs: 61 and 95; SEQ ID NOs: 62 and 96; SEQ ID NOs: 63 and 97; SEQ ID NOs: 64 and 65; NO:64 and 98; SEQ ID NO:65 and 99; SEQ ID NO:66 and 100; SEQ ID NO:67 and 101; SEQ ID NO:68 and 102; SEQ ID NO:69 and 103; SEQ ID NO:70 and 104; SEQ ID NO:71 and 105; SEQ ID NO:72 and 106; SEQ ID NO:73 and 107; SEQ ID NO:74 and 108; SEQ ID NO:85 and 109; SEQ ID NO:86 and 110; SEQ ID NO:77 and 111; SEQ ID NO:78 and 112; SEQ ID NO:79 and 113 or SEQ ID NO:80 and SEQ ID NO:114.

[0247] It will be understood by those skilled in the art that various modifications and / or additions may be made to the antibodies or their antigen-binding fragments as disclosed herein to adapt the antibodies or their antigen-binding fragments to specific applications without departing from the scope of this disclosure. For example, an antibody or its antigen-binding fragment may have an amino acid sequence that is extended and / or comprises additional amino acids at the C-terminus and / or N-terminus, such as the C-terminus and / or N-terminus of its heavy or light chain. Therefore, an antibody or its antigen-binding fragment may comprise any suitable number of additional amino acid residues, such as at least one additional amino acid residue. Each additional amino acid residue may be added individually or collectively to, for example, improve and / or simplify the production, purification, in vivo or in vitro stabilization, coupling or detection of a polypeptide. These additional amino acid residues may comprise one or more amino acid residues added for chemical coupling purposes. An example is the addition of cysteine ​​residues. Additional amino acid residues may also provide "tags" for purification or detection of antibodies or their antigen-binding fragments, such as His6 tags, (HisGlu)3 tags, "myc" (c-myc) tags or FLAG tags.

[0248] The isolated antibody or antigen-binding fragment thereof of the present invention can be selected from a full-length antibody, a combination of CDR sequences, a single-chain variable fragment, a Fab fragment, a F(ab')2 fragment, a F(ab')3 fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, an isolated complementarity determining region (CDR) and a nanobody, but is not limited thereto.

[0249] In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, and a chimeric antibody or antigen-binding fragment thereof.

[0250] It may be desirable to reduce or eliminate the effector function of an antibody or antigen-binding fragment thereof, for example to prevent target cell death or unwanted cytokine secretion. This may be particularly appropriate when the antibody or antigen-binding fragment thereof is intended to engage a cell surface receptor and prevent receptor-ligand interaction, i.e., an antagonist. Other examples of effector functions that may warrant reduction include preventing antibody-drug conjugates from interacting with Fc receptors (FcγRs), leading to off-target cytotoxicity.

[0251] Thus, in one embodiment, the isolated antibody or antigen-binding fragment thereof comprises at least one Fc silent mutation that inhibits interaction with FcγR. For example, an antibody or antigen-binding fragment thereof based on the IgG1 isotype class may comprise at least one, preferably at least two, and more preferably all three Fc silent mutations L234A, L235A, and P329G.

[0252] Moreover, when the effector function required to be reduced, the antibody of IgG4 isotype or its antigen-binding fragment is considered as the potential candidate of immunotherapy. Known IgG4 antibody is a dynamic molecule that can undergo a process called Fab arm exchange (FAE), and is not bound by theory, and it is believed that this leads to functional monovalent bispecific antibodies (bsAbs) with unknown specificity and therefore potentially reduced therapeutic efficacy. This may introduce undesirable pharmacodynamic unpredictability into human immunotherapy.

[0253] Thus, in one embodiment, the isolated antibody or antigen-binding fragment thereof comprises at least one stabilizing mutation that prevents or reduces Fab arm exchange in vivo. For example, it has been suggested in the art that a single amino acid mutation (S228P) in the IgG4 core hinge region is sufficient to prevent FAE in vivo [8]. In a specific embodiment, the isolated antibody or antigen-binding fragment thereof is of the IgG4 isotype subclass, and the at least one stabilizing mutation is S228P.

[0254] In one embodiment, the isolated antibody or antigen-binding fragment thereof has an isotype class selected from the group consisting of IgG, IgA, IgM, IgD and IgE. In a specific embodiment, the isotype class is IgG. For example, the isolated antibody or antigen-binding fragment thereof can be selected from the group consisting of isotype subclasses IgG1 and IgG4.

[0255] In one embodiment, the isolated antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof. The monoclonal antibody or antigen-binding fragment thereof is preferably a humanized monoclonal antibody or antigen-binding fragment thereof.

[0256] Currently preferred antibodies or antigen-binding fragments thereof are designated S-SL048-11 (also designated herein as clone 11, heavy chain SEQ ID NO: 119 and light chain SEQ ID NO: 120), S-SL048-46 (also designated herein as clone 46, heavy chain SEQ ID NO: 121 and light chain SEQ ID NO: 122), S-SL048-106 (also designated herein as clone 106, heavy chain SEQ ID NO: 123 and light chain SEQ ID NO: 124), S-SL048-116 (also designated herein as clone 116, heavy chain SEQ ID NO: 125 and light chain SEQ ID NO: 126), and S-SL048-118 (also designated herein as clone 118, heavy chain SEQ ID NO: 127 and light chain SEQ ID NO: 128).

[0257] The stability of these five candidates was evaluated by size exclusion chromatography (SEC), SDS-PAGE, nano-differential scanning fluorimetry (nano-DSF), and differential light scattering. Combining these data with the overall ranking, it can be concluded that the most stable candidates in the hIgG4 S228P format are candidates 106, 118, and 116.

[0258] Binding affinity

[0259] In one embodiment, an isolated antibody or antigen-binding fragment thereof according to the present invention may have a K of no more than D 1×10 -7 M, preferably not more than K D 1×10 -8 For example, an isolated antibody or antigen-binding fragment thereof may have an affinity for hBSSL of no more than K. D 5nM, e.g. not exceeding K D 3 nM affinity for hBSSL.

[0260] As shown in the experimental section, an isolated antibody or antigen-binding fragment thereof may have a K of no more than D 1.7nM, i.e. not exceeding K D 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7 or no more than 0.6 nM affinity for hBSSL. In one example, an isolated antibody or antigen-binding fragment thereof that binds hBSSL according to the present disclosure may have a K D 0.6-1.7nM, such as K D 0.6-1.0, 0.7-0.9, 0.8-1.6, 0.9-1.5, 1.0-1.7, 1.1-1.6, 1.2-1.7, 1.3-1.5, 1.0-1.4, 0.7-1.5, 0.7-1.6, or 1.0-1.7 nM affinity for hBSSL.

[0261] Pharmaceutical composition

[0262] The term "pharmaceutical composition" refers to a formulation that is in such a form that the biological activity of the active ingredient contained therein is effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. The pharmaceutical composition herein comprises an antibody and / or antigen-binding fragment thereof (e.g., scFv) as defined herein and a pharmaceutically acceptable carrier or excipient.

[0263] The antibodies or antigen-binding fragments thereof, such as scFv, as defined herein can be formulated by methods known in the art in the form of, for example, tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols for inhalation and for parenteral use (including intravenous, subcutaneous or intramuscular infusion), sterile aqueous or oily solutions or suspensions, or sterile emulsions.

[0264] Thus, provided herein are compositions comprising an isolated antibody or antigen-binding fragment thereof as described herein and at least one pharmaceutically acceptable excipient or carrier. For example, the excipient can be a diluent. In one example, the pharmaceutical composition can further comprise at least one additional active agent, such as at least two additional active agents, such as at least three additional active agents. A non-limiting example of an additional active agent that may prove useful in such a combination is an immune response modifier.

[0265] "Pharmaceutically acceptable carriers" refer to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0266] As used herein, pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for oral administration, as well as intravenous, intramuscular, subcutaneous, spinal or epidermal administration (e.g., by injection or infusion).

[0267] The pharmaceutical compositions disclosed herein may further comprise a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0268] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

[0269] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Can be ensured to prevent the presence of microorganisms by sterilization procedures and by comprising various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol sorbic acid etc.). May also need to include isotonic agents in the composition, such as sugar, sodium chloride etc. In addition, the prolonged absorption of injectable pharmaceutical forms can be realized by comprising an agent that delays absorption such as aluminum monostearate and gelatin.

[0270] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.

[0271] Also provided herein are kits of components comprising an antibody or antigen-binding fragment thereof or pharmaceutical composition according to the invention, a device for administering the antibody or antigen-binding fragment thereof or pharmaceutical composition, and optionally a package insert comprising instructions for use. The device for administering the antibody or antigen-binding fragment thereof or pharmaceutical composition can be, for example, a syringe. The term "package insert" is used to refer to instructions typically included in commercial packaging of a therapeutic product that contain information regarding indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0272] Medical uses of isolated antibodies or antigen-binding fragments thereof

[0273] The antibodies or antigen-binding fragments thereof according to the present invention can be used to effectively reduce the pro-inflammatory effects of BSSL in a subject, such as a human.

[0274] An advantage of the antibodies and antigen-binding fragments of the present invention is that they do not bind to the active site of the BSSL protein responsible for the lipase activity of BSSL. This is demonstrated and further elaborated in the experimental section. Therefore, the risk of negative side effects is reduced because the lipase activity is not significantly affected.

[0275] Thus, the present invention relates to an isolated antibody or antigen-binding fragment thereof such as a scFv or a pharmaceutical composition as defined herein for use as a medicament.

[0276] The present invention also relates to an antibody or antigen-binding fragment thereof or a pharmaceutical composition as defined herein for use in treating and / or preventing an inflammatory disease. The present invention also relates to the use of an antibody or antigen-binding fragment thereof, such as an scFv, or a pharmaceutical composition as defined herein for use in the manufacture of a medicament for treating and / or preventing an inflammatory disease. The present invention also relates to a method for treating and / or improving and / or preventing and / or preventing an inflammatory disease. The method comprises administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof, such as an scFv, or a pharmaceutical composition to a subject in need thereof.

[0277] Various in vivo models can be used to predict the effects of antibodies and antigen-binding fragments in treating and / or preventing inflammatory diseases. These models typically use mice and inject different substances to elicit an immune response. Thus, the effects of the antibody or antigen-binding fragment on this immune response can be studied after administration of the antibody / antigen-binding fragment.

[0278] A model that can be used is the so-called "collagen-induced arthritis" (CIA) model. In this model, type II collagen (CII) in complete Freund's adjuvant (CFA) is injected, and CII in incomplete Freund's adjuvant (IFA) is usually boosted on day 21. This model induces autoimmune arthritis. Arthritis usually occurs 21-28 days after the first injection of CII. This model relies on B cells and T cells (adaptive immunity).

[0279] Another model is " collagen antibody induced arthritis " (CAIA). In this model, a CII antibody mixture is injected, usually with a booster injection of lipopolysaccharide (LPS) on day 5. This model is independent of B cells and T cells (innate immunity). The protocol for using the CAIA model to test the in vivo efficacy of the antibodies and antigen-binding fragments thereof described herein in the treatment and / or prevention of inflammatory diseases is disclosed in the experimental section herein.

[0280] Yet another model is the "glucose-6-phosphate isomerase-induced arthritis" model. Here, a peptide corresponding to a sequence in glucose-6-phosphate isomerase is injected to elicit an immune response. This model relies on T cells.

[0281] Another model is the pristane-induced arthritis (PIA) model, which involves injection of pristane. This model relies on T cells.

[0282] Additionally, a "dextran sulfate sodium-induced colitis" model can be used, in which dextran sulfate sodium (DSS) is administered in the drinking water.

[0283] All of the above methods are well known to those skilled in the art.The methods can be used to test the in vivo efficacy of the antibodies or antigen-binding fragments thereof herein.

[0284] The inflammatory disease to be treated and / or prevented according to the present invention may for example be a chronic inflammatory disease.The inflammatory disease may be a local or a systemic inflammatory disease.

[0285] Inflammatory diseases can be, for example, autoimmune diseases or autoinflammatory diseases. Another type of inflammatory disease is a natural killer (NK) cell-mediated inflammatory disease. Such NK cell-mediated inflammatory diseases include rheumatoid arthritis (RA), systemic juvenile idiopathic arthritis (sJIA), macrophage activation syndrome (MAS), systemic lupus erythematosus (SLE), systemic sclerosis, multiple sclerosis (MS), Sjögren's syndrome, and inflammatory bowel disease (IBD).

[0286] In one embodiment, the inflammatory disease is selected from the group consisting of RA, JIA, psoriatic arthritis, IBD such as Crohn's disease or ulcerative colitis (UC), hepatic steatosis (also known as hepatic steatosis), and excessive inflammation.

[0287] In a specific embodiment, the inflammatory disease is an inflammatory condition induced by a pathogen such as a bacterium or a virus. Examples of such viruses include coronaviruses, such as severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) or SARS-CoV-2. The latter virus causes coronavirus disease 2019 (COVID-19). Severe COVID-19 patients often suffer from systemic hyperinflammation, including elevated levels of various inflammatory cytokines, such as interleukin 2 (IL-2), IL-7, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1-α (MIP-1α) and TNF-α.

[0288] RA primarily affects the joints but can also be a systemic inflammatory disease that can give extra-articular manifestations in several organs. RA can therefore be considered a systemic inflammatory disease.

[0289] In addition, the isolated antibodies or antigen-binding fragments thereof, such as scFv fragments, or pharmaceutical compositions disclosed herein can be alternatives to current biological treatments for patients who do not respond or respond transiently to current tumor necrosis factor alpha (TNFα) inhibitors, thereby reducing the need to administer corticosteroids and / or immunosuppressants and / or drugs. Thus, the use of the isolated antibodies or antigen-binding fragments thereof, such as scFv fragments, disclosed herein, prevents and / or reduces the adverse effects and / or side effects of alternative treatment regimens in patients, which are often a critical issue in qualitative care, particularly in young patients and children, as well as immunosuppressed and / or elderly patients.

[0290] Treatment and / or prevention using the isolated antibodies and / or antigen-binding fragments thereof or pharmaceutical compositions disclosed herein is typically passive immunotherapy, meaning that the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising such an antibody and / or antigen-binding fragment thereof, is administered to a subject in need thereof. However, other types of immunotherapy approaches, such as gene therapy, may also be used, wherein rather than directly administering an antibody or antigen-binding fragment thereof to a subject, a gene construct capable of expressing such an antibody or antigen-binding fragment thereof is administered.

[0291] According to the subject of the present disclosure, it can be any human or non-human animal.The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Mammals include, but are not limited to, domesticated animals (such as cattle, sheep, cats, dogs and horses), primates (such as humans and non-human primates, such as monkeys), rabbits and rodents (such as mice and rats).The term "subject" can be used interchangeably with the term "patient" in this article.The subject can be a human.

[0292] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of a disease in the individual being treated, and can be used prophylactically or performed during clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms (improving quality of life), reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. Antibodies or antigen-binding fragments thereof according to the present invention can be used to delay the development of the disease or slow the progression of the disease.

[0293] "Reduce" or "inhibit" refers to the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more. Reduce or inhibit can refer to the symptoms of the condition being treated. Reduce or inhibit also includes delaying the onset of a disease, particularly an inflammatory disease.

[0294] Administration

[0295] The isolated antibodies or antigen-binding fragments thereof, such as scFv, or pharmaceutical compositions according to the present invention can be administered in a standard manner for the condition in need of treatment and / or prevention, for example, by oral, topical, parenteral, intravenous, subcutaneous, buccal, nasal, or rectal administration or by inhalation. For example, the antibodies or antigen-binding fragments thereof, such as scFv, or pharmaceutical compositions used as described herein can be formulated for parenteral administration, such as intravenous or subcutaneous administration, in particular subcutaneous administration.

[0296] Typically, the isolated antibody or antigen-binding fragment thereof, such as scFv, or pharmaceutical composition as defined herein is administered systemically. The mode of administration may be, for example, parenteral, such as by intravenous or subcutaneous administration, in particular subcutaneous administration.

[0297] Although the administration schedule can be adjusted depending on the specific disease and subject to be treated, the isolated antibody or antigen-binding fragment thereof, such as scFv, or pharmaceutical composition as defined herein is typically administered 1-3 times per week, such as 1-2 times per week, for example once per week, although other administration schedules are possible.

[0298] The antibodies, antigen-binding fragments thereof and / or pharmaceutical compositions of the present invention can also be used in combination therapy, i.e., in combination with other agents. For example, a combination therapy can include an antibody according to the present invention or an antigen-binding fragment thereof, such as a scFv, in combination with at least one other anti-inflammatory agent or immunosuppressant. It should be understood that combination therapy includes sequential administration as well as simultaneous administration. The term "simultaneously" is used herein to refer to the administration of two or more therapeutic agents, wherein at least some of the administrations overlap in time. Therefore, simultaneous administration includes a dosing regimen when the administration of one or more agents continues after the administration of one or more other agents is interrupted.

[0299] The dosage regimen may be adjusted to provide the optimal desired response, e.g., a therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

[0300] The therapeutically effective amount of an antibody or antigen-binding fragment thereof, such as an scFv, can vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the antibody or antigen-binding fragment thereof, such as an scFv, to elicit a desired response in the subject. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the administered substance. When an antibody or antigen-binding fragment thereof, such as an scFv, is used to prevent a condition (prophylactic purposes), the prophylactic effective amount is typically, but not necessarily, less than the therapeutically effective amount because the prophylactic dose is used in the subject before or at an early stage of the disease.

[0301] The pharmaceutically effective amount (i.e., dosage) of an antibody or antigen-binding fragment thereof, such as an scFv, according to the present invention is typically in the range of about 0.0001 to 100 mg / kg, more typically 0.01 to 5 mg / kg, of the host body weight. However, the precise dosage must be adjusted depending on, for example, the condition to be treated or prevented, the age and / or sex of the subject, and whether it is intended to treat or prevent a condition.

[0302] Expression system

[0303] The present invention also relates to polynucleotides, such as isolated polynucleotides, wherein the polynucleotide encodes an antibody or antigen-binding fragment thereof according to the present invention.

[0304] Examples of polynucleotides according to the embodiments are shown in SEQ ID NOs: 174 to 202, which show DNA sequences encoding the HC and LC of five different antibody fragments: S-SL048-11 HC (SEQ ID NOs: 174; 185; 196) and LC (SEQ ID NOs: 175; 186; 197), S-SL048-46 HC (SEQ ID NOs: 176; 187) and LC (SEQ ID NOs: 177; 188), S-SL048-106 HC (SEQ ID NOs: 178; 189; 198) and LC (SEQ ID NOs: 179; 190; 199), S-SL048-116 HC (SEQ ID NOs: 180; 191; 200) and LC (SEQ ID NOs: 181; 192; 201), and S-SL048-118 HC (SEQ ID NOs: 182; 193; 204). ID NO: 180; 191; 200) and LC (SEQ ID NO: 182; 193; 202), and AS20 HC (SEQ ID NO: 183) and LC (SEQ ID NO: 184), and CDR-graft HC (SEQ ID NO: 194) and LC (SEQ ID NO: 195).

[0305] Thus, in one embodiment, the polynucleotide is selected from the group consisting of SEQ ID NOs: 174 to 202 and any combination and / or variants thereof. As used herein, variants of any one of SEQ ID NOs: 174 to 202 include polynucleotides encoding the same antibody or antigen-binding fragment thereof as the polynucleotide defined in any one of SEQ ID NOs: 174 to 202, but may have at least one synonymous substitution, i.e., at least one base is substituted for another such that the resulting amino acid sequence is not modified. Thus, such a synonymous substitution changes at least one base in a codon in the polynucleotide to another codon, both of which encode amino acid residues. For example, a polynucleotide according to any one of SEQ ID NOs: 174 to 202 or a combination thereof can be codon-optimized for expression in a specific host cell.

[0306] Polynucleotides encoding antibodies or antigen-binding fragments thereof as disclosed herein can be introduced into expression vectors. Expression vectors allow for the propagation of the polynucleotides introduced therein. Vectors can be self-replicating nucleic acid structures, as well as vectors that integrate into the genome of the host cell into which they are introduced. Therefore, the present invention also relates to expression vectors comprising polynucleotides encoding antibodies or antigen-binding fragments thereof.

[0307] The expression vector preferably comprises a polynucleotide encoding an antibody or antigen-binding fragment thereof operably linked to at least one regulatory element. In one embodiment, the regulatory element is or comprises a promoter. A promoter is a DNA sequence to which a protein binds, initiating transcription of an RNA molecule from the downstream DNA (gene). Another example of a regulatory element is an enhancer. An enhancer is a short region of DNA that can be bound by an activator to increase the likelihood that transcription of a specific gene will occur.

[0308] Examples of expression vectors include DNA molecules, RNA molecules, plasmids, episomal plasmids, and viral vectors. Non-limiting but illustrative examples of viral vectors include lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, Semliki Forest virus, poliovirus, and hybrid vectors.

[0309] The expression vector can be introduced into a host cell for expression and / or proliferation of the vector comprising the polynucleotide. In particular, the expression vector is used to treat and / or prevent inflammatory diseases by expressing the vector in a subject to produce antibodies or antigen-binding fragments thereof in the subject.

[0310] Therefore, host cells comprising expression vectors are also provided herein. The host cell used can be any type of host cell, including eukaryotic and prokaryotic host cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, without considering the number of passages.

[0311] The present invention also relates to a cell comprising the antibody or antigen-binding fragment thereof according to the invention, the polynucleotide according to the invention and / or the expression vector according to the invention.

[0312] The cell may be an isolated cell, including a cell of a cell line. The cell may be selected from bacterial cells, eukaryotic cells, such as yeast cells, mammalian cells, human cells or non-human cells.

[0313] Antibodies or antigen-binding fragments thereof can be produced by introducing their sequences into an expression vector and allowing the expression vector to express the antibody or antigen-binding fragment thereof in a host cell, after which the produced antibody or antigen-binding fragment thereof is isolated / purified and then used for medical treatment purposes as disclosed elsewhere herein or for diagnostic purposes. In addition, the vector itself can be introduced into a subject to directly express the antibody or antigen-binding fragment thereof in the subject to be treated. The expression vector preferably contains a promoter that controls the expression of the polynucleotide encoding the antibody or antigen-binding fragment thereof.

[0314] Therefore, the present invention also relates to a method for producing an antibody or an antigen-binding fragment thereof. The method comprises culturing a cell according to the present invention comprising an expression vector according to the present invention under conditions in which the antibody or antigen-binding fragment thereof is expressed by the cell. In one embodiment, the method optionally comprises isolating the antibody or antigen-binding fragment thereof from the cell or the culture medium in which the cell is cultured.

[0315] Diagnostic uses of isolated antibodies or antigen-binding fragments thereof

[0316] The antibodies or antigen-binding fragments thereof of the invention can also be used to detect BSSL, eg, hBSSL, in a sample using standard techniques such as, but not limited to, ELISA, Western blot, RIA, surface plasmon resonance (SPR), and flow cytometric analysis.

[0317] An advantage of using the antibodies or antigen-binding fragments thereof of the present invention is that they do not bind to the active site of BSSL and therefore do not inhibit the protein's lipase activity. Therefore, the antibodies or antigen-binding fragments thereof can be used to study BSSL proteins without significantly affecting lipase activity, as demonstrated in the experimental section. Thus, the antibodies or antigen-binding fragments thereof can be used as molecular tools when studying BSSL proteins and / or their enzymatic activity in vitro / ex vivo and / or in vivo.

[0318] Thus, the present invention discloses a method for detecting the presence of BSSL in a sample and / or for quantifying the amount of BSSL, such as hBSSL, in a sample. The method comprises contacting the sample with an isolated antibody or antigen-binding fragment thereof according to the present invention. The method further comprises detecting the presence of BSSL in the sample and / or quantifying the amount of BSSL in the sample based on the amount of the isolated antibody or antigen-binding fragment thereof that binds to BSSL. Detection or quantification can be performed, for example, using ELISA, Western blot, RIA, surface plasmon resonance (SPR), proximity ligation assay (PLA), or flow cytometry analysis. One or more, i.e., at least two, antibodies or antigen-binding fragments thereof of the present invention can be used for such detection.

[0319] The above method can be in the form of an ex vivo or in vitro method. In this case, the method comprises contacting the sample with the isolated antibody or antigen-binding fragment thereof according to the present invention ex vivo or in vitro.

[0320] In one embodiment, the method further comprises providing a sample that may contain BSSL.

[0321] The present invention also discloses a method for diagnosing a BSSL-related disorder. The method comprises a) contacting a sample with an isolated antibody or antigen-binding fragment thereof according to the present invention, and b) detecting the presence of BSSL in the sample and / or quantifying the amount of BSSL in the sample based on the amount of the isolated antibody or antigen-binding fragment thereof that binds to BSSL. Detection or quantification can be performed, for example, using ELISA, Western blot, RIA, SPR, PLA, or flow cytometry analysis. The method further comprises c) determining, based on the result of step b), whether the subject is diagnosed with a BSSL-related disorder.

[0322] In one embodiment, the method further comprises providing a sample from a subject suspected of having a BSSL-related disorder.

[0323] In certain embodiments, the method comprises comparing the quantified amount of BSSL in the sample to a threshold value. In such certain embodiments, step c) comprises determining whether the subject is diagnosed as having a BSSL-related disorder based on the comparison between the quantified amount of BSSL in the sample and the threshold value. For example, if the amount of BSSL in the BSSL exceeds the threshold value, then the subject is diagnosed as having a BSSL-related disorder or not.

[0324] The value of the threshold value depends on the specific BSSL-associated disorder and can be defined by quantifying the amount of BSSL in a sample taken from a subject who has been diagnosed with the specific BSSL-associated disorder and / or by quantifying the amount of BSSL in a sample taken from a healthy subject who does not have the specific BSSL-associated disorder. The threshold value can then be determined based on these quantified amounts of BSSL from subjects with the specific BSSL-associated disorder and, preferably, based on the quantified amounts of BSSL from healthy subjects.

[0325] BSSL-related disorders are typically inflammatory disorders as disclosed elsewhere herein. The inflammatory disorder can, for example, be a chronic or systemic inflammatory disorder, such as an inflammatory disease, an autoinflammatory disease, and / or an autoimmune disease. The inflammatory disorder can, for example, be rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, atherosclerosis, Crohn's disease, or ulcerative colitis.

[0326] A sample that may contain BSSL can be any type of sample, such as a sample obtained from a subject. Thus, in one embodiment, the sample is a biological sample. An example of such a biological sample is a body fluid sample, such as a blood sample, a plasma sample, or a serum sample. Another example of a biological sample is a body tissue sample, such as a biopsy. The sample can be a natural sample or an in vitro sample that may contain BSSL. Methods for detecting BSSL and / or diagnosing BSSL-related disorders include in vitro methods and in vivo methods, such as in situ hybridization.

[0327] As described elsewhere herein, antibodies or antigen-binding fragments thereof may be humanized or their CDR sequences (or portions thereof) may be grafted onto a non-human scaffold. The latter may be advantageous when using antibodies or antigen-binding fragments thereof as molecular tools to study BSSL proteins in species other than humans, for example to reduce negative immunogenic responses to the antibodies and / or antigen-binding fragments thereof.

[0328] Illustrative embodiments

[0329] One embodiment relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to bile salt-stimulated lipase (BSSL), e.g., human BSSL (hBSSL). The antibody or antigen-binding fragment thereof binds to at least one of a first epitope and a second epitope identified on BSSL. The first epitope comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and the second epitope comprises an amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence that is at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

[0330] In one embodiment, the first epitope comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence that is at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3.

[0331] In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to both the first epitope and the second epitope.

[0332] In one embodiment, the isolated antibody or antigen-binding fragment thereof further specifically binds to an amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence that is at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0333] In one embodiment, the isolated antibody or antigen-binding fragment thereof further specifically binds to an amino acid sequence according to SEQ ID NO: 5, or an amino acid sequence that is at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0334] In one embodiment, the isolated antibody or antigen-binding fragment thereof further specifically binds to an amino acid sequence according to SEQ ID NO: 6, or an amino acid sequence that is at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0335] One embodiment relates to an isolated antibody or antigen-binding fragment thereof. The isolated antibody or antigen-binding fragment thereof comprises three complementarity determining regions (CDRs) (HCDRs) of a heavy chain variable region (HCVR). The first HCDR comprises or consists of an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence having at least 87% identity thereto, the second HCDR comprises or consists of an amino acid sequence according to SEQ ID NO: 8, or an amino acid sequence having at least 75% identity thereto, and the third HCDR comprises or consists of an amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence having at least 90% identity thereto. The isolated antibody or antigen-binding fragment thereof comprises three CDRs (LCDRs) of a light chain variable region (LCVR). The first LCDR comprises or consists of the amino acid sequence according to SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 10, the second LCDR comprises or consists of the amino acid sequence ATS, or an amino acid sequence that is at least 66% identical to the amino acid sequence ATS, such as AAS, and the third LCDR comprises or consists of the amino acid sequence according to SEQ ID NO: 11, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 11.

[0336] In one embodiment, the first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 18 and 19, and the third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the first LCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 20, the second LCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of ATS and AAS, and the third LCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 21 and 22.

[0337] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of the HCVR. The first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 18, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of the LCVR. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 10, the second LCDR comprises, preferably consists of, the amino acid sequence ATS, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 21.

[0338] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of a HCVR. The first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 8, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of a LCVR. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 10, the second LCDR comprises, preferably consists of, the amino acid sequence ATS, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11.

[0339] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of the HCVR. The first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 19, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of the LCVR. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 20, the second LCDR comprises, preferably consists of, the amino acid sequence ATS, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 22.

[0340] In one embodiment, the antibody or antigen-binding fragment thereof comprises three HCDRs of the HCVR. The first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 8, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of the LCVR. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 20, the second LCDR comprises, preferably consists of, the amino acid sequence AAS, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11.

[0341] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises the three HCDRs of the HCVR. The first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 23, or an amino acid sequence that is at least 77% identical to SEQ ID NO: 23, and the third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence that is at least 83% identical to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises the three LCDRs of the LCVR. The first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, the second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 15, or an amino acid sequence that is at least 66% identical to SEQ ID NO: 15, and the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 21, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 21.

[0342] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR). The first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 24, or an amino acid sequence that is at least 77% identical to SEQ ID NO: 24, and the third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence that is at least 83% identical to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three CDRs of a light chain variable region (LCVR). The first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 27, or an amino acid sequence that is at least 70% identical to SEQ ID NO: 27, the second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 29, or an amino acid sequence that is at least 50% identical to SEQ ID NO: 29, and the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 11, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 11.

[0343] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises the three HCDRs of the HCVR. The first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 25, or an amino acid sequence that is at least 83% identical to SEQ ID NO: 25, and the third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence that is at least 83% identical to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises the three LCDRs of the LCVR. The first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 26, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 26, the second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 28, or an amino acid sequence that is at least 66% identical to SEQ ID NO: 28, and the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 22, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 22.

[0344] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of a HCVR. The first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 12, or an amino acid sequence that is at least 77% identical to SEQ ID NO: 12, and the third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence that is at least 83% identical to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of a LCVR. The first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 14, or an amino acid sequence that is at least 70% identical to SEQ ID NO: 14, the second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 15, or an amino acid sequence that is at least 66% identical to SEQ ID NO: 15, and the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 11, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 11.

[0345] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of a HCVR. The first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 12, or an amino acid sequence that is at least 77% identical to SEQ ID NO: 12, and the third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9, or an amino acid sequence that is at least 83% identical to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of a LCVR. The first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, the second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 17, or an amino acid sequence that is at least 50% identical to SEQ ID NO: 17, and the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 11, or an amino acid sequence that is at least 87% identical to SEQ ID NO: 11.

[0346] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of the HCVR. The first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 23, 24 and 25, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of the LCVR. The first LCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 26 and 27, the second LCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 17, 28 and 29, and the third LCDR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 21 and 22.

[0347] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of a HCVR. The first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 23, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of a LCVR. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 16, the second LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 15, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 21.

[0348] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of a HCVR. The first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 24, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of a LCVR. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 27, the second LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 29, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11.

[0349] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of a HCVR. The first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 25, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of a LCVR. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 26, the second LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 28, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 22.

[0350] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of a HCVR. The first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 12, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of a LCVR. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 14, the second LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 15, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11.

[0351] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of a HCVR. The first HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 12, and the third HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of a LCVR. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 16, the second LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 17, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11.

[0352] In one embodiment, the HCVR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 32, 34 and 36, or an amino acid sequence that is at least 98% identical thereto; for example, the group consisting of SEQ ID NOs: 30, 34 and 36, or an amino acid sequence that is at least 96% identical thereto; for example, the group consisting of SEQ ID NOs: 34 and 36, or an amino acid sequence that is at least 96% identical thereto.

[0353] In one embodiment, the HCVR comprises or consists of an amino acid sequence according to SEQ ID NO: 36, or an amino acid sequence that is at least 96% identical thereto.

[0354] In one embodiment, the LCVR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 33, 35, 37 and 38, or an amino acid sequence that is at least 96% identical thereto; for example, the group consisting of SEQ ID NOs: 31, 35, 37 and 38, or an amino acid sequence that is at least 96% identical thereto; for example, the group consisting of SEQ ID NOs: 35, 37 and 38, or an amino acid sequence that is at least 96% identical thereto; for example, the group consisting of SEQ ID NOs: 37 and 38, or an amino acid sequence that is at least 96% identical thereto.

[0355] In one embodiment, the LCVR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 37, or an amino acid sequence that is at least 96% identical thereto.

[0356] In one embodiment, the HCVR comprises, preferably consists of, an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 30, 32, 34, and 36, or an amino acid sequence with at least 96% identity thereto, and the LCVR comprises, preferably consists of, an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 31, 33, 35, 37, and 38, or an amino acid sequence with at least 96% identity thereto. In a specific embodiment, the HCVR comprises, preferably consists of, an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 30, 34, and 36, or an amino acid sequence with at least 96% identity thereto, and the LCVR comprises, preferably consists of, an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 31, 35, 37, and 38, or an amino acid sequence with at least 96% identity thereto. In another specific embodiment, the HCVR comprises, preferably consists of, an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 34 and 36, or an amino acid sequence with at least 96% identity thereto, and the LCVR comprises, preferably consists of, an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 35, 37 and 38, or an amino acid sequence with at least 96% identity thereto. In another specific embodiment, the HCVR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 36, or an amino acid sequence with at least 96% identity thereto, and the LCVR comprises, preferably consists of, an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 37 and 38, or an amino acid sequence with at least 96% identity thereto.

[0357] In one embodiment, the isolated antibody or antigen-binding fragment comprises, preferably consists of, a HCVR comprising an amino acid sequence according to SEQ ID NO: 36, or an amino acid sequence that is at least 96% identical thereto, and a LCVR comprising an amino acid sequence according to SEQ ID NO: 37 or 38, or an amino acid sequence that is at least 96% identical thereto.

[0358] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises an HCVR and a LCVR. The HCVR and LCVR are an amino acid sequence pair that is at least 96% identical to an amino acid sequence pair selected from the group consisting of amino acid sequence pair SEQ ID NOs: 30 and 31, amino acid sequence pair SEQ ID NOs: 32 and 33; amino acid sequence pair SEQ ID NOs: 34 and 35; amino acid sequence pair SEQ ID NOs: 36 and 37; and amino acid sequence pair SEQ ID NOs: 36 and 38; for example, an amino acid sequence pair selected from the group consisting of amino acid sequence pair SEQ ID NOs: 36 and 37; and amino acid sequence pair SEQ ID NOs: 36 and 38.

[0359] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequence ZH1-[CDR-H1]-ZH2-[eCDR-H2]-ZH3-[CDR-H3]-ZH4, wherein ZH1, ZH2, ZH3 and ZH4 each represent zero, one or several independently selected amino acid residues. In one embodiment, the heavy chain variable region consists of an amino acid sequence selected from the group consisting of: i) ZH1-[GYTFTSYN]-ZH2-[X 53 GVIX 57 PGDGX 64 TSYX 68 QKFX 72 ]-ZH3-[ARDYYGSSPLGY]-ZH4, where independently of each other, X 53 Selected from I and M; X 57 Selected from N and Y; X 64 Selected from A and S; X 68 is selected from A and N; and X 72 selected from K and Q, and ii) an amino acid sequence having at least 92% identity to the sequence defined in i). The light chain variable region comprises the amino acid sequence ZL1-[eCDR-L1]-ZL2-[eCDR-L2]-ZL3-[CDR-L3]-ZL4, wherein ZL1, ZL2, ZL3 and ZL4 each represent zero, one or several independently selected amino acid residues. In one embodiment, the light chain variable region consists of an amino acid sequence selected from the group consisting of: iii) ZL1-[X 24 ASX 27 SISYX 39 N]-ZL2-[AX 57 SX 66 LX 68 ]-ZL2-[HQRSSX 115 PT]-ZL4, where independently of each other, X 24Selected from S and R; X 27 Selected from S and P; X 39 Choose from M and L; X 57 Selected from A and T; X 66 Selected from K and S; X 68 selected from A and P; and X 115 An amino acid sequence selected from the group consisting of S, T and Y, and iv) having at least 87% identity with the sequence defined in iii).

[0360] In one embodiment, ZH1 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 39, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 39.

[0361] In one embodiment, ZH2 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 40, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 40.

[0362] In one embodiment, ZH3 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 41, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 41.

[0363] In one embodiment, ZH4 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 42, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 42.

[0364] In one embodiment, ZL1 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 43, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 43.

[0365] In one embodiment, ZL2 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 44, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 44.

[0366] In one embodiment, ZL3 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 45, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 45.

[0367] In one embodiment, ZL4 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 46, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 46.

[0368] In one embodiment, the antibody is a full-length antibody.

[0369] In one embodiment, the antibody is selected from the group consisting of a human antibody, a humanized antibody, and a chimeric antibody.

[0370] In one embodiment, the antigen binding fragment is an antigen binding fragment, such as a single chain variable fragment, a Fab fragment, a F(ab')2 fragment, a F(ab')3 fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, an isolated complementarity determining region (CDR) and a nanobody. In a specific embodiment, the antigen binding fragment is a scFv fragment.

[0371] In one embodiment, the isolated antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof. In a specific embodiment, the monoclonal antibody or antigen-binding fragment thereof is a humanized monoclonal antibody or antigen-binding fragment thereof.

[0372] In one embodiment, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of isotype classes IgG, IgA, IgM, IgD and IgE; for example, IgG. In a specific embodiment, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of isotype subclasses IgG1 and IgG4.

[0373] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises one or more Fc silent mutations. In a specific embodiment, the IgG1 comprises the Fc silent mutations L234A, L235A and P329G.

[0374] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises one or more stabilizing mutations that prevent or reduce Fab arm exchange in vivo. In a specific embodiment, the IgG4 comprises the stabilizing mutation S228P.

[0375] In one embodiment, the isolated antibody or antigen-binding fragment thereof is a single-chain variable fragment (scFv) that specifically binds hBSSL and comprises a HCVR domain comprising a first HCDR, a second HCDR, and a third HCDR comprising, or consisting of, an amino acid sequence at least 80% identical to SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively, and a LCVR domain comprising a first LCDR, a second LCDR, and a third LCDR comprising, or consisting of, an amino acid sequence at least 80% identical to SEQ ID NO: 10, amino acid sequence ATS, and SEQ ID NO: 11, respectively.

[0376] In one embodiment, the first HCDR, the second HCDR and the third HCDR consist of the amino acid sequence according to SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, and the first LCDR, the second LCDR and the third LCDR consist of the amino acid sequence according to SEQ ID NO: 10, the amino acid sequence ATS and SEQ ID NO: 11, respectively.

[0377] In one embodiment, the antibody is a humanized antibody.

[0378] In one embodiment, the isolated antibody or antigen-binding fragment thereof has an affinity for hBSSL of no more than K D 1.7nM.

[0379] In one embodiment, the isolated antibody or antigen-binding fragment thereof is capable of displacing hBSSL from monocytes, preferably CD14 + Binding of monocytes.

[0380] One embodiment relates to a pharmaceutical composition comprising an isolated antibody and / or antigen-binding fragment thereof according to the present invention and a pharmaceutically acceptable carrier or excipient.

[0381] One embodiment relates to an isolated antibody and / or antigen-binding fragment thereof or a pharmaceutical composition according to the invention for use as a medicament.

[0382] One embodiment relates to an isolated antibody and / or antigen-binding fragment thereof or a pharmaceutical composition according to the present invention for use in the treatment and / or prevention of an inflammatory disease.

[0383] One embodiment relates to the use of an isolated antibody and / or antigen-binding fragment thereof or a pharmaceutical composition according to the present invention for the manufacture of a pharmaceutical composition for the treatment and / or prevention of an inflammatory disease.

[0384] One embodiment relates to a method for treating and / or ameliorating and / or preventing and / or prophylactic inflammatory diseases. In the method, a therapeutically effective amount of an isolated antibody and / or antigen-binding fragment thereof or a pharmaceutical composition according to the present invention is administered to a subject in need thereof.

[0385] In one embodiment, the inflammatory disease is a chronic inflammatory disease.

[0386] In one embodiment, the inflammatory disease is a systemic inflammatory disease.

[0387] In one embodiment, the inflammatory disease is an autoimmune disease. In a specific embodiment, the autoimmune disease is rheumatoid arthritis or juvenile rheumatoid arthritis. In another specific embodiment, the autoimmune disease is inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis.

[0388] In one embodiment, the inflammatory disease is an autoinflammatory disease. In a specific embodiment, the autoinflammatory disease is psoriatic arthritis.

[0389] In one embodiment, the inflammatory disease is hepatic steatosis.

[0390] In one embodiment, the isolated antibody and / or antigen-binding fragment thereof or pharmaceutical composition is administered systemically.

[0391] In one embodiment, the isolated antibody and / or its antigen-binding fragment or pharmaceutical composition is administered parenterally, e.g., subcutaneously. Thus, in a specific embodiment, the isolated antibody and / or its antigen-binding fragment or pharmaceutical composition is formulated for parenteral administration, e.g., subcutaneous administration.

[0392] In one embodiment, the isolated antibody and / or antigen-binding fragment thereof or pharmaceutical composition is administered 1-3 times per week, such as 1-2 times per week, such as once per week.

[0393] In one embodiment, treatment and / or prevention is performed by passive immunotherapy.

[0394] Embodiments relate to a polynucleotide encoding an isolated antibody or antigen-binding fragment thereof as defined according to the invention, an expression vector comprising a polynucleotide according to the invention and a host cell comprising an expression vector according to the invention.

[0395] One embodiment relates to a method for producing an isolated antibody or antigen-binding fragment thereof according to the present invention. The method comprises culturing a host cell according to the present invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.

[0396] One embodiment relates to a method for detecting the presence or absence of BSSL in a sample and / or quantifying the amount of BSSL in a sample. The method comprises the steps of: a) providing a sample that may contain BSSL, b) contacting the sample with an isolated antibody or antigen-binding fragment thereof according to the invention, and c) detecting the presence or absence of BSSL in the sample and / or quantifying the amount of BSSL in the sample.

[0397] One embodiment relates to a method for diagnosing a BSSL-associated disorder. The method comprises the steps of: a) providing a sample from a subject suspected of having a BSSL-associated disorder, b) contacting the sample with an isolated antibody or antigen-binding fragment thereof according to the present invention, c) detecting the presence or absence of BSSL in the sample and / or quantifying the amount of BSSL in the sample, and d) based on the result of step c), determining whether the subject is diagnosed with a BSSL-associated disorder.

[0398] In one embodiment, the BSSL-associated disorder is an inflammatory disease, such as a chronic inflammatory disease; a systemic inflammatory disease; an autoimmune disease, such as rheumatoid arthritis, juvenile rheumatoid arthritis, an inflammatory bowel disease, such as Crohn's disease and ulcerative colitis; an autoinflammatory disease, such as psoriatic arthritis; or hepatic steatosis.

[0399] One embodiment relates to a method for determining the enzymatic activity of BSSL. The method comprises the following steps: a) providing a sample containing BSSL, b) contacting the sample with an isolated antibody or antigen-binding fragment thereof according to the present invention, and c) determining the enzymatic activity of BSSL in the sample.

[0400] One embodiment relates to a BSSL epitope comprising or consisting of a first epitope and a second epitope. The first epitope comprises or consists of an amino acid sequence according to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, such as 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. The second epitope consists of a second surface comprising or consisting of an amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence that is at least 80%, such as 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2.

[0401] In one embodiment, the first epitope comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 3, or an amino acid sequence that is at least 80%, e.g. 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0402] In one embodiment, the epitope further comprises an amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence at least 80%, such as 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0403] In one embodiment, the epitope further comprises an amino acid sequence according to SEQ ID NO: 5, or an amino acid sequence at least 80%, such as 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0404] In one embodiment, the epitope further comprises an amino acid sequence according to SEQ ID NO: 6, or an amino acid sequence at least 80%, such as 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0405] Although the present invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for its elements without departing from the scope of the present invention. Furthermore, many modifications may be made to adapt a particular situation or molecule to the teachings of the present invention without departing from the basic scope of the present invention. Therefore, the present invention is not limited to any particular embodiment contemplated, but rather the present invention encompasses all embodiments falling within the scope of the appended claims.

[0406] Example

[0407] Table 2 - BSSL materials used in the examples

[0408]

[0409] Example 1 - Binding of AS20 IgG to human and mouse BSSL (SPR)

[0410] In this example, the binding of the antibody AS20 mouse-IgG1 (AS20mIgG) to human and mouse BSSL was investigated by surface plasmon resonance (SPR). AS20 mIgG (heavy chain variable region (HCVR) SEQ ID NO: 80 and light chain variable region (LCVR) SEQ ID NO: 114) was raised in mice against the full-length BSSL protein (SEQ ID NO: 138) purified from human milk. Reactivity to mouse BSSL is unknown.

[0411] Materials and methods

[0412] hBSSL and mBSSL (Table 2) were used in the experiments described in this example together with AS20 mouse IgG1 (AS20 mlgG1) (produced in-house, HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114).

[0413] use SPR measurements were performed using a T200 instrument (GE Healthcare). To minimize avidity effects, antibodies were immobilized on the sensor surface and BSSL was injected as the analyte. AS20 mIgG1 was immobilized on a CM5 (carboxylated dextran surface) sensor chip. The chip was activated by injecting a 1:1 mixture of 0.2 M N-ethyl-N'-[(dimethylamino)propyl]carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS) with a contact time of 7 minutes. The antibody was injected over 0.2-2.8 minutes and diluted to 14-50 μg / ml in 10 mM acetate-HCl pH 5.0 (Group 1) or pH 6.0 (Group 2) to achieve a final immobilization level of 560 to 830 RU. Remaining activated carboxyl groups on the sensor surface were inactivated by injecting 1 M ethanolamine for 7 minutes.

[0414] The running buffer in the first set of experiments was PBS buffer pH 7.4 (10 mM phosphate, 2.5 mM KCl, 137 mM NaCl) supplemented with 0.05% (v / v) Tween 20. In the second set of experiments, the running buffer was 25 mM TrisHCl, pH 7.5, 150 mM NaCl. 146 mM H3PO4 was used as the standard regeneration solution. Kinetic studies and nonlinear regression analysis were performed using the single-cycle kinetics (SCK) method and evaluation software on the T200 instrument. The interaction between mBSSL and AS20 mIgG1 was analyzed using a steady-state affinity model.

[0415] In the first set of experiments, three SCK experiments were performed with the highest hBSSL concentration in the concentration series being 300, 100 and 50 nM. The concentration series was prepared at 1:3, 1:3.16 (half log) and 1:2 dilutions.

[0416] In the second set of experiments, hBSSL was diluted to a starting concentration of 20 nM in running buffer and then serially diluted 1:1 in the same buffer to obtain a five-point concentration range from 20 nM to 1.25 nM. mBSSL was diluted to a starting concentration of 2000 nM in running buffer and then serially diluted 1:1 in the same buffer to obtain a five-point concentration range from 2000 nM to 125 nM.

[0417] result

[0418] AS20 mIgG1 was found to bind to both human and mouse BSSL. The affinity for human BSSL was strong with low nanomolar affinity. The interaction was well characterized by a 1:1 binding model ( Figure 1). The association and dissociation rate constants and the equilibrium dissociation constant from the nonlinear regression analysis of the SCK experiments are presented in Table 3. In the first set of experiments, the measurements were performed in triplicate, so the mean values ​​and standard deviations are presented.

[0419] Mouse BSSL was also observed to interact with immobilized AS20 mIgG1, however, the affinity was nearly 100-fold weaker. Steady-state analysis was performed to determine the affinity ( Figure 2 Table 3 - Kinetic parameters of the interactions between AS20 mIgG1 and hBSSL and between AS20 mIgG1 and mBSSL

[0420]

[0421] *Single cycle kinetics

[0422] ** Steady-state analysis

[0423] ND not determined

[0424] 1 The first set of experiments

[0425] 2 The second set of experiments

[0426] Example 2 - Generation and Binding Characterization of AS20 scFv to Human and Mouse BSSL (ELISA, SPR and )

[0427] In this example, a single-chain variable fragment (scFv) format based on AS20 mIgG1, denoted AS20scFv (comprising HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114), was generated that retained binding to human BSSL as determined by enzyme-linked immunosorbent assay (ELISA), SPR, and Evaluate.

[0428] Materials and methods

[0429] Small-scale production and purification

[0430] The gene encoding the corresponding scFv construct was formed by fusing the HCVR (SEQ ID NO: 80) to the LCVR (SEQ ID NO: 114) via a glycine-serine linker. The scFv gene was subcloned into the pHAT-6 screening vector (SciLifeLab, Stockholm, Sweden), which provides the scFv secretion signal and a triple FLAG tag and a hexahistidine (His) tag at the C-terminus. The construct was then transformed into TOP10 Escherichia coli. The bacterial supernatant of the lysed cells was purified using α-FLAG antibody-conjugated magnetic beads (Sigma Aldrich, #M8823). The purified scFv was analyzed by gel electrophoresis under reducing conditions to determine its purity and integrity, and the protein concentration was determined using a BCA (bicinchoninic acid) assay kit (Pierce).

[0431] ELISA

[0432] Non-biotinylated human BSSL (hBSSL) and biotinylated human BSSL (b-hBSSL) were coated directly or via streptavidin at two different concentrations, 1 μg / ml and 0.5 μg / ml, in PBS, in 384-well ELISA plates overnight at 4°C. Purified AS20 scFv was serially diluted three-fold in blocking buffer (phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA) and 0.05% Tween 20) to concentrations ranging from 1 μg / ml to 4 ng / ml. Binding was detected by horseradish peroxidase (HRP)-conjugated α-FLAG M2 (Sigma-Aldrich) followed by incubation with the chromogenic substrate Ultra 3,3',5,5'-tetramethylbenzidine (TMB) ELISA. Signal generation was terminated by the addition of 1 M sulfuric acid, and absorbance was measured at 450 nm.

[0433] In a second ELISA assay, 1 μg / ml of human and mouse BSSL and a negative control protein were coated directly into a 384-well ELISA plate and incubated overnight at 4°C. Purified AS20 scFv and a negative control scFv were added at two different concentrations, 1 μg / ml and 0.2 μg / ml. Binding signals were detected as described above. In both ELISA settings, all samples were assayed in duplicate. hBSSL, b-hBSSL, and mBSSL (Table 2) were used in the experiments performed in this example together with AS20 scFv (produced in-house, HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114).

[0434] SPR measurements

[0435] use Affinity assessment of scFv clones was performed using SPR on a T200 (GE Healthcare). The α-FLAG M2 antibody was immobilized on a CM5 S chip via primary amine coupling using NHS-EDC chemistry, allowing capture of the AS20 scFv via its 3xFLAG tag. A 3-fold dilution series consisting of 5 different concentrations (200 nM to 2 nM) of hBSSL and b-hBSSL was sequentially injected onto the flow cell to allow binding to the captured AS20 scFv. Surface regeneration was achieved under acidic conditions using 10 mM glycine-HCl at pH 2.2. The obtained single-cycle kinetic data were fitted to a 1:1 Langmuir binding model, and the kinetic parameter k was retrieved using the software BIAevaluation. a (1 / Ms), k d (1 / s) and K D (M).

[0436] analyze

[0437] Biotinylated hBSSL was conjugated to neutravidin. The beads were incubated and mixed with 30 different bead IDs, each conjugated to an unrelated protein. The mixed bead pool was incubated with AS20 scFv present in bacterial supernatant diluted 1:10 in assay buffer (PBS supplemented with 3% BSA, 0.05% Tween20 and 10 μg / ml neutravidin). A positive scFv control was also included, i.e., an scFv expected to bind to beads coated with an unrelated protein. The scFv clones were bound to specific protein-conjugated beads by R-PE-conjugated anti-FLAG M2 antibody and subsequently analyzed on the FlexMAP 3D instrument.

[0438] result

[0439] Small-scale production and purification

[0440] Gel electrophoresis of bacterially expressed and purified AS20 scFv showed high purity, with one major protein band corresponding exactly to the expected molecular weight of the scFv (data not shown).

[0441] ELISA

[0442] AS20 scFv showed concentration-dependent binding to non-biotinylated and biotinylated human BSSL ( Figure 3a At a specific scFv concentration, the signal intensity for biotinylated BSSL was much higher than that for non-biotinylated BSSL, which may be due to the difference in coating conditions.

[0443] AS20 scFv also showed binding to mouse BSSL, but the signal intensity was much weaker than that to human BSSL ( Figure 3b ), which may indicate that the affinity of AS20 scFv for mouse BSSL is weak. No binding of AS20 scFv to the negative control was detected.

[0444] SPR measurements

[0445] The affinity of AS20 scFv for non-biotinylated and biotinylated human BSSL was determined using a single cycle kinetic method. D =0.6 nM) and biotinylated human BSSL (K D = 0.8 nM) showed very similar affinities (Table 4).

[0446] Table 4 - Kinetic parameters for the interaction between AS20 scFv and native and biotinylated hBSSL

[0447]

[0448] Luminex analysis

[0449] To determine whether AS20 scFv is prone to nonspecific interactions with unrelated proteins, a Luminex assay was performed in which AS20 scFv was analyzed against 30 different unrelated proteins as well as their cognate targets. AS20 scFv showed binding only to human BSSL, with no binding or very low binding to all other proteins included in the assay ( Figure 4 ).

[0450] in conclusion

[0451] AS20 scFv was found to bind with similar affinity (similar K D The K value of non-biotinylated BSSL was obtained by combining non-biotinylated human BSSL and biotinylated human BSSL. D The K values ​​were similar to those of the full-length IgG antibodies reported in Example 1. D The values ​​were completely consistent. When assayed in a Luminex-based assay, the AS20 scFv also showed low off-target binding to 30 unrelated proteins. As shown by the ELISA results, the AS20 scFv showed binding to mouse BSSL, as also demonstrated for full-length IgG in Example 1.

[0452] Example 3 - Characterization of AS20 scFv binding to mouse BSSL by HTRF

[0453] Homogeneous time-resolved fluorescence (HTRF) is a spectrophotometric method based on the phenomenon of fluorescence resonance energy transfer (FRET) between two different molecules, termed donor and acceptor, respectively. This example describes the development and use of an HTRF-based competition assay as an alternative method to characterize the interaction between AS20 scFv and mouse BSSL.

[0454] Materials and methods

[0455] To investigate the interaction between AS20 scFv and native mouse BSSL, a competition assay was developed. Binding was detected using a donor molecule, a terbium-conjugated α-FLAG antibody (Cisbio #611FG2TL), which interacts with the C-terminally located FLAG tag of the scFv, and an acceptor molecule, streptavidin-conjugated XL665 (Cisbio #610SAXL), which interacts with the biotin moiety on human BSSL. Experiments were performed using a range of concentrations of non-biotinylated protein: 0-500 nM for mBSSL and 0-80 nM for hBSSL. hBSSL, b-hBSSL, and mBSSL (Table 2) were used in experiments performed in this example together with AS20 scFv (produced in-house, HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114).

[0456] 2.5 nM AS20 scFv was preincubated with mouse or human BSSL orthologs for 2 hours, followed by the addition of 5 nM b-hBSSL and FRET donor and acceptor molecules. The mixture was incubated at room temperature for 16 hours, and the binding signal (665 nm) and background / noise signal (615 nm) were measured using EnVision (PerkinElmer). The experimental output ΔR for each point was calculated using four replicates and two blanks.

[0457] result

[0458] The data showed that mouse BSSL competed with human BSSL-biotin for binding to AS20 scFv in a concentration-dependent manner. Figure 5 The same observations were made for human native BSSL. A 50% reduction in ΔR was achieved with 150-200 nM mouse BSSL and approximately 2 nM human BSSL, indicating that the affinity of AS20 scFv for mouse BSSL is approximately 100-fold lower than that of the human ortholog.

[0459] in conclusion

[0460] The data obtained showed that the affinity of AS20 scFv for mouse BSSL was approximately 100-fold lower than that of the human ortholog. This is in good agreement with the affinities obtained using SPR for the full-length antibody forms of the same clone; AS20 mIgG1 (Example 1) and chimeric AS20 (Example 4).

[0461] Example 4 - Generation of Chimeric AS20

[0462] In this example, chimeric AS20 was generated. More specifically, a chimera of the human IgG4 subclass was constructed.

[0463] Materials and methods

[0464] The production of materials was outsourced to GenScript (Piscataway, NJ, USA). The sequences of the variable domains of the heavy chain (VH) and light chain (VL) of the mouse AS20 antibody corresponding to SEQ ID NO: 80 and SEQ ID NO: 114, respectively, were used. The genes encoding the heavy and light chains were synthesized and cloned into a vector encoding the human IgG4 subclass. The constructs were transfected and transiently expressed in FreeStyle 293-F cells. The expressed antibodies were then purified by affinity chromatography using protein A, followed by preparative size exclusion chromatography (SEC), and the purity was determined by high-performance liquid chromatography (HPLC) and the concentration was determined spectrophotometrically at 280 nm.

[0465] result

[0466] The purity was >98% as determined by HPLC.The sequence of the chimeric AS20 was determined to be SEQ ID NO: 139 for the heavy chain and SEQ ID NO: 140 for the light chain.

[0467] Chimeric AS20 retained the same binding affinity for mouse and human BSSL as the AS20 mouse IgG1 antibody in Example 1 (data not shown).

[0468] Example 5 - Design and Construction of AS20 CDR-Grafted Antibodies and AS20 Humanized Libraries

[0469] AS20 is a mouse antibody, AS20 mIgG, see Example 1. Non-human antibodies have been shown to induce human immune responses, which can lead to neutralization of the administered antibody and, in turn, limit the antibody's effectiveness in treating disease. To overcome this potential problem, humanization of the antibody was performed. This example describes two strategies for the humanization of AS20: complementary determining region (CDR) grafting and a library-based approach. The resulting CDR-grafted antibody is referred to herein as an AS20 CDR graft or CDR graft, and the resulting library is referred to herein as an AS20 humanized library. The library is subsequently used to select and isolate AS20-binding scFv fragments using phage display (see Example 6).

[0470] Materials and methods

[0471] Figure 15A and 15B is an overview of combinatorial scFv library design of heavy chain variable regions, and Figure 16A and 16B An overview of the design of a combinatorial scFv library of light chain variable regions.

[0472] The scFv format was chosen as a scaffold for CDR grafts and humanized libraries. The data presented in Example 2 show that the AS20 scFv fully retained the binding capacity of its full-length parental IgG counterpart, indicating that the scFv gene is a good scaffold format for both CDR grafts and construction of combinatorial libraries.

[0473] Based on IMGT / DomainGapAlign (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi), the human immunoglobulin heavy chain variable region germline gene (IHGV), IGHV1-46, with the highest sequence homology to the AS20 heavy chain variable region, with an identity of 73.5% (positions 1-104), was selected as the IHGV framework. For the selection of light chain sequences, homology was considered, but also consideration was given to heavy and light chain pairings with favorable biophysical properties

[10] . Overall, this led to the selection of the human germline gene IGKV1-39. Also based on IMGT / DomainGapAlign domain searches, IGHJ4 and IKVJ2 were selected as connecting segments to obtain the complete variable heavy and light chain domains, respectively.

[0474] The AS20 CDR graft was created by grafting six mouse CDR loops into human germline genes. For the heavy chain, the following regions were grafted into the IGHV1-46 framework: heavy chain complementary determining region 1 (HCDR1) (SEQ ID NO: 7); extended HCDR2 (eHCDR2) (SEQ ID NO: 141); HCDR3 (SEQ ID NO: 9). This resulted in a CDR graft with an HCVR according to SEQ ID NO: 144. For the light chain, the following regions, extended light chain complementary determining region 1 (eLCDR1) (SEQ ID NO: 142), eLCDR2 (SEQ ID NO: 143), and LCDR3 (SEQ ID NO: 21), were grafted into the IGKV1-39 framework, resulting in an LCVR according to SEQ ID NO: 145. Genes encoding the corresponding scFv constructs were formed by fusing the HCVR to the LCVR via a glycine-serine linker ((Gly4Ser)3). Two additional amino acids (Arg and Thr, both part of the CL domain) were added to the ends of the LCVR to include a BsiWI restriction site. Synthesis and subcloning of the scFv gene were outsourced to GenScript (Piscataway, NJ, USA). After synthesis, the scFv gene was cloned into an in-house phagemid using the restriction enzymes SfiI and BsiWII.

[0475] The same HCVR and LCVR frameworks used in the CDR graft were used to construct the AS20 humanized library scaffold ( Figure 6 The mutagenesis strategy for the AS20 humanized library is summarized in Table 5. HCDR3 is considered the most important region for antigen binding. It is reasonable to assume that this loop is also likely important for the AS20-BSSL interaction and therefore remained constant. In contrast, 22 positions in the other five CDR regions that differed between AS20 and the AS20 humanized scaffold were selected for mutation ( Figure 6). Here, dual diversity was primarily attempted, i.e., residues found in AS20 and human germline genes at specific positions to construct humanized scaffolds were allowed to be found. However, not all amino acid pairs can be established by NNS oligonucleotides without introducing additional amino acids, so additional chemical diversity was added at 6 positions (three in VH: 62, 64, 68 and three in VL: 27, 66, 68). In LCDR3, another strategy was adopted. Here, the diversity found in the rearranged functional antibodies encoded by the germline genes IGVK1-39 / IKV1D-39 found in the IMGT database (http: / / www.imgt.org / ligmdb / ) was considered. More specifically, antibody sequences with an LCDR3 length of 8 amino acids (the length of LCDR3 in AS20) were used as a guide for what diversity to introduce. The consensus sequence obtained was QQSYSTPT (aa 105-117, SEQ ID NO: 173). Based on the mouse AS20 and human consensus sequences, double diversity was introduced at positions 105, 107, and 108. At position 115, four amino acids were introduced, again due to the limitations of the NNS oligonucleotides. Due to the VJ gene joining process, most of the diversity in LCDR3 is found at position 116. To mimic this variability, but also subject to the limitations of NNS codon usage, six amino acids (P, H, L, Y, S, and F) were allowed here. This strategy allowed the capture of >50% of the diversity found at this position in antibodies. In summary, the above process generated approximately 1.2x10 9 Theoretical diversity of combinations of different variants. Table 5 - Targeted positions for mutagenesis in the AS20 humanized library. Positions in the HCVR and LCVR are listed at the top and bottom of the table, respectively. The amino acids marked in bold are those found in AS20, while the underlined amino acids are the corresponding diversity found in the human germline gene. Numbering is as defined by IMGT nomenclature, and for codon definitions, the IUPAC nucleotide code is used. Separate primers for the five targeted regions (HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3) were used to introduce diversity (Table 6).

[0476] Target location HCVR Design diversity codon 38 N,Y WAT 53 I,M ATR 55 V,I RTA 57 Y,N WAC 59 G,S RGT 62 N,G,S,D RRT 64 D,S,A,Y KMC 68 N,A,T,D RMT 72 K,Q MAG Target position LCVR Design diversity codon 24 S,R AGK 27 S,Q,P,STOP YMG 39 M,L WTG 40 H,N MAT 56 D,A GMT 57 T,A RCA 66 K,S,N,R ARS 68 A,Q,P,E SMA 105 H,A CAS 107 R,S AGK 108 S,Y TMT 115 Y,T,N,S WMT 116 P,H,L,Y,S,F YHC

[0477] Using the optimized Kunkel mutagenesis method essentially as described in

[11] , the AS20 humanized library scaffold gene ( Figure 6) and five mutagenic oligonucleotides (Table 6) were used to introduce diversity into the library scaffold genes. To assess whether the expected diversity was introduced, TOP10 E. coli cells were chemically transformed with a small portion of the DNA generated by the Kunkel mutagenesis method, and 96 clones were selected and sent for sequencing (GATC, Germany). The remaining DNA was then electroporated into SS320 cells (Lucigen, Middleton, WI, USA) to generate a library containing approximately 1.7 × 10 10 The highly diverse library of individual clones was prepared, as measured by the number of bacterial colonies obtained after transformation. The transformed SS320 cells were harvested and stored at -80°C with 15% glycerol. A total of 600 ml 2 × YT was inoculated with bacterial glycerol stock, which contained antibiotics that were selective for both phagemids and F' appendages. The bacteria were grown to exponential phase and then infected with M13KO7 helper phage (New England Biolabs, Ipswich, MA, USA) at a multiplicity of infection of 5. The culture was propagated overnight and the phage displaying scFv was harvested by standard polyethylene glycol PEG / NaCl precipitation. The final library stock was dissolved in PBS supplemented with 0.5% BSA, 0.05% Tween-20.

[0478] Table 6 - Oligonucleotide primers used to construct the AS20 humanized library. Sequences formatted using the IUPAC nucleotide code.

[0479]

[0480] result

[0481] The genes encoding the AS20 CDR graft and the AS20 humanized library scaffold were synthesized and cloned into the pHAT4 phagemid vector. The AS20 humanized library was constructed by using the optimized Kunkel procedure, generating 1.7 × 10 10 Sequencing of 96 randomly selected clones confirmed the introduction of the expected diversity (data not shown).

[0482] in conclusion

[0483] AS20 CDR grafts and AS20 humanized libraries were successfully constructed. In both cases, IGHV1-46 and IGKV1-39 were used as human framework scaffold genes. The binding of the AS20 CDR grafts to BSSL was evaluated in scFv (Example 6) and IgG formats (Examples 9 and 11). The AS20 humanized library was used to isolate humanized BSSL-binding scFv fragments by phage display and various binding screening assays (Example 6). Several selected clones showed binding to the cognate target (human BSSL) with affinity and specificity comparable to the parental IgG, and even better affinity to the mouse ortholog (Example 9). When the sequences of the selected clones were analyzed (Example 11), it was clear that specific residues were enriched at certain positions. Interestingly, at several positions (e.g., VH:62, 64 and VL:115), amino acids outside of the double diversity were preferentially selected, indicating that introducing additional diversity was a successful strategy.

[0484] Example 6 - Phage display selections on human and mouse BSSL and subsequent screening and sequencing

[0485] In this example, phage display selections were performed to enable the isolation of scFv fragments specific for human and mouse BSSL.

[0486] Materials and methods

[0487] antigen

[0488] During phage display selections, mouse BSSL and non-biotinylated and biotinylated human BSSL were used as target antigens. More specifically, two variants with varying degrees of biotinylation and coupling chemistries were prepared. These were BSSL-b-amine and BSSL-b-sugar. hBSSL, b-hBSSL, hBSSL-b-amine, and mBSSL (Table 2) were used as targets for phage display selections in this example.

[0489] Phage display selection

[0490] For all antigens, phage display was performed using four rounds of enrichment using two in-house constructed human synthetic scFv phage libraries, SciLifeLib2 and the AS20 humanized library (see Example 5). SciLifeLib2 is a natural human synthetic scFv library, whose design and construction are similar to those previously reported

[12] . The selection pressure was increased by gradually reducing the amount of antigen and increasing the number and intensity of washes between different rounds. For the two biotinylated human BSSL samples, selection was performed by immobilizing them on streptavidin-coated paramagnetic beads (Dynabeads M-280, ThermoFisher Scientific, #11206D), and most steps in the selection process were automated and performed using a Kingfisher Flex robot. Selection of natural antigens was performed by coating the natural antigens on 96-well plates (NUNC Maxisorp #442404). In some tracks, in order to preferentially select cross-species reactive scFvs, the antigens were alternated between human and mouse BSSL in different rounds. Phage were eluted with trypsin or by competitive elution using mouse BSSL for selection on human BSSL, and vice versa. The combination of these different parameters produced schemes covering a total of five different selection tracks for ScilifeLib2 and nine different selection tracks for the AS20 humanized library. The recovered phages were propagated in Top10F' Escherichia coli on agar plates at 37°C overnight (rounds 1 and 2) or in solution at 30°C overnight (rounds 3 and 4). Phage stock solutions were prepared by infection with an excess of M13K07 helper phage (New England Biolabs, #N0315S) and induction of scFv expression by the addition of IPTG. Overnight cultures were precipitated with PEG / NaCl, resuspended in selection buffer and used for the next round of selection. Table 7 summarizes the phage display selection tracks.

[0491] Table 7 - Phage display loci

[0492]

[0493]

[0494] 1- Selection on biotinylated BSSL (amine coupled, 10X) coupled to magnetic SAV beads (M280)

[0495] 2- Selection on biotinylated BSSL (for carbohydrates, BH8520) coupled to magnetic SAV beads (M280)

[0496] 3-Selection on natural antigens coated on the surface of immunotubes

[0497] 4- Phage elution using competition with other species (mouse or human) via BSSL (trypsin will be used for elution in all other cases)

[0498] Recloning

[0499] To produce soluble scFv, phagemid DNA was isolated from the third and fourth rounds of each selection track. In the pool, the gene encoding the scFv fragment was subcloned into a screening vector, providing the scFv secretion signal and a triple FLAG tag and a hexahistidine (His) tag at the C-terminus. The construct was then transformed into TOP10 E. coli.

[0500] Primary ELISA and sequencing

[0501] For each selection trajectory, a total of 89-222 colonies were selected from rounds 3 and 4 and cultured in 96-well plates and grown overnight. The expressed scFvs (supernatants) were screened for binding to the native form of the respective selected target by ELISA. Experiments in this screening method included the previously constructed, produced, and characterized AS20 scFv for binding to human and mouse BSSL (see Example 2). The AS20 CDR graft was also included as an scFv (see Example 5). Clones that were positive in the ELISA were subjected to DNA sequencing (GATC Biotech, Cologne, Germany).

[0502] Second ELISA and HTRF

[0503] All sequence-unique clones identified for each selection trajectory were further analyzed in a secondary screen using ELISA. Here, the number of antigens was increased to include native human and mouse BSSL as well as biotinylated human BSSL. Streptavidin was used as a non-relevant antigen. Binding of all scFvs was also assessed by HTRF (homogeneous time-resolved fluorescence).

[0504] result

[0505] A total of 14 phage selection tracks were performed in parallel against four versions of BSSL using SciLifeLib 2 and the AS20 humanized library. From each of the 14 tracks, 89 to 222 clones were picked and analyzed. ELISA and HTRF screening combined with sequencing yielded a total of 68 unique scFv clones capable of binding to the orthologs of the BSSL for which they were selected. Unexpectedly, no binding was detected for the AS20 CDR-grafted scFv.

[0506] in conclusion

[0507] A total of 2,365 clones were initially screened by ELISA, yielding a total of 467 scFv fragments with potential binding affinity to both human and mouse BSSL, which were then sent for sequencing. A secondary ELISA screen, followed by HTRF and resequencing, yielded a total of 68 sequence-unique scFv clones. Binding data from these clones revealed that their relative binding to human and mouse BSSL could be divided into three groups, characterized by recognition of one or both of these orthologs.

[0508] 64 isolates of scFv were derived from the AS20 humanized library, whereas only 4 were derived from SciLifeLib2. This suggests that BSSL is a challenging target for phage display selection using naive antibody libraries.

[0509] Example 7-68 ELISA of anti-BSSL scFv and affinity ranking by SPR

[0510] In this example, the 68 unique scFvs generated in Example 6 were analyzed in ELISA and further ranked based on affinity using SPR. Together with earlier binding data (ELISA and HTRF), the results were used as decision points for selecting candidates for further development.

[0511] Materials and methods

[0512] hBSSL and mBSSL (Table 2) were used as BSSL reagents in this example.

[0513] ELISA

[0514] Human and mouse BSSL were coated into 384-ELISA well plates at 1 μg / ml in PBS at 4°C overnight. Two negative control proteins, streptavidin and BSA, were also included. FLAG-tagged scFv clones present in bacterial supernatants were diluted 1:2 and 1:20 in assay buffer (PBS + 0.5% BSA + 0.05% Tween20) and allowed to bind to the coating protein. All samples were assayed in duplicate. Binding was detected by incubation with HRP-conjugated α-FLAG M2 antibody (Sigma-Aldrich #A8592) followed by TMB ELISA substrate (ThermoFisher Scientific #34029). Colorimetric signal generation was terminated by the addition of 1 M sulfuric acid, and the plates were analyzed at 450 nm.

[0515] SPR

[0516] exist Kinetic screening was performed on a T200 biosensor instrument (GE Healthcare).α-FLAG M2 antibody (Sigma-Aldrich #F1804) as capture ligand was immobilized on all four surfaces of a CM5-S amine sensor chip according to the manufacturer's recommendations.

[0517] FLAG-tagged scFv clones in bacterial supernatant were injected and captured onto the chip surface, followed by injection of 50 nM and 200 nM human or mouse BSSL, respectively. The surface was regenerated with 10 mM glycine-HCl, pH 2.2. All experiments were performed at 25°C in running buffer (PBS + 0.1% BSA + 0.05% Tween 20, pH 7.5 for human BSSL and 25 mM Tris-HCl + 150 mM NaCl, pH 7.5 for mouse BSSL).

[0518] result

[0519] ELISA

[0520] For most clones, binding of 68 scFv clones to directly coated human and mouse BSSL could be confirmed. As observed in Example 6, BSSL-binding clones could be divided into three groups with characteristics of recognizing human BSSL, mouse BSSL, or both human and mouse BSSL. Most clones showed preferential binding to human BSSL (data not shown).

[0521] SPR

[0522] Data analysis was performed by visual inspection of the sensorgrams (not shown). When examining these, it was apparent that most clones had considerably higher affinity for human BSSL than for mouse BSSL, with lower K values. DInspection of the sensorgrams also showed that many of the humanized clones displayed affinities in the same range as observed for the AS20 scFv. Examples of such clones are S-SL048-11 (comprising HCVR SEQ ID NO:30 and LCVR SEQ ID NO:31), S-SL048-14 (comprising HCVR SEQ ID NO:50 and LCVR SEQ ID NO:84), S-SL048-106 (comprising HCVR SEQ ID NO:34 and LCVR SEQ ID NO:35), S-SL048-108 (comprising HCVR SEQ ID NO:72, LCVR SEQ ID NO:106), S-SL048-109 (comprising HCVR SEQ ID NO:73 and LCVR SEQ ID NO:107), S-SL048-116 (comprising HCVR SEQ ID NO:36 and LCVR SEQ ID NO:37), and S-SL048-125 (comprising HCVR SEQ ID NO:77, LCVR SEQ ID NO:111). The affinity of these particular clones for mouse BSSL was also similar to that seen for AS20.

[0523] A few clones derived from the phage display selection locus panned against mouse BSSL showed preferential binding to mouse BSSL compared to human BSSL, such as clone S-SL048-66 (comprising HCVR SEQ ID NO: 61 and LCVR SEQ ID NO: 95).

[0524] in conclusion

[0525] In this example, binding of 68 previously identified scFv clones to BSSL was confirmed by ELISA. All 68 clones were also kinetically screened using a single concentration of human and mouse BSSL. As found for AS20, most clones showed a higher estimated binding affinity, defined as the dissociation equilibrium constant, K, for human BSSL than for mouse BSSL. D (M). Estimated affinity for human BSSL is in the low to nanomolar range, with the reference AS20 scFv showing a K of 2 nM. D A small group of scFv clones showed higher affinity for mouse BSSL than for human BSSL, with the highest affinity corresponding to a K of 43 nM D value.

[0526] From all the collected data, 38 clones (comprising HCVR SEQ ID NOs: 30, 32, 34, 36, and 47-79 and LCVR SEQ ID NOs: 31, 33, 35, 37, 38, and 81-113) and a reference AS20 scFv (HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114) were selected for conversion to human IgG4 S228P. All candidate clones were derived from the AS20 humanization library, and none were derived from SciLifeLib.

[0527] Example 8-38 Conversion of humanized BSSL-specific antibodies into hIgG4 S228P format and small-scale transient expression

[0528] In this experiment, the 38 most promising humanized scFv clones (comprising HCVR SEQ ID NOs: 30, 32, 34, 36, and 47-79 and LCVR SEQ ID NOs: 31, 33, 35, 37, 38, and 81-113) from the phage selection and subsequent binding screening in Example 7 were converted to human IgG4 S228P antibody format. In addition, AS20 (parental clone) (comprising HCVR SEQ NO: 80 and LCVR SEQ ID NO: 114) and the AS20 CDR graft (comprising HCVR SEQ NO: 144 and LCVR SEQ ID NO: 145) were similarly converted to IgG4 S228P.

[0529] Briefly, human IgG4 is considered the most Fc-silent natural IgG subclass in humans, meaning that it does not mediate its primary effector functions through the Fc portion of the antibody. Similar to IgG1, IgG4 has a serum half-life of 21 days. However, IgG4 naturally tends to dissociate into half IgG4 molecules in vivo, which can then combine with other circulating IgG4 molecules. This half-molecule exchange can be avoided by introducing a stabilizing mutation in the hinge region, namely S228P (Eu numbering; this is equivalent to Kabat numbering S241P)

[13] .

[0530] The genes encoding the VH and VL of 38 scFv clones, AS20, and the AS20 CDR graft were successfully transferred into a vector encoding the human IgG4 S228P subclass. Antibodies were transiently transfected into ExpiHEK293 cells, expressed on a small scale (4 ml), and purified using Protein A. Purity and integrity / monomer content were analyzed by SDS-PAGE and analytical size exclusion chromatography (SEC).

[0531] Materials and methods

[0532] Sequence analysis

[0533] The amino acid sequences of the HCVR and LCVR of the scFvs selected for IgG conversion are shown in the Sequence Listing and, for clarity, are listed in Table 8 along with an anti-hapten (4-hydroxy-3-nitrophenylacetyl, NP) antibody (anti-NP).

[0534] Table 8 - Antibodies used in this example

[0535]

[0536]

[0537] Figure 14 Sequence differences between 38 humanized clones converted to hIgG4 S228P are shown. In the AS20 humanized library, a total of 20 positions were targeted for diversification in the CDR1 and CDR2 of the heavy chain and the CDR1, CDR2, and CDR3 of the light chain. For comparison, the AS20 and CDR-grafted constructs are included in the figure.

[0538] Seamless cloning

[0539] Plasmid DNA for 38 BSSL-specific scFvs and AS20 was purified from bacterial cultures using standard miniprep procedures. The gene for the AS20 CDR graft was synthesized by Genscript. The VH and VL regions were PCR amplified using the In-Fusion HD Plus Cloning Kit (Clontech #638909) and inserted into the in-house constructed vector pHAT-hIgG4-S241P. A representative example of the generated full-length IgG sequence is the S-SL048-11 hIgG4 S228P heavy chain (VH-CH1-hinge-CH2-CH3) corresponding to SEQ ID NO: 119 and the S-SL048-11 hIgG4 S228P light chain (VL-CL) corresponding to SEQ ID NO: 120.

[0540] Transfection into HEK293, expression and purification

[0541] Plasmid DNA was transfected into expiHEK293 cells using the ExpiFectamine™ 293 transfection kit (ThermoFisher Scientific #A14525) in 4 ml cultures in 24 deep-well plates. After 5 days of cultivation at 37°C, 6% CO₂, 80% rH₄, and 400 rpm, the culture supernatant was mixed with protein A-conjugated magnetic beads and purified on a KingFisher Flex instrument. After elution in 0.1 M glycine pH 2.7, the cells were immediately neutralized by adding 1 M Tris-HCl, pH 8.8, and the buffer was exchanged for PBS using a 96-well rotating desalting plate. SDS-PAGE was performed to determine the purity and integrity of the purified IgG, and the concentration was determined using an Implen NP80 UV-Vis spectrophotometer (Fisher Scientific).

[0542] result

[0543] Seamless cloning

[0544] As confirmed by sequencing, 38 unique scFv, AS20 and AS20 CDR grafts were successfully converted into full-length human IgG4 antibodies.

[0545] Transfection into HEK293, expression and purification

[0546] Antibodies were expressed in expi HEK293 cells and purified from the supernatant by protein A purification. The purity and integrity of the purified IgG were confirmed by SDS-PAGE (data not shown).

[0547] in conclusion

[0548] All BSSL-binding antibodies were successfully recloned into hIgG4 format, expressed in HEK293 cells, and purified by protein A-conjugated magnetic beads on a Kingfisher Flex instrument. All showed acceptable levels of purity as assessed by SDS-PAGE.

[0549] Example 9-38 hIgG4 S228P clones binding to human and mouse BSSL

[0550] This example describes target binding analysis by surface plasmon resonance (SPR) of 38 hIgG4 S228P clones (Example 8, Table 8) to verify that binding to human and mouse BSSL was retained after conversion from scFv to IgG format.

[0551] Materials and methods

[0552] use Kinetic parameters of hIgG4 S228P clone were determined by SPR using a T200 (GE Healthcare). Single cycle kinetics were used to measure the affinity of purified hIgG4 molecules to human and mouse BSSL. Anti-Fab antibodies (GE Healthcare, #28958325) were immobilized on a CM5 S sensor chip via primary amine coupling using NHS-EDC chemistry. The hIgG4 was captured by the anti-Fab antibody and five different concentrations of hBSSL (1:5 dilution starting from 50 nM) or mBSSL (1:5 dilution starting from 500 nM) were then injected onto the surface. The sensor chip surface was regenerated with 10 mM glycine-HCl pH 2.1. BSSL reagents as listed in Table 2 were used. For binding to human BSSL, single cycle kinetic data were fitted to a 1:1 binding model and kinetic parameters were retrieved using the software BIAevaluation. For mouse BSSL, steady state analysis was performed by plotting the response level at equilibrium against each concentration and K was retrieved using the BIAevaluation software. D value.

[0553] result

[0554] The affinity of the converted antibodies for non-biotinylated human BSSL and mouse BSSL was determined using a single-cycle kinetic method. The resulting equilibrium dissociation constant (K D ) are listed in Table 9. For hBSSL, the data were generally successfully fitted to a 1:1 binding model. For mouse BSSL, the data did not always fit a 1:1 binding model well. Instead, steady-state analysis was used to analyze the data. The K values ​​for binding to human and mouse BSSL were determined. D The values ​​were comparable to the K values ​​determined for the same clones in scFv format. D The values ​​were in the same range (see Example 7). For the AS20 CDR-grafted clone, low binding to human BSSL was observed (data not shown). However, the model fit was considered inaccurate, so the K value for this particular clone was not retrieved. D value.

[0555] Table 9 - Measured equilibrium dissociation constants (K D ) In some cases, the quality of the data was considered too low to be reliable and is indicated here as "nd" (not determined).

[0556]

[0557]

[0558] in conclusion

[0559] In summary, the results indicate that the antibody's binding affinity for human and murine BSSL was unaffected by recloning into the hIgG4 format. However, the AS20 CDR graft behaved differently. As shown in Example 6, the AS20 CDR graft did not show any binding to BSSL when expressed in an scFv format. However, in the IgG format, a binding signal to human BSSL was observed.

[0560] Example 10 - Functional testing of 28 hIgG4 S228P clones using flow cytometry displacement assay

[0561] In this example, 28 hIgG4 S228P antibodies (comprising HCVR SEQ ID NOs: 30, 32, 34, 36, 47, 50-56, 59-65, 68, 69, 71-73, 75, 77, and 78 and LCVR SEQ ID NOs: 31, 33, 35, 37, 38, 81, 84-90, 93-99, 102, 103, 105-107, 109, 111, and 112) with the highest binding affinity to human and / or mouse BSSL in Example 9 were tested for their ability to block the binding of human BSSL to CD14 using a flow cytometry-based displacement assay. + Monocyte binding ability. Five antibodies were included as reference: AS20mIgG1 (HC SEQ ID NO: 135 and LC SEQ ID NO: 136), AS20 hIgG4 (HC SEQ ID NO: 129 and LC SEQ ID NO: 130), AS20 CDR graft (HC SEQ ID NO: 131 and LC SEQ ID NO: 132), anti-human α-synuclein mIgG1, and anti-NP hIgG4 (HC SEQ ID NO: 133 and LC SEQ ID NO: 134).

[0562] Materials and methods

[0563] Preparation of buffy coat

[0564] Human blood was drawn from a healthy donor in vacutainers supplemented with citrate anticoagulant (BD Vacutainer).The buffy coat consisting of leukocytes and platelets was separated after centrifugation at 1300 xg for 10 minutes at room temperature in a swinging bucket rotor.

[0565] Flow cytometry displacement assay

[0566] Various concentrations of 28 BSSL-specific hIgG4 antibodies and five reference antibodies (0.5 μg to 3.0 μg per reaction; see Table 10) were added to b-hBSSL (1 μg per reaction, see Table 2) in round-bottom polystyrene tubes. 1× PBS (pH 7.4) was added to a final volume of 20 μl, and the antibody / b-hBSSL mixture was incubated at +4°C for 30 minutes to promote antibody binding to BSSL. Buffy coat (50 μl) was then added to each antibody / b-hBSSL mixture, and incubation was continued at +4°C for an additional 30 minutes. Thereafter, 2 ml of FACS lysis buffer (BD Biosciences) was added, and the cells were incubated at room temperature for 10 minutes to lyse red blood cells and fix white blood cells. The cells were then centrifuged at 200 × g for 5 minutes, and the resulting pellet was washed by adding 2 ml of FACS buffer (1× PBS supplemented with 1% FCS and 0.1% NaN3) and centrifuged again. Finally, the supernatant was discarded and the cells were resuspended in a final drop of approximately 50 μl of liquid.

[0567] 5 μl of BV421-labeled anti-human CD14 (BD Biosciences) and 5 μl of BB515-labeled streptavidin (BD Biosciences) were added to each tube and incubated at +4°C in the dark for 30 minutes. Afterwards, the cells were washed twice with FACS buffer, resuspended in 500 μl FACS buffer, and run on a BD LSRII flow cytometer (BD Biosciences). Finally, the data were analyzed using FlowJo software (BD Biosciences).

[0568] result

[0569] First, CD14 + Cells were gated to depict the monocyte population. Then, b-hBSSL was detected by BB515-labeled streptavidin and gated on CD14 + The binding of b-hBSSL to monocytes was quantified as median fluorescence intensity (MFI) in the BB515 channel. + Monocyte binding capacity was quantified as the reduction in BB515 MFI in monocytes after incubation with increasing concentrations of BSSL-specific hIgG4 or a reference antibody.

[0570] Table 10 - 28 BSSL-specific hIgG4 S228P and reference antibodies block BSSL binding to human CD14 + Monocyte binding capacity. Note that not all antibodies were tested at all concentrations.

[0571]

[0572]

[0573] in conclusion

[0574] The molecular weight of hBSSL (76 kD) is approximately half that of an IgG molecule (150 kD). Thus, in this example, 1 μg of BSSL and 2 μg of IgG correspond roughly to a 1:1 molar ratio. Using the highest antibody concentration (3 μg per reaction), we showed that 9 of 28 BSSL-specific hIgG4 S228P antibodies inhibited (displaced) at least 60% of BSSL (1 μg per reaction) binding to monocytes. The most potent antibody at displacing binding was AS20 mIgG1, while AS20 CDR-graft hIgG4, anti-NP hIgG4, and anti-α-synuclein mIgG1 had no effect on binding at all.

[0575] Example 11 - Generation of 5 Pre-named hIgG4 S228P Antibodies and Controls

[0576] Based on the results obtained from the binding assays performed in Example 9 and the in vitro functional studies in Example 10 and the sequence content, five humanized hIgG4 S228P clones were selected for larger scale production (10 mg), namely S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116 and S-SL048-118.

[0577] Two controls were also included: AS20 and an AS20 CDR graft clone. Furthermore, an isotype control was included. For this purpose, an anti-hapten (4-hydroxy-3-nitrophenylacetyl, NP) antibody (clone B1-8) was ordered from Absolute Antibody. The subclass format selected was hIgG4 S228P, the same as that used previously in Example 8 to evaluate the 38 BSSL-specific clones.

[0578] Materials and methods

[0579] Antibody production

[0580] Material production was outsourced to Absolute Antibody (Oxford, UK). Sequence information for seven cloned VH and VL proteins was sent to the company, where the genes were synthesized and cloned into a vector encoding the human IgG4-S228P subclass. The antibodies were transiently expressed in mammalian HEK293 cells and subsequently purified by affinity chromatography using protein A. Purity and integrity were assessed by SDS-PAGE, and endotoxin levels were determined by the LAL chromogenic endotoxin assay.

[0581] The amino acid sequences of the eight antibodies correspond to the SEQ ID NOs shown in Table 11.

[0582] Table 11 - Antibodies used in this example

[0583]

[0584] Surface plasmon resonance (SPR)

[0585] use Kinetic parameters of the hIgG4 S228P clone were determined by SPR on a T200 (GE Healthcare). Anti-Fab antibodies (GE#28958325) were immobilized on a CM5 S sensor chip using primary amine coupling using NHS-EDC chemistry. The hIgG4 antibody was captured by the anti-Fab antibody and five different concentrations of hBSSL (1:5 dilution, 0.08-50 nM) or mBSSL (1:2 dilution, 50-800 nM) were then injected onto the surface. The sensor chip surface was regenerated with 10 mM glycine-HCl pH 2.1. For binding to human BSSL, single cycle kinetic data were fitted to a 1:1 binding model and kinetic parameters were retrieved using the software BIAevaluation. For mouse BSSL, steady-state analysis was performed by plotting the response level at equilibrium against each concentration and K was retrieved using the BIAevaluation software. D value.

[0586] result

[0587] Antibody production

[0588] Receive 10 mg of eight antibodies at a concentration of 4.6-5 mg / ml in 25 mM histidine, 150 mM NaCl, 0.02% P80 (pH 6.0) from Absolute Antibody. Purity was >98% as determined by SDS-PAGE, and endotoxin levels were <0.05 EU / mg as determined by the LAL chromogenic endotoxin assay. Monomeric content of each clone was >98% as determined by analytical size exclusion chromatography.

[0589] SPR

[0590] SPR was used to determine the affinity of the antibodies to human and mouse BSSL. Table 12 summarizes the different K values ​​of the pre-named clones for hBSSL and mBSSL binding. D For comparison, K values ​​determined for the same clone produced in-house were also included. D values ​​(these experiments are described in Example 9).

[0591] Table 12 - Summary of SPR analysis results for hIgG4 S228P versions of antibodies. *These values ​​were obtained from the experiments reported in Example 9. Numbers marked with # are considered unreliable and should be viewed with caution. In some cases, the quality of the data was considered too low to be reliable and is indicated here as "nd" (not determined).

[0592]

[0593] in conclusion

[0594] Five pre-named candidates and three controls produced by Absolute Antibody were analyzed for BSSL binding in ELISA (data not shown) and SPR. The results showed that binding to BSSL was generally very similar to that observed for individual clones of the same IgG format produced in-house (see Example 9). The results also showed that the isotype control anti-NP hIgG4 S228P did not bind to BSSL and should therefore be suitable for use as a negative control in future analyses. The functionality of these antibody batches was also evaluated in a displacement assay (Example 10). As reported in Example 10, very similar results were obtained for different clones (data not shown).

[0595] In this example, we were able to measure the affinity of CDR grafts for the first time. Here, the AS20 CDR graft hIgG4 S228P (comprising HC SEQ ID NO: 131 and LC SEQ ID NO: 132) showed significant binding to hBSSL at higher concentrations, although binding was greatly reduced compared to AS20 hsIgG S228P (comprising HC SEQ ID NO: 129 and LC SEQ ID NO: 130). The affinity for hBSSL measured here was approximately 100-fold lower, while the affinity for mBSSL was too low to be determined. Thus, when CDRs are grafted onto a new framework, affinity for the target is significantly reduced. The observed stronger BSSL binding of the AS20 CDR graft hIgG4 S228P compared to the scFv AS20 CDR graft (comprising HCVR SEQ ID NO: 144 and LCVR SEQ ID NO: 145, see Example 6) is likely due to the different antibody formats.

[0596] Example 12 - Stability study of five candidate antibodies

[0597] This example describes a stability study of hIgG4 S228P forms S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116, and S-SL048-118 to investigate the biophysical stability of the antibodies. For comparison, AS20 (see Example 17) was included as hIgG4 S228P and hIgG1 LALA-PG. AS20 CDR grafts hIgG4 S228P and anti-NP hIgG1 LALA-PG isotype controls were also included. Analysis was performed by SDS-PAGE, analytical SEC, nano-DSF, and DLS.

[0598] Materials and methods

[0599] The nine different antibodies included in the stability study and their corresponding SEQ ID NOs are listed in Table 13. Antibodies were aliquoted into vials and incubated at -80°C, +4°C, and +40°C at 5 mg / ml in 25 mM histidine, 150 mM NaCl, 0.02% P80, pH 6.0. Samples were drawn and analyzed according to the schedule in Table 14, with Day 0 being the starting day.

[0600] Table 13 - Antibodies used in this example

[0601]

[0602]

[0603] Table 14 - Sample Analysis Schedule

[0604]

[0605] A BioSEC column (300A, 7.8×300 mm; πP.N. 5190-2511, Agilent) connected to an Agilent 1100 system was used and 0.15 M sodium phosphate (Na x H y Analytical size exclusion chromatography (SEC) was performed with a running buffer of pH 6.8 at a flow rate of 1 ml / min. 280 and A 220Protein was detected by absorbance detection at 400 nm. 20 μg of each sample was loaded onto the column. NuPAGE 4-12% Bis-Tris gel (InVitrogen NP0321BOX) was used, and SDS-PAGE was performed according to the manufacturer's instructions using NuPAGE MES SDS running buffer (InVItrogen NP000202). Samples were run under reducing or non-reducing conditions. Simply Blue staining (Invitrogen LC6065) was used to visualize the bands, and optical density scanner (Oddyssey, Li-cor) was used to quantify the bands. 5 μg of each sample was loaded into each well.

[0606] Nano-DSF (differential scanning fluorimetry) was performed using a Prometheus instrument (NanoTemper) with a temperature gradient of 20-95°C at a heating rate of 1 degree / minute. Fluorescence emission at 300 nm and 350 nm and backscattered signals were recorded. 10 μL of 5 mg / ml sample was loaded per sample. Data were analyzed using PR.Stabilityanalysis v1.02 from Nanotemper.

[0607] Dynamic light scattering was performed using a Zetasizer Pro (Malvern). Scattered light was recorded and analyzed using ZS Explorer software v1.0.0.436 and built-in algorithms. Samples were analyzed at 5 mg / ml.

[0608] result

[0609] Size Exclusion Chromatography (SEC)

[0610] Chromatograms for each sample were obtained according to the protocol presented in Table 14. Note that additional peaks appeared over time with lower molecular weight, indicating degradation of the material. Plotting the total integrated area over time indicated no loss of material on the prefilter or column.

[0611] The size exclusion chromatography data are shown in Figure 7. Looking at the percentage of the total area made up of the main peak, only a small change can be observed at +4°C. A more pronounced effect can be seen at +40°C.

[0612] The greatest reduction was observed for S-SL048-46 hIgG4 S228P and anti-NP hIgG1 LALA-PG. S-SL048-118 hIgG4 S228P, AS20 of hIgG4 S228P, and AS20 hIgG1 LALA-PG showed moderate reduction, while the CDR graft showed little reduction.

[0613] SDS-PAGE

[0614] As shown in Table 14, samples were analyzed under reducing and non-reducing conditions. The -80°C sample showed no additional bands except for S-SL048-46 hIgG4S228P and anti-NP hIgG1 LALA-PG, which showed a weak band under reducing conditions, each constituting less than 1%. This is almost identical to what was observed for the day 0 sample. In fact, the day 0 sample showed an additional band not seen at -80°C analyzed on day 30, reflecting the variability of the assay. The non-reducing samples were almost identical to the day 0 sample. At +4°C, no additional significant bands were observed on days 9 or 30 under reducing or non-reducing conditions. At +40°C, all samples showed additional bands on days 9 and 30. Under reducing conditions, all candidates showed lower molecular weight bands, i.e., below 25 kDa, while AS20 hIgG4 S228P, AS20 hIgG1 LALA-PG, and anti-NP hIgG1 LALA-PG all showed higher Mw bands (i.e., greater than 50 kDa at days 9 and 30). In both cases, these bands became more pronounced over time. Under non-reducing conditions, a clear difference was seen, with additional bands appearing in all samples. The results suggest that candidates S-SL048-46 and S-SL048-106 may be more susceptible to degradation.

[0615] Nano DSF

[0616] The samples were analyzed according to the schedule in Table 14 and the results are presented in Figure 8 The main observations that can be made from the nanoDSF data are the changes that occur in AS20 hIgG4 S228P, AS20 hIgG1 LALA-PG format and anti-NP hIgG1 LALA-PG, where the main melting temperature T m The shift is visible when stored at +40°C.

[0617] For the candidate, the main observation was that the second T m The magnitude of the decrease (data not shown) may reflect a major contribution from the Fab portion of the antibody. Candidates S-SL048-46 and S-SL048-106 showed slightly larger changes, while S-SL048-11, S-SL048-116, and S-SL048-118 showed similar but slightly smaller effects. For all candidates, these differences were considered minor. The CDR grafts appeared to be the most stable of the samples tested.

[0618] Dynamic Light Scattering (DLS)

[0619] The samples were analyzed by DLS on day 30. The results for each sample at three different temperatures are shown in Figure 9At both -80 and +4°C, all samples showed a peak with an apparent diameter of approximately 10-11 nm, consistent with the expected result for monomeric antibodies. No larger particles were observed in any sample, indicating that only protein molecules were detected.

[0620] At +40°C, larger particles were clearly visible to varying degrees. Using the Z-average and polydispersity numbers as indicators of size uniformity (data not shown), S-SL048-106 showed the lowest amount of higher Mw particles. In contrast, AS20hIgG1 LALA-PG and anti-NP hIgG1 LALA-PG showed the highest amount of higher Mw particles, indicating that these two molecules are more prone to aggregation.

[0621] in conclusion

[0622] In summary, combining the data with the overall ranking, the most stable candidates for the hIgG4 S228P form appear to be: S-SL048-106, S-SL048-118, and S-SL048-116, in that order.

[0623] Example 13 - Epitope mapping of AS20 by HDX-MS

[0624] In this example, chimeric AS20 IgG4 antibody was subjected to hydrogen deuterium exchange mass spectrometry (HDX-MS) along with human BSSL to map its epitope.

[0625] Materials and methods

[0626] 40 μL of a 2 mg / mL solution of human BSSL (SEQ ID NO: 138) in PBS was mixed with 92.5 μL of a 1.7 mg / mL chimeric AS20 hIgG4 antibody (containing HC SEQ ID NO: 139 and LC SEQ ID NO: 140) at a 1:1 molar ratio. The BSSL / antibody complex was concentrated to 40 μL using a 10K centrifugal filter device (Amicon Ultra, Merck). Simultaneously, samples containing only BSSL without added antibody were similarly prepared. Samples were analyzed in an automated HDX-MS system (CTC PAL / Biomotif HDX), where samples were automatically labeled, quenched, digested, cleaned, and separated at 2°C. More specifically, samples were labeled by mixing 3 μL of BSSL (or BSSL / antibody complex) with 22 μL of deuterated PBS and incubated at 4°C for four labeling time points: 5 minutes, 30 minutes, 90 minutes, and 180 minutes. The labeling reaction was stopped / quenched by adding 20 μL of a solution containing 6 M urea, 100 mM TCEP, and 0.5% TFA to lower the pH to approximately 2.3 and the temperature to approximately 4°C. The samples were digested using an immobilized pepsin column (2.1 column (2.1×30 mm) at 250 μl / min, followed by an online desalting step using a 2 mm ID×10 mm length C-18 precolumn (ACE HPLC Columns, Aberdeen, UK) with 0.05% TFA at 350 μl / min for 3 minutes. Pepsin peptides were then separated using a 2 mm ID×50 mm length HALO C18 / 1.8 μm analytical column running at 95 μl / min with an 18 min linear gradient of 8-55% ACN in 0.1% formic acid. Analysis was performed using an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific) operating at m / z 400 with a resolution of 70,000. Mascot software was used for peptide identification, and HDExaminer (Sierra Analytics, USA) was used to process all HDX-MS data. Statistical analysis was performed using 95% confidence intervals.

[0627] result

[0628] The deuteration kinetics of 66 peptides were followed by HDX-MS, covering 57.9% of the protein. Deuterium labeling (5, 30, 90, and 180 minutes) and differential deuteration uptake kinetics between BSSL alone and in the presence of AS20 were calculated. Several peptides near the N-terminus showed statistically lower deuterium uptake in the presence of the AS20 antibody, clearly mapping the epitope to this region. By taking into account overlapping peptides, the spatial resolution could be reduced, resulting in the three peptide regions shown in Table 15.

[0629] Table 15 - List of AS20 epitope regions proposed by HDX-MS experiments

[0630]

[0631] in conclusion

[0632] In summary, the AS20 epitope was successfully mapped to the N-terminal region of BSSL. Three discrete clusters of peptide regions with significantly lower deuterium uptake in the presence of AS20 were identified. Mapping these regions together in the three-dimensional structure of BSSL revealed that these peptides together constitute a conformational epitope for AS20. In Example 21, we describe the crystal structure of the AS20 Fab fragment in complex with hBSSL. Analysis of the three-dimensional structure confirmed the results from the HDX-MS data and allowed the specific definition of the amino acids involved in the interaction.

[0633] Example 14 - Epitope Mapping of Pre-named Antibody Candidates by HDX-MS

[0634] In this example, hydrogen deuterium exchange mass spectrometry (HDX-MS) was performed to characterize the five pre-named candidate antibodies S-SL048-11 hIgG4 S228P (mAb11, HC SEQ ID NO: 119, LC SEQ ID NO: 120), S-SL048-46 hIgG4 S228P (mAb46, HC SEQ ID NO: 121, LC SEQ ID NO: 122), S-SL048-106 hIgG4 S228P (mAb106, HC SEQ ID NO: 123, LC SEQ ID NO: 124), S-SL048-116 hIgG4 S228P (mAb116, HC SEQ ID NO: 125, LC SEQ ID NO: 126), and S-SL048-118 hIgG4 S228P (mAb11, HC SEQ ID NO: 127, LC SEQ ID NO: 128). NO:128) was mapped.

[0635] Materials and methods

[0636] A 2 mg / mL solution of BSSL in PBS was mixed with the various antibodies (5 mg / mL in 25 mM histidine, 150 mM NaCl, 0.02% P80, pH 6.0) at a 1:1 molar ratio. The sample was concentrated using a 10K centrifugal filter device (Amicon Ultra, Merck) and the buffer was exchanged into PBS. Simultaneously, the same procedure was performed on samples containing only BSSL without the addition of antibodies.

[0637] Samples were analyzed in an automated HDX-MS system (CTC PAL / Biomotif HDX), where samples were automatically labeled, quenched, digested, cleaned, and separated at 2°C. More specifically, samples were labeled by mixing 3 μL of BSSL (or BSSL / antibody complex) with 22 μL of deuterated PBS and incubated at 4°C for four labeling time points: 5 minutes, 30 minutes, 90 minutes, and 180 minutes. The labeling reaction was stopped / quenched by adding 20 μL of a solution containing 2 M urea, 100 mM TCEP, and 0.5% TFA to lower the pH to approximately 2.3 and the temperature to approximately 4°C. The samples were digested using an immobilized pepsin column (2.1 column (2.1×30 mm) at 250 μl / min, followed by an online desalting step using a 2 mm ID×10 mm length C-18 precolumn (ACE HPLC Columns, Aberdeen, UK) with 0.05% TFA at 350 μl / min for 3 minutes. Pepsin peptides were then separated using a 2 mm ID×50 mm length HALO C18 / 1.8 μm analytical column running at 95 μl / min with a 15 min 8-60% linear gradient of ACN in 0.1% formic acid. Analysis was performed using an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific) operating at m / z 400 with a resolution of 70,000. Mascot software was used for peptide identification, and HDExaminer (Sierra Analytics, USA) was used to process all HDX-MS data. Statistical analysis was performed using 95% confidence intervals.

[0638] result

[0639] Deuteration kinetics of 54 peptides were tracked by HDX-MS covering 64% of the protein. Deuterium labeling (5, 30, 90, and 180 minutes) and differential deuteration uptake kinetics between BSSL alone and in the presence of any of the five antibodies were calculated. It was observed that several peptides near the N-terminus of BSSL showed statistically lower deuterium uptake in the presence of each of the pre-named candidate antibodies. By taking into account overlapping peptides, the spatial resolution could be reduced, resulting in the two peptide regions shown in Table 16.

[0640] Table 16 - List of common epitope regions proposed by HDX-MS experiments

[0641]

[0642] In addition to the epitope region common to all pre-named antibodies, one additional peptide was identified that had statistically lower deuterium uptake among the three antibodies, namely peptide 10 (aa 84-101; NIWVPQGRKQVSRDLPVM (SEQ ID NO: 4)) of S-SL048-46, peptide 24 (aa 174-180; VKRNIAA (SEQ ID NO: 5)) of S-SL048-116, and peptide 39 (aa 283-295; HYVGFVPVIDGDF (SEQ ID NO: 6)) of S-SL048-11 (Table 17). However, these signals were relatively weak.

[0643] It can be noted that although spread out in the primary sequence, the two epitope regions (aa 1-12 and aa 42-55) common to all five antibodies are clustered together in the three-dimensional structure ( Figure 10 Peptide 24 (aa 174-180) is relatively close to this core region, while peptides 10 (aa 84-101) and 39 (aa 283-295) are more dispersed.

[0644] Table 17 - Summary of peptides with differential deuteration uptake kinetics between BSSL alone and in the presence of any of the five antibodies

[0645]

[0646]

[0647] in conclusion

[0648] The epitopes of five pre-named candidate antibodies were successfully mapped to the N-terminal region of BSSL. Two discrete peptide regions were identified with significantly lower deuterium uptake in the presence of all five antibodies.

[0649] Overlapping peptides can allow for reduced spatial resolution. For example, in the AS20 experiment (Example 13), it was found that the peptide corresponding to aa 1-6 was unchanged by deuterium exchange, while a longer overlapping peptide was altered (AKLGAVYTEGGF, aa 1-12, SEQ ID NO: 3). In other words, the epitope residues could be reduced to YTEGGF (aa 7-12) (SEQ ID NO: 1). In contrast, in this example, no peptide corresponding to aa 1-6 was detected, so this reduction could not be performed.

[0650] In addition to the two common epitope regions, in the three pre-named candidates (S-SL048-46, S-SL048-116 and S-SL048-11), an additional peptide was each identified as a potential epitope region. Although statistically significant, the amplitude of the signal changes of these peptides is relatively low and may not be important for the interaction. Moreover, the fact that the peptide VKRNIAA (aa 174-180, SEQ ID NO: 5) is concentrated in the core region of three-dimensional space indicates that this region is also part of the epitope. It is worth noting that this peptide also overlaps with a peptide detected in the original mapping of AS20. The signal changes of the other two peripheral peptides 10 (aa 84-101, SEQ ID NO: 4) and peptide 39 (aa 283-295, SEQ ID NO: 6) can be explained by stabilization ("allosteric" effect) after antibody binding.

[0651] Taken together, these data strongly suggest a shared common core consisting of two peptide regions somewhere in the sequences 1-12 (SEQ ID NO: 3) and 42-55 (SEQ ID NO: 2) of all previously named candidates. These regions were also identified in previous epitope mapping experiments of AS20 as described in Example 13 and are thought to be important for interactions in the crystal structure of the AS20 complex described in Example 21.

[0652] Example 15 - Immunogenicity Assessment

[0653] This example describes the immunogenicity evaluation of candidates SL048-11 (HC SEQ ID NO: 119 and LC SEQ ID NO: 120), S-SL048-46 (HC SEQ ID NO: 121 and LC SEQ ID NO: 122), S-SL048-106 (HC SEQ ID NO: 123 and HC SEQ ID NO: 124), S-SL048-116 (HC SEQ ID NO: 125 and LC SEQ ID NO: 126), and S-SL048-118 (HC SEQ ID NO: 127 and HC SEQ ID NO: 128) and AS20 hIgG4 S228P (HC SEQ ID NO: 129 and LC SEQ ID NO: 130) performed by Abzena.

[0654] Materials and methods

[0655] Using Abzena's iTope TM MHC class II computer prediction algorithm, analyzing the immunogenic potential of sequences provided by Lipum. iTope TMThe software predicts favorable interactions between the amino acid side chains of the peptide and specific binding pockets (pocket positions; p1, p4, p6, p7 and p9) within the open binding grooves of 34 human MHC class II alleles. These alleles represent the most common HLA-DR alleles found worldwide, with no weight attributed to those most commonly found in any ethnic group. 20 alleles contain an "open" p1 configuration, and 14 contain a "closed" configuration in which the glycine at position 83 is replaced by valine. The positioning of key binding residues is achieved by computer-generated 9-mer peptides that overlap with 8 amino acids spanning the test protein sequence.

[0656] The results should be evaluated in light of the fact that all prediction methods for MHC class II binding inherently over-predict the number of T-cell epitopes because they do not allow for other important processes during antigen presentation, such as protein / peptide processing, T-cell receptor recognition, or T-cell tolerance to the peptide.

[0657] The position of the pi anchor residue (comprising the first residue of the MHC class II core 9-mer ligand) is highlighted in hIgG4 S228P forms S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116, S-SL048-118, and AS20.

[0658] If ≥50% of the MHC class II binding peptides (ie, ≥17 out of 34 alleles) had high binding affinity (score >0.6), such peptides were defined as "promiscuous high affinity" MHC class II binding peptides.

[0659] MHC class II binding peptides that bound ≥50% of alleles with a score >0.55 (but mostly not >0.6) were defined as "promiscuous intermediate affinity."

[0660] These criteria changed in the case of the presence of large aromatic amino acids (i.e., F, W, Y) at the P1 anchor position, where the open P1 pocket allowed binding of large aromatic residues in 20 of the 34 alleles. When this occurred, promiscuous peptides were defined as binding to 10 or more of the 20 allele subsets.

[0661] The pi anchor positions of germline intermediate and high affinity binding peptides were also analyzed, and these are not expected to cause problems due to T cell tolerance in healthy individuals.

[0662] TCED for known positive peptides TM Database for positive iTope TMThe hits were BLAST searched and regions representing closely homologous peptides from the T cell epitope database (i.e., known to be expressed in ex vivo EpiScreen) were indicated. TM Peptides that induce T cell activation in T cell epitope mapping assays). Kabat numbering is used to label antibody sequences.

[0663] result

[0664] Previously in vitro EpiScreen TM TCED of peptides tested in T cell assays TM Analysis revealed that many iTope TM Strong homology to MHC binding peptides. In Table 18, + indicates mismatch residues, where the replacement is an amino acid with similar physiochemical properties, and - indicates other mismatch residues. TM and TCED TM The locations of the key MHC class II pocket positions of the sequences are shown in the bottom row of Table 18.

[0665] Table 18 - TCED TM Results of the analysis

[0666]

[0667] Table 19 shows the iTope of each candidate sequence TM Promiscuous intermediate- and high-affinity MHC class II binding peptides and TCED TM Total number of hits (AS20 shown for reference only).

[0668] Table 19 - Summary results of immunogenicity analysis

[0669]

[0670] All five candidate humanization clones contained fewer non-germline promiscuous MHC class II peptides when compared to AS20 hIgG4 S228P. All variant sequences contained at least two TCEDs. TM Hits, and Known in EpiScreen TM The peptides that induced T activation in the ex vivo assay had partial homology (minimum 6 of 9 positions). Of the 5 variants, S-SL048-106 hIgG4 S228P represented the lowest immunogenicity risk based on the number of non-germline promiscuous high and medium affinity MHC class II binding peptides. Candidates were ranked as follows: fewer promiscuous hits 106 < 46 < 11 = 118 < 116 more promiscuous hits.

[0671] It is important to note that in silico immunogenicity analysis is inherently over-predictive.

[0672] Example 16 - Manufacturability Assessment

[0673] This example describes the in silico manufacturability assessments performed by Abzena of the hIgG4 S228P format candidates S-SL048-11 (HC SEQ ID NO: 119 and LC SEQ ID NO: 120), S-SL048-46 (HC SEQ ID NO: 121 and LC SEQ ID NO: 122), S-SL048-106 (HC SEQ ID NO: 123 and LC SEQ ID NO: 124), S-SL048-116 (HC SEQ ID NO: 125 and LC SEQ ID NO: 126), and S-SL048-118 (HC SEQ ID NO: 127 and LC SEQ ID NO: 128).

[0674] Materials and methods

[0675] The amino acid sequences of hIgG4 S228P forms S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-, and 116S-SL048-118 were analyzed using Abzena's computational responsibility prediction algorithm. The determined responsibility was then analyzed in the context of the structure. Briefly, for each V domain sequence, the following items were analyzed:

[0676] The presence of a deamidation site;

[0677] The presence of isomerization sites;

[0678] Potential oxidation sites;

[0679] Presence of N-linked glycosylation sites

[0680] Presence of free cysteine.

[0681] Throughout this document, IMGT CDR definitions and numbering are used unless otherwise stated.

[0682] Deamidation site

[0683] Deamidation of asparagine residues can lead to changes in structure, pharmacokinetics and efficacy, and potential immunogenicity. Asparagine residues were analyzed as potential deamidation sites in the context of amino and carboxyl adjacent amino acids using a method based on

[14] .

[0684] Isomerization sites exist

[0685] Aspartate isomerization sites were predicted by analyzing the sequences of known isomerization motifs (DG, DS, DT, or DD), with an emphasis on the CDR regions.

[0686] Potential oxidation sites

[0687] Structural models of the heavy and light chain variable regions were generated, and methionine and tryptophan residues were identified and evaluated to determine if they were likely surface-exposed and therefore candidates for oxidation. Oxidation of single residues may affect the biological activity of the antibody and may have biological consequences such as reduced potency or altered pharmacokinetics.

[0688] Presence of N-linked glycosylation sites

[0689] The VH and Vκ sequences were analyzed based on the consensus N-linked glycosylation motif -NXS / T-, where X can be any amino acid except proline.

[0690] Presence of free cysteine

[0691] The sequences were analyzed to identify unpaired cysteine ​​residues. The VH and Vκ domains typically each contain two typical cysteines that form intrachain disulfide bonds in the folded molecule. Additional cysteines are expected to be detrimental to folding and may cause problems such as aggregation.

[0692] result

[0693] The results of the present invention were as follows: 1. The present invention relates to a novel antibody candidate comprising a VH or Vκ sequence, wherein the VH or Vκ sequence of the five candidate antibodies contains no potential N-linked glycosylation sites; 2. The present invention relates to a novel antibody candidate comprising a VH or Vκ sequence, wherein the VH or Vκ sequence of the five candidate antibodies contains no potential N-linked glycosylation sites; 3. The present invention relates to a novel antibody candidate comprising a VH or Vκ sequence, wherein the VH or Vκ sequence of the five candidate antibodies contains no potential N-linked glycosylation sites; 4. The present invention relates to a novel antibody candidate comprising a VH or Vκ sequence, wherein the VH or Vκ sequence of the five candidate antibodies contains no potential N-linked glycosylation sites; 5. The present invention relates to a novel antibody candidate comprising a VH or Vκ sequence, wherein the VH or Vκ sequence of the five candidate antibodies contains no potential N-linked glycosylation sites; 6. The present invention relates to a novel antibody candidate comprising a VH or Vκ sequence, wherein the VH or Vκ sequence of the five candidate antibodies contains no potential N-linked glycosylation sites; 7. The present invention relates to a novel antibody candidate comprising a VH or Vκ sequence, wherein the VH or Vκ sequence of the five candidate antibodies contains no potential N-linked glycosylation sites; 8. The present invention relates to a novel antibody candidate comprising a VH or Vκ sequence, wherein the VH or Vκ sequence of the five candidate antibodies contains no potential N-linked glycosylation sites; 9. The present invention relates to a novel antibody candidate comprising a VH or Vκ sequence, wherein the VH or Vκ sequence of the five candidate antibodies contains no potential N-linked glycosylation sites; 10. The present invention relates to a novel antibody candidate comprising a VH or Vκ sequence, wherein the VH or Vκ sequence of the five candidate antibodies contains no potential N-linked glycosylation sites; 11. The present invention relates to a novel antibody candidate comprising a VH or Vκ sequence, wherein the VH or Vκ sequence 1 / 2 <10 days) or moderate (t 1 / 2 <25 days) sites with potential for deamidation.

[0694] Two potential isomerization sites were identified in the CDRs or regions close to the CDRs in the VH of all five clones.

[0695] Asp54 (DG) is located within VH CDR2, so isomerization can affect antigen binding;

[0696] • Asp72 is located adjacent to a CDR in a region sometimes referred to as "CDR4," so isomerization may affect antigen binding.

[0697] Table 20 - Summary of Sequence Responsibilities

[0698]

[0699] Representation of S-SL048-106 Fv with potential post-translational liabilities highlighted is shown in Figure 11 middle.

[0700] In summary, no free cysteines, oxidation sites, or N-linked glycosylation sites were identified in any of the five lead candidates. No high potential deamidation sites were identified in S-SL048-11, S-SL048-116, or S-SL048-118. S-SL048-46 and S-SL048-106 contained a potential deamidation site in the VH CDR2. Two potential isomerization sites were identified in the heavy chain of each of the five lead variants.

[0701] Example 17 - Generation and Binding Characterization of AS20 hIgG1 LALA-PG and Anti-NP hIgG1 LALA-PG

[0702] In this example, the generation and binding characterization of anti-NP (clone B1-8) antibodies of the AS20 and human IgG1-LALA-PG subclasses (hereinafter referred to as AS20 hIgG1 LALA-PG (comprising HC SEQ ID NO: 115 and LC SEQ ID NO: 116) and anti-NP hIgG1 LALA-PG (comprising HC SEQ ID NO: 117 and LC SEQ ID NO: 118)) are described. An anti-NP antibody was included as an isotype control.

[0703] Materials and methods

[0704] Expression and purification

[0705] IgG4 is the most inert natural human subclass of Fc. However, several publications have shown that IgG4 can interact with FcRs and complement in mice

[15] and humans. Therefore, human IgG1 with mutations at three positions, L234A, L235A, and P329G (abbreviated as hIgG1 LALA-PG), was selected for this study because it is reported to be the most silent Fc variant [7, 16], i.e., without immune effector function.

[0706] Material production was outsourced to Absolute Antibody (Oxford, UK). The VH and VL sequence information for AS20 was sent to the company, where the genes were synthesized and cloned into a vector encoding the human IgG1-LALA-PG subclass. The antibodies were transiently expressed in mammalian HEK293 cells and subsequently purified by affinity chromatography using protein A. Purity and integrity were assessed by SDS-PAGE, and endotoxin levels were determined using the LAL chromogenic endotoxin assay.

[0707] Surface plasmon resonance (SPR)

[0708] Single cycle kinetics was used to measure the affinity of the antibodies. AS20hIgG1 LALA-PG and anti-NP hIgG1 LALA-PG were immobilized on a CM5 S sensor chip by primary amine coupling using NHS-EDC chemistry. Five different concentrations of antigen were injected on the surface (for hBSSL, 1:3 dilution starting from 50nM, for mBSSL, 1:2 dilution starting from 800nM). The sensor chip surface was regenerated with 10mM glycine-HCl pH2.1. The obtained single cycle kinetic data were fitted to a Langmuir 1:1 binding model, and the kinetic parameters were retrieved using the software BIAevaluation. For mBSSL, steady-state analysis was also performed by plotting the response level at equilibrium against each concentration, and K was retrieved by the BIAevaluation software. D value.

[0709] result

[0710] AS20 hIgG1 LALA-PG and anti-NP hIgG1 LALA-PG were successfully produced (data not shown).

[0711] The affinity of IgG was determined using a single-cycle kinetic SPR method. For the binding of AS20 hIgG1 LALA-PG to hBSSL, an equilibrium dissociation constant (K) of 0.91 nM was calculated. D For mBSSL, the K D As expected, no binding to BSSL was observed for anti-NP hIgG1 LALA-PG.

[0712] in conclusion

[0713] This example describes the quality checks performed to verify that AS20 hIgG1 LALA-PG is functional, ie, binds to BSSL comparable to the hIgG4 S228P form of AS20.

[0714] SPR analysis of AS20 hIgG1 LALA-PG showed that the binding to BSSL was very similar to that observed for other IgG forms of AS20. D The K values ​​of AS20 were 0.91 nM, which is consistent with the K values ​​of 0.6 nM-3 nM determined for other IgG forms of AS20. D The affinity of AS20 hIgG1 LALA-PG for mBSSL was estimated to be K in the steady-state affinity analysis. DThe K value was 361 nM, which is consistent with the K estimated previously by the same analysis. D (155 nM, Example 1) are of the same order of magnitude, and the appearance of the sensorgrams is similar (not shown).

[0715] Thus, the data show that binding of AS20 hIgG1 LALA-PG is not affected by subclass changes. The results also show that the isotype control anti-NP hIgG1 LALA-PG does not bind to BSSL and may therefore be suitable for use as a negative control in future analyses.

[0716] Example 18 - Verification of the efficacy of AS20 hIgG1 LALA-PG in a rheumatoid arthritis mouse model

[0717] In this example, the effects of AS20 hIgG1 LALA-PG (heavy chain SEQ ID NO: 115 and light chain SEQ ID NO: 116) were investigated in an in vivo mouse model of rheumatoid arthritis (RA), collagen antibody-induced arthritis (CAIA). Anti-NP hIgG1 LALA-PG antibody (heavy chain SEQ ID NO: 117 and light chain SEQ ID NO: 118) was included in the study as an isotype control.

[0718] Model Description - CAIA in Mice

[0719] Collagen antibody induced arthritis (CAIA) in mice is a model of arthritis that is independent of B and T cells. The disease is induced with antibodies against type II collagen (CII) administered intravenously (iv), followed by intraperitoneal (ip) administration of LPS 3-5 days later to promote disease development. The injected antibodies bind to the cartilage, thereby activating the immune system and recruiting macrophages and granulocytes to the joints. Booster injections of LPS are required to achieve significant severity and morbidity of disease development. The disease course is highly predictable and onset occurs after the LPS boost. The disease reaches maximum severity around day 15, after which the severity decreases until it is finally cured. The study was conducted in Malmö / Lund, Sweden. Approval was obtained from the local Animal Ethics Committee (M118-15).

[0720] Materials and methods

[0721] Disease induction

[0722] DBA / 1 mice (male, 8-9 weeks) were injected intravenously with 2 mg / mouse of a monoclonal anti-CII antibody cocktail (CIA-MAB-50, MD Bioproducts) on day 0. On day 5, mice were injected intraperitoneally with LPS (50 μg / mouse) to aggravate the disease.

[0723] Experimental groups and administration of test items

[0724] AS20 hIgG1 LALA-PG and isotype control antibodies were delivered at a concentration of 5 mg / ml and further diluted in vehicle (25 mM histidine, 150 mM NaCl, 0.02% P80, pH 6.0). The test item (antibody) was administered intraperitoneally every 4 days starting one day before disease induction (day -1), and then on days 3, 7, 11 and 15. Based on the average body weight of the animals on day -2, AS20 hIgG1 LALA-PG was administered at three different doses, namely 10, 30 and 90 mg / kg, and the isotype control (anti-NPhIgG1 LALA-PG) was administered at 90 mg / kg. The relatively high dose was selected to compensate for the low affinity of AS20 hIgG1 LALA-PG for mouse BSSL compared to human BSSL, as described in Example 17. The experimental groups are summarized in Table 21. The first administration on day -1 was a bolus dose, i.e., all test items were given as a double dose divided into two injections, the first injection was given in the morning and the second injection was given in the afternoon. The following administrations (days 3, 7, 11 and 15) were given as single doses. Animals were weighed before each administration, and the dose volume of 20 ml / kg was based on the individual weight of the animal.

[0725] Table 21 - Experimental Groups

[0726]

[0727] *Two satellite animals per group.

[0728] Disease evaluation

[0729] Disease was assessed daily in a blinded manner starting on day 3 using a macroscopic limb scoring system ranging from 0 to 15 (1 point for each swollen or red toe, 1 point for a swollen or red middle toe or knuckle, and 5 points for a swollen ankle), resulting in a maximum total score for each mouse of 60. Animals with a score of more than 45 were removed from the experiment due to ethical restrictions.

[0730] Blood sampling

[0731] The study was terminated on day 19, and blood from all mice was collected into microtubes containing LiHeparin by cardiac puncture under deep anesthesia (96 hours after the last injection on day 15). The samples were immediately placed on ice and centrifuged within 20 minutes after sampling (2000 × g, 5 minutes, 4°C). Plasma samples were aliquoted and frozen on dry ice. The aliquots were stored at -80°C until analysis of AS20 IgG1 LALA-PG exposure and anti-drug antibodies (ADA) (all groups, n=70) and a panel of safety biomarkers (groups 1-3, n=42).

[0732] PK sampling of satellite animals

[0733] To obtain information about PK profiles and ADA, two satellite animals were included in each dosing group of antibody-treated animals. Blood samples were taken from the sublingual vein 1 hour before administration and 24 hours after administration (i.e., on day 8) on day 7 (two animals per dosing group). Blood samples were also collected 1 hour before administration and 24 hours after administration (i.e., on day 16) on day 15 (two animals per dosing group).

[0734] Tissue collection

[0735] At study termination, spleens from all animals were dissected out and weighed.Fourteen spleens from 14 animals in the isotype control treatment group (Group 2) and from 90 mg / kg AS20 hIgG1 LALA-PG group (Group 3) were homogenized and analyzed by FACS as described in Example 19.

[0736] Drug exposure in plasma

[0737] Exposure to AS20 IgG1 LALA-PG was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) in plasma samples collected from satellite animals (see above) and from all animals at study termination (Day 19).

[0738] Anti-drug antibodies (ADA)

[0739] Immunogenicity assessment was performed by analyzing ADA in plasma samples collected from satellite animals (see above) and from all animals at study termination (Day 19).

[0740] ADA was measured using an enzyme-linked immunosorbent assay (ELISA). Briefly, maxisorp plates were coated with AS20hIgG1 LALA-20 or an isotype control antibody and blocked with 1× PBS containing 5% BSA, 0.05% Tween-20. Plasma samples or mouse plasma spiked with a positive control antibody were added, and the plates were incubated for 2 hours at room temperature. The plates were washed, and the secondary antibody, peroxidase AffiniPure goat anti-mouse IgG+IgM (H+L) (Jackson ImmunoResearch), was added and incubated for 1 hour at room temperature. The plates were then washed thoroughly and detected using TMD substrate (Sigma-Aldrich).

[0741] Measurement of safety biomarkers

[0742] Plasma samples collected from Group 1 (vehicle control), Group 2 (isotype control, 90 mg / kg), and Group 3 (AS20 hIgG1 LALA-PG, 90 mg / kg) were analyzed for 14 safety biomarkers (albumin, alanine aminotransferase, alkaline phosphatase, amylase, bilirubin, blood urea nitrogen, calcium, creatinine, globulin, glucose, phosphate, potassium, sodium, and total protein) using clinical chemistry methods at the Unit of Chemical and Pharmaceutical Safety, RISE, Södertälje, Sweden. Sweden) and analyzed using the Abaxis Vetscan system with cartridge #500-0038.

[0743] result

[0744] Severity of arthritis

[0745] CAIA-induced and vehicle-treated animals developed moderate to severe disease with an incidence of 100%. Similar results were observed in isotype control-treated animals. The efficacy of AS20 hIgG1 LALA-PG administered intraperitoneally every 4 days from day -1 was evaluated at three doses (10, 30, and 90 mg / kg). Compared to the isotype control, AS20 hIgG1 LALA-PG at 90 mg / kg and 30 mg / kg showed significant ameliorative effects on the disease on days 7-14, 16, 18, and 7-12, respectively. A small reduction in disease severity was observed when AS20 hIgG1 LALA-PG was administered at 90 mg / kg compared to the vehicle, although not statistically significant ( Figure 18 and 19 ).

[0746] For ethical reasons (high score), two animals, one in the AS20 hIgG1LALA-PG 10 mg / kg group and one in the isotype control group, were removed before termination (day 12). These animals were included in the maximum score, but not in the mean, AUC, and % inhibition ( Figure 19 , Table 22).

[0747] Disease parameter statistics are summarized in Table 22.

[0748] Table 22 - Statistics of CAIA severity parameters

[0749]

[0750] 1 Cumulative incidence: Mice were considered to have developed disease if the score was 1 or higher on 2 consecutive scoring days. 2The mean CAIA score across all scoring time points. 3 Area under the curve. 4 Percent change in overall disease burden for each animal relative to the isotype control-treated group. Calculated by determining the difference between the mean AUC of the isotype control group and the AUC of each individual animal, dividing by the isotype control group AUC and multiplying by 100*(-1). *p<0.05; **p<0.01 compared to isotype control.

[0751] Health Assessment

[0752] From day 3 until termination, general health assessments were performed daily in conjunction with disease assessments. Mice were weighed twice weekly as part of general health assessments. No side effects of treatment were observed in the animals (data not shown).

[0753] Drug exposure in plasma

[0754] At the end of the study on day 19, plasma exposure of AS20 hIgG1 LALA-PG was generally within the expected concentration range based on previous single-dose pharmacokinetic assessments. Mean concentrations were determined to be 199 μg / mL (approximately 1.3 μM, 58-309 μg / mL) for the 10 mg / kg dose group, 614 μg / mL (approximately 4.1 μM, 229-970 μg / mL) for the 30 mg / kg dose group, and 1408 μg / mL (approximately 9.4 μM, 520-2557 μg / mL) for the 90 mg / mL dose group. Thus, a slightly nonlinear dose-exposure relationship was observed. No evidence of altered plasma exposure over the treatment period due to immunogenicity was detected. Mean hIgG1 LALA-PG concentrations in plasma samples from mice receiving the isotype control were determined to be 1815 μg / mL (approximately 12 μM, 1224-2511 μg / mL), 614 μg / mL (approximately 4.1 μM, 229-970 μg / mL), and 1408 μg / mL (approximately 9.4 μM, 520-2557 μg / mL).

[0755] Anti-drug antibodies (ADA)

[0756] No worrisome ADA was detected in the study samples. Although most samples tested positive, the titers in the AS20 hIgG1LALA-PG group were no higher than in the control group. Given the early onset of the reaction (around day 7) and the unchanged drug exposure over time, the anti-drug signals detected in the samples were likely caused primarily by nonspecific / low-affinity IgM.

[0757] Safety biomarkers

[0758] Plasma glucose increased after treatment with AS20 hIgG1 LALA-PG and an isotype control antibody. High-dose AS20 hIgG1 LALA-PG did not increase biomarkers of liver injury. There was a trend toward increased plasma creatinine in the AS20 hIgG1 LALA-PG-treated group compared to the isotype control group, although this trend was not statistically significant (p < 0.06). Other renal markers, namely blood urea, electrolytes, or total protein, did not differ between AS20 hIgG1 LALA-PG- and isotype control-treated mice.

[0759] in conclusion

[0760] CAIA-induced isotype control-treated mice developed moderate to severe disease with a 100% morbidity rate. Similar results were observed in vehicle-treated mice.

[0761] AS20 hIgG1 LALA-PG at 90 mg / kg and 30 mg / kg showed significant amelioration of disease on days 7-14, 16-18, and 7-12, respectively, compared to isotype control. A small, although not statistically significant, reduction in disease severity was observed when AS20 hIgG1 LALA-PG was administered at 90 mg / kg compared to vehicle.

[0762] Plasma exposures of AS20 IgG1 LALA-PG were generally within the expected concentration range at the end of the study. No evidence of altered plasma exposures due to immunogenicity was detected during the treatment period. No ADA of concern was detected in the study samples.

[0763] High-dose AS20 IgG1 LALA-PG did not increase biomarkers of liver injury. There was a trend toward increased plasma creatinine in the AS20hIgG1 LALA-PG treatment group, although this was not statistically significant. Other renal markers, namely blood urea, electrolytes, or total protein, did not differ between AS20 hIgG1 LAL and AS20 hIgG1 LAL groups.

[0764] Example 19 - FACS analysis of spleens from CAIA experiments

[0765] In this example, the effects of AS20 hIgG1 LALA-PG (heavy chain SEQ ID NO: 115 and light chain SEQ ID NO: 116) on cell subsets in the spleens of mice with collagen antibody-induced arthritis (CAIA) were investigated by fluorescence-activated cell sorting (FACS). Anti-NP hIgG1 LALA-PG (heavy chain SEQ ID NO: 117 and light chain SEQ ID NO: 118) was included as an isotype control.

[0766] Materials and methods

[0767] This study is part of the experimental in vivo study (Example 18) described above. In brief, CAIA was induced in DBA / 1 mice (male, 8-9 weeks) by intravenous (iv) administration of antibodies against type II collagen, followed by intraperitoneal (ip) administration of LPS to promote disease progression. By intraperitoneal administration of AS20 hIgG1 LALA-PG or isotype control antibody (anti-NP hIgG1LALA-PG), mice were treated every 4 days starting the day before disease induction (day -1). At the end of the study (day 19), the spleens of animals treated with the highest dose of AS20 hIgG1 LALA-PG (90 mg / kg) or isotype control (90 mg / kg) were dissected out, weighed, and homogenized as described below. FACS plates were designed to identify T cells, B cells, NKT cells, neutrophils, eosinophils, NK cells, dendritic cells, monocytes, and macrophages.

[0768] Single cell suspension

[0769] The spleen was passed through a 70-μm cell strainer in RPMI-1640 medium (Thermo Fisher, HyClone) to obtain a single-cell suspension. The cells were pelleted and resuspended in 1 ml of MilliQ water to lyse the red blood cells (10 seconds), 1 ml of 2× PBS was added, and then 10 ml of 1× PBS was added. The cells were washed with 10 ml of HBSS (Thermo Fisher, HyClone) and finally resuspended in an appropriate volume of HBSS to obtain 10×10 6 cells / ml.

[0770] FACS protocol

[0771] 1×10 6 Cells were seeded into each well of a 96-well round-bottom plate and centrifuged at 850 × g, 4°C for 1 minute, washed with FACS buffer (1 × PBS, 3% FBS, 2mM EDTA), and pelleted again. Purified rat anti-mouse CD16 / CD32 (Fc blocking) diluted 1:50 in FACS buffer was added to each well, and the cells were incubated for 15 minutes and then washed again with FACS buffer. The antibody mixture was prepared by adding the following antibodies to an appropriate volume of FACS buffer:

[0772] FITC hamster anti-mouse CD3ε (1:100)

[0773] PE rat anti-mouse CD86 (1:100)

[0774] PE-CF594 rat anti-mouse Ly-6C (1:50)

[0775] PE-Cy7 rat anti-mouse Ly-6G (1:200)

[0776] Biotin hamster anti-mouse CD49b (1:200)

[0777] APC-Cy7 hamster anti-mouse CD11c (1:50)

[0778] Alexa Fluor 647 Rat Anti-Mouse IA / IE (MHCII) (1:200)

[0779] Alexa Fluor 700 mouse anti-mouse CD45.2 (1:100)

[0780] BV421 rat anti-mouse Siglec-F (1:200)

[0781] BV605 rat anti-CD11b (1:200)

[0782] BV650 rat anti-mouse CD19 (1:100)

[0783] BV786 hamster anti-mouse CD80 (1:200)

[0784] eF506 Fixable Viability Dye (1:400)

[0785] 25 μl of the antibody mixture was added to the cells in each well and incubated on ice for 20 minutes in the dark. The cells were washed with FACS buffer, and 25 μl of the secondary antibody mixture (PerCP-Cy5.5 streptavidin, diluted 1:200 in FACS buffer) was added, and the cells were incubated on ice for 20 minutes in the dark. The cells were then washed twice with FACS buffer, resuspended in 250 μl of FACS buffer, transferred to FACS tubes, and finally analyzed on a CytoFLEX flow cytometer platform (Beckman Coulter).

[0786] Gating strategy

[0787] Cell subsets were identified as follows:

[0788] T cells: CD45.2 + 、CD11b - 、CD3 +

[0789] B cells: CD45.2 + 、CD11b - 、CD19 +

[0790] NKT cells: CD45.2 + 、CD11b - 、CD3 + 、CD49b +

[0791] Neutrophils: CD45.2 + 、CD11b + 、Ly6G +

[0792] Eosinophils: CD45.2 + 、CD11b + 、Ly6G - 、Siglec F +

[0793] NK cells: CD45.2 + 、CD11b + 、Ly6G - 、Siglec F - 、CD49b +

[0794] Dendritic cells: CD45.2 + 、CD11b + 、Ly6G - 、Siglec F - 、CD11c + , MHCII +

[0795] Monocytes: CD45.2 + 、CD11b + 、Ly6G - 、Siglec F - 、CD11C - 、Ly6C +

[0796] Macrophages: CD45.2 + 、CD11b + 、Ly6G - 、Siglec F - 、CD11C - 、Ly6C - , MHCII +

[0797] In addition, the expression of CD80 and CD86 on dendritic cells was analyzed.

[0798] result

[0799] There were no significant differences in body weight between the AS20 hIgG1 LALA-PG treated group (Group 3, Example 18) and the isotype control group (Group 2, Example 18), either two days before disease induction (AS20, 23.9 ± 1.4 g; isotype control, 23.8 ± 1.2 g) or at study termination on day 19 (AS20, 22.5 ± 1.7 g; isotype control, 22.1 ± 1.0 g). However, spleen weight was significantly higher in the AS20 hIgG1 LALA-PG treated group compared to the isotype control group (110 ± 23 mg vs. 88 ± 21 mg; p = 0.02), and CD45 β-actin was significantly higher in the AS20 group compared to the isotype control group. + The total white blood cell count was also higher (44.5×10 6 With 39.0×10 6 ; p = 0.008).

[0800] The total number of B cells in the spleen of AS20 hIgG1 LALA-PG-treated mice was higher (27.1 ± 3.5 × 10 6 and 21.0±5.8×10 6 ; p = 0.004). In contrast, the total number of NK cells was reduced in AS20hIgG1 LALA-PG treated mice compared with isotype control treated mice (0.44 ± 0.09 × 10 6 and 0.54±0.09×10 6 The total number of NKT cells was also reduced in AS20 hIgG1 LALA-PG treated mice compared to isotype control treated mice (0.14 ± 0.03 × 10 6 and 0.16±0.03×10 6 ; p = 0.08). No significant differences were found in the total number of neutrophils, eosinophils, monocytes, macrophages, dendritic cells, or T cells between AS20 IgG1 LALA-PG-treated mice and isotype control-treated mice ( Figure 20 ).

[0801] CD45 in spleen was decreased in AS20 IgG1 LALA-PG treated mice compared with isotype control treated mice + In contrast, AS20 hIgG1 LALA-PG treated mice showed a higher proportion of B cells (60.9±5.4% vs. 53.0±10.0%, p=0.02) than mice treated with isotype control. +The proportions of T cells, NKT cells, and NK cells were lower in the AS20 IgG1 LALA-PG treated mice (T cells: 15.2±3.3% vs. 18.9±4.1%, p=0.02; NKT cells: 0.31±0.05% vs. 0.40±0.07%, p=0.0003; NK cells: 0.99±0.22% vs. 1.4±0.23%, p=0.0001). No difference in CD45 expression was found between AS20 IgG1 LALA-PG treated mice and isotype control treated mice. + There were significant differences in the proportions of neutrophils, eosinophils, monocytes, macrophages, or dendritic cells among leukocytes ( Figure 21 ). In CD45 + There were no significant differences in either the total number or the proportion of cells expressing CD80 or CD86 between AS20 IgG1 LALA-PG-treated mice and isotype control-treated mice (data not shown).

[0802] in conclusion

[0803] Treatment with AS20 hIgG1 LALA-PG (90 mg / kg dose) significantly reduced CD45 in the spleens of mice with CAIA. + The total number of NK cells and the ratio of T cells, NKT cells and NK cells in the cells.

[0804] Example 20 - Analysis of the Effects of Pre-named Antibody Candidates on BSSL Enzyme Activity

[0805] In this example, the effects of five pre-named candidate antibodies in the hIgG4 S228P format on BSSL enzyme activity were investigated, namely, S-SL048-11 (heavy chain SEQ ID NO: 119 and light chain SEQ ID NO: 120), S-SL048-46 (heavy chain SEQ ID NO: 121 and light chain SEQ ID NO: 122), S-SL048-106 (heavy chain SEQ ID NO: 123 and light chain SEQ ID NO: 124), S-SL048-116 (heavy chain SEQ ID NO: 125 and light chain SEQ ID NO: 126) and S-SL048-118 (heavy chain SEQ ID NO: 127 and light chain SEQ ID NO: 128). Comparisons were made with the hIgG4S228P version of the AS20 CDR graft (heavy chain SEQ ID NO: 131 and light chain SEQ ID NO: 132), chimeric AS20 (heavy chain SEQ ID NO: 129 and light chain SEQ ID NO: 130), and an isotype control anti-NP antibody (heavy chain SEQ ID NO: 133 and light chain SEQ ID NO: 134).

[0806] Materials and methods

[0807] Pre-incubation of BSSL with pre-named candidate antibodies

[0808] In this example, native human BSSL (SEQ ID NO: 138; Table 2) was used. The BSSL stock solution was diluted 10× in MilliQ (MQ)-H2O to a concentration of 0.42 mg / ml.

[0809] Antibodies were diluted in MQ-H2O to a starting concentration of 0.3 μg / μl and then serially diluted 1:1 in MQ-H2O to yield six concentration points ranging from 0.3 μg / μl to 0.009 μg / μl. From each antibody dilution, 20 μl was added to 2.4 μl of BSSL (1 μg), and the antibody / BSSL mixture was incubated at +4°C for 1 hour. Following incubation, 20 μl of MQ-H2O was added to each BSSL / antibody reaction.

[0810] Triglyceride hydrolysis assay

[0811] A triglyceride (TG) emulsion was prepared by mixing 25 mg of unlabeled triolein (Sigma catalog number 92860) with 50 μl of 3H-labeled triolein (triolein [9,10-3H(N)], 91 CI / mmol) NET431001MC Perkin Elmer (Waltham, MA) was mixed in a round-bottom 30 mm diameter glass container suitable for sonication; the solvent was evaporated at room temperature under nitrogen (N2); 1.0 ml of 10% gum arabic (Sigma catalog number G-9752), 1.25 ml of 1.0 M Tris-HCl pH 9.0, and 2.0 ml of MQ-H2O were added to the container; the container was cooled in ice water and sonicated at a medium setting of 50% pulse mode for 10 minutes using a Soniprep 150 (MSE, UK) with a 9 mm diameter flat-tip probe, the probe being placed a few millimeters below the surface of the liquid until an emulsion was obtained; and 2.5 ml of 18.7% BSA (Sigma catalog number A7906), 2.5 ml of 1.0 M NaCl, and 3.25 ml of MQ-H2O. The emulsion was used on the day of preparation. Next, 10 μl of the pre-incubated BSSL / antibody solution (see above) was mixed with 150 μl of TG emulsion with 10 mM sodium cholate (Sigma catalog number C-1254) and MQ-H2O in a 13×100 mm glass tube for a total volume of 200 μl. Samples were prepared in duplicate. The tubes were incubated at 37°C for 15 minutes, and the reaction was subsequently stopped by adding 3.25 ml of methanol / chloroform / heptane (vol / vol / vol, 760 / 680 / 540) and 1.0 ml of 0.1 M sodium carbonate, pH 10.5, followed by centrifugation at 3500×g for 10 minutes. 1400 μl of the upper aqueous phase containing the hydrolyzed free fatty acids was removed and mixed with 2.0 ml of Optiphase Hisafe 3 scintillation cocktail (PerkinElmer, Waltham, MA) in a 6 ml polyethylene vial (Perkin Elmer), and the hydrolyzed free fatty acids were measured by liquid scintillation counting on a WinSpectral 1414 (Wallac, Turku, Finland). 3 The amount of H-labeled free fatty acids.

[0812] Cholesterol ester hydrolysis assay

[0813] Cholesterol ester (CE) emulsion was prepared by mixing 40 μl 14C-labeled cholesterol oleate (oleate-1-14C, NEC6380050UC, Perkin Elmer) was placed in a round-bottomed 30 mm diameter glass container suitable for sonication, and the solvent was evaporated at room temperature under nitrogen. 2.0 ml of 0.2 M Tris-HCl, pH 7.5, and 0.85 ml of MQ-H2O were added to the container. The container was cooled in ice water and sonicated for 10 minutes at a medium setting of 50% pulse mode using a Soniprep 150 (MSE) with a 9 mm diameter flat-tip probe placed a few millimeters below the liquid surface until an emulsion was obtained. 1.65 ml of 200 mM sodium cholate and 1.0 ml of MQ-H2O were added to the emulsion. The emulsion was used on the day of preparation. Next, 10 μl of the pre-incubated BSSL / antibody solution (see above) was mixed with 100 μl of CE emulsion and MQ-H2O in a 13×100 mm glass tube for a total volume of 200 μl. Samples were prepared in duplicate. The tubes were incubated at 37°C for 30 min and the reaction was subsequently stopped by the addition of 3.25 ml of methanol / chloroform / heptane (vol / vol / vol, 760 / 680 / 540) and 1.0 ml of 0.1 M sodium carbonate pH 10.5, followed by centrifugation at 3500×g for 10 min. 400 μl of the upper aqueous phase containing the hydrolyzed free fatty acids was removed and mixed with 2.0 ml of Optiphase Hisafe 3 scintillation cocktail (Perkin Elmer) in a 6 ml polyethylene vial (PerkinElmer) and the hydrolyzed free fatty acids were measured by liquid scintillation counting on a WinSpectral 1414 (Wallac). 14 The amount of C-labeled free fatty acids.

[0814] result

[0815] Enzyme activity was assessed by measuring the release of free fatty acids (radiolabeled) after 15 min (triglyceride hydrolysis assay) or 30 min (cholesterol ester hydrolysis assay) incubation, respectively. Figure 17 For technical reasons, samples had to be analyzed in two consecutive sets, but both sets included both antibodies (AS20 and isotype control anti-NP antibody). The data are expressed as relative values, where the value obtained without adding antibodies to the reaction was set to 100% (Tables 23 and 24). Table 23 - Effect of five BSSL-specific hIgG4 S228P and control antibodies of the hIgG4 S228P subtype on BSSL enzyme activity (hydrolysis of triglycerides)

[0816]

[0817] Table 24 - Effects of five BSSL-specific hIgG4 S228P and hIgG4 S228P subtype control antibodies on BSSL enzyme activity (hydrolysis of cholesterol esters)

[0818]

[0819] in conclusion

[0820] None of the antibodies tested, ie, the five pre-named drug candidates, AS20, AS20 CDR graft, and anti-NP in the hIgG4 S228P format, showed any significant effect on the enzymatic activity of BSSL.

[0821] Example 21 - Epitope mapping of AS20 by X-ray crystallography

[0822] The three-dimensional structure of the complex of AS20 and hBSSL was determined using X-ray crystallography. For this experiment, the antibody was cleaved to generate Fab fragments and a new C-terminally truncated hBSSL (t-hBSSL) construct was prepared, which corresponds to amino acids 1 to 530 of hBSSL followed by AHHHHHH (SEQ ID NO: 146).

[0823] Materials and methods

[0824] Cloning, expression and purification of t-hBSSL

[0825] Human BSSL had previously crystallized as a truncated form lacking the flexible C-terminal portion. Therefore, to enable the generation of crystals of AS20 Fab and hBSSL, a new truncated BSSL construct was prepared that included a C-terminal his-tag for purification. The construct gp67-BSSL-6×H, consisting of amino acids 1-530 + AHHHHHH (BSSL numbering is based on the sequence after removal of the signal peptide), was ordered from GeneArt and cloned into the pFastBac tGFP Dual vector, which had been prepared for ligation-independent cloning (LIC). LIC was performed using the InFusion cloning kit and transformed into Stellar competent cells.

[0826] Recombinant bacmid DNA was produced in DH10Bac E. coli cells. The bacmid was transfected into Sf9 cells at 27°C for 120 hours. P1 virus was harvested from the growth medium and subsequently used to produce P2 virus stock. After 96 hours, the P2 virus stock was harvested by centrifugation. 400 ml of Sf9 cells (1.5 × 10 6Cells / ml) were infected with 2 ml of P2 virus stock and grown for 72 hours post infection. The culture medium (P3 stock) was harvested by centrifugation and filtered. For large-scale expression, 35 ml of P3 virus stock was added to 2130 ml of Sf9 cells (1.6 × 10 cells / ml) in a Thomson optimal growth flask (5 L). 6 The expression was carried out at 27°C for 72 hours. At the time of harvest, the cell density was 2.34×10 6 1 cell / ml, 75% expressed GFP, and 89% were viable. The culture was centrifuged to remove cells. t-hBSSL was purified from the culture medium by batch IMAC using Ni-agarose fast-flow resin and eluted with 50 mM Tris pH 7.5, 500 mM NaCl, 500 mM imidazole. It was further purified by size exclusion chromatography using a Superdex 200 16 / 60 column in 50 mM Hepes pH 7.0, 500 mM NaCl. The protein eluted as a single monomeric peak, which was pooled and concentrated.

[0827] Complex formation between AS20 Fab fragment and t-hBSSL

[0828] The production of AS20 antibodies was carried out at Absolute Antibody (Oxford, UK). Fab fragments were produced by following the papain digestion protocol from an immobilized papain supplier (Thermo Scientific, product number 20341). A total of 15 mg of AS20 was concentrated to 17 mg / ml, and the buffer was exchanged with 20 mM sodium phosphate, 10 mM EDTA pH 7, 20 mM cysteine. The antibody was incubated with immobilized papain (0.6 ml slurry) at 37 ° C for 3 hours, then incubated overnight at 4 ° C. Because the cutting was not completed, the sample was incubated at 37 ° C for another 3 hours, then incubated at room temperature over the weekend. The antibody was eluted with 10 mM Tris pH 7.5 and purified t-hBSSL was added. The mixture was incubated at room temperature for approximately 30 minutes, after which it was loaded onto a Superdex 200 column in 50 mM Hepes pH 7.0, 500 mM NaCl. The first major peak with an elution volume of approximately 60 ml contained the complex of t-hBSSL and AS20 Fab fragment. The relevant fractions were concentrated (10K MWCO) and the salt concentration was reduced to 250 mM by dilution with 50 mM Hepes pH 7.0 buffer.

[0829] Crystallization and structural elucidation of the AS20 Fab-t-hBSSL complex

[0830] After purification of the Fab t-hBSSL complex, the sample was kept on ice at 4°C for approximately 36 hours during transport between laboratories. The complex was then concentrated from 4 mg / mL to a final concentration of 17.3 mg / mL using 500 μL of Vivaspin with a 30,000 MWCO (Vivaspin 500 from Sartorius, VS0122). Concentration was measured on a Nanodrop at A280 using an extinction coefficient of 174,375 and an Mw of 106.242 kDa.

[0831] Crystallization experiments were set up in Swiss CI XTAL SD-3 3-well plates using a Mosquito liquid handling robot, with 35 μL of reservoir solution and 150 + 50, 100 + 100, and 50 + 150 nL of protein + reservoir solution droplets. Screening software Morpheus from Molecular Dimensions yielded crystals under conditions B9 and B10 at 20 degrees Celsius. Rod-shaped crystals appeared within the first 12 hours and grew larger over the next 24 hours. After rapid transfer to a reservoir solution containing an additional 10% glycerol, they were cryogenically cooled by plunging into liquid nitrogen. Data were collected at the Biomax beamline on MaxIV. The automatically processed data files were used for structure elucidation by molecular replacement in Phaser, using 1f6w.pdb (hBSSL) and 4n0y.pdb (Fab fragment) as search models. Water molecules were removed, as was the C-terminal portion of BSSL, and for 4n0y.pdb, only the Fab chain modeled as polyalanine was retained. Manual model building was performed in Coot and model refinement was performed using Refmac5 (all CCP4 packages).

[0832] result

[0833] Crystallization and structure determination of AS20 Fab t-hBSSL

[0834] AS20 Fab fragments can be crystallized with t-hBSSL in Morpheus Condition B9 containing 30 mM NaBr, 30 mM NaFl, 30 mM NaI, 0.1 M Tris (base), 0.1 M Bicine (the buffer is set to pH 8.5 with these two buffers), 20% PEG550 MME, and 10% PEG 20 K. The crystals have unit cell dimensions of 323, 67, 123, 90, 101.7, 90 and diffraction patterns of The structure was solved by molecular replacement, revealing two complete complexes in the asymmetric unit. The crystal packing around the two copies differs, resulting in minor structural variations in the t-hBSSL. In particular, the final residues encompassing the 6-His tag are visible in molecule A but not in molecule B, as B has fewer crystal contacts in this region and more room for C-terminal flexibility. The region between residues 272 and 283 is also highly disordered in molecule B and could not be modeled. Despite these differences, the two t-hBSSL models superimpose very well.

[0835] Analysis of the interaction interface between AS20 Fab and t-hBSSL

[0836] The BSSL structure has been described as having a large core region consisting of a twisted 11-stranded β-sheet surrounded by α-helices and connecting loops [9]. There is a smaller 3-stranded β-sheet at the N-terminus. The structure has been likened to a left-hand oven glove, with the palm containing the active site triad near the "thumb". Similarly, the small N-terminal β-sheet is located on the back of the hand near the "pinky finger", see Figure 12 The part of the BSSL structure that interacts with the Fab molecule is located in the small N-terminal β-sheet and the C-terminal part of αC

[17] , which is the third α-helix in the structure. In other words, the binding region of the antibody is not close to the active site, but on the opposite side of the antigen. Figure 13 Shown is how the variable chain of AS20 binds to t-hBSSL, with the epitope sequence highlighted in light grey.

[0837] The epitope region is listed in Table 25 and includes residues 7-12 (chains 1 and 2, SEQ ID NO: 1), 42-55 (a loop region introduced into the fold of chain 3, part of SEQ ID NO: 2), and 174-180 (the C-terminus of αC, SEQ ID NO: 5). The epitope is quite flat, with only a few characteristic residues protruding, namely Tyr7, Phe12, and Gln52 (the major interactions listed in Table 245). The loop region 47-54 is well defined and forms a uniform surface. Proline 47 is important for stacking interactions with Tyr31 of the Fab, but overall, the surface here is flat. Many residues within the epitope sequence are important for BSSL folding but do not interact with AS20 in a specific manner.

[0838] Table 25 - List of BSSL residues involved in the interaction between BSSL and Fab AS20, ordered by the most important interactions in the top row. BSSL residues important for BSSL folding in the epitope region are also listed.

[0839]

[0840] in conclusion

[0841] This example describes the crystallization and structure elucidation of AS20 Fab and t-hBSSL. The structures revealed that the epitope region is located in the same region previously identified by HDX-MS as described in Example 13. It is a three-dimensional epitope composed of a small beta-sheet, an ordered loop region of strand 3 that introduces the fold, and the C-terminal portion of the adjacent helix. The epitope sequences are 7-12 (YTEGGF, SEQ ID NO: 1), 42-55 (LENPQPHPGWQGTL, SEQ ID NO: 2), and 174-180 (VKRNIAA, SEQ ID NO: 5); they are arranged sequentially but close together in the structure. The most well-defined residues are Tyr7, Phe12, and Gln52, which protrude from the surface.

[0842] The sequences of five previously named antibodies were analyzed in the context of the AS20 Fab t-hBSSL structure. Sequence differences (primarily conserved) and results from HDX-MS mapping (Example 14) indicated that all five antibodies would bind to the same epitope on BSSL.

[0843] Example 22 - Complex formation, crystallization, structure determination and epitope analysis of the S-SL048-116 Fab-BSSL complex

[0844] Materials and methods

[0845] S-SL048-116 cleavage with FabRICATOR

[0846] The S-SL048-116 antibody (heavy chain SEQ ID NO: 125 and light chain SEQ ID NO: 126) was lysed in PBS and the F(ab')2 was purified using the FragIT kit (Genovis) according to the manufacturer's instructions.

[0847] Reduction of F(ab')2 fragments and purification of Fab' fragments

[0848] Large-scale reduction of the purified F(ab')2 fragment was performed at room temperature for 2 hours using cysteamine at a final concentration of 50 mM in PBS, pH 7.2, containing 5 mM EDTA. Four-fifths of the resulting sample was purified on a HiLoad 26 / 60 Superdex 200 preparative grade (GE Healthcare) using PBS, pH 7.2, 2 mM EDTA as the mobile phase. Fractions from the peak of interest were pooled, concentrated, and stored at -80°C for downstream applications. The total yield was 7.2 mg.

[0849] Alkylation of Fab' fragments and subsequent purification

[0850] The remaining one-fifth of the reduced sample was treated with an equal volume of 375 mM iodoacetamide at room temperature for 30 minutes to block free cysteines by alkylation. The resulting alkylated sample was purified identically to the unalkylated sample. The total yield was 2 mg.

[0851] Expression and purification of BSSL

[0852] The P2 virus stock for expression was obtained from SciLifeLab (Stockholm, Sweden). The ExpiSf9 insect cell line was used in ExpiSf CD culture. Expression was performed using the ExpiSf Protein Expression Kit (ThermoFisher Scientific) according to the manufacturer's recommendations using the viral load recommended by SciLifeLab.

[0853] The culture supernatant from 2L expression was supplemented with EDTA-free protease inhibitor tablets (Merck Millipore), NiSO4 and imidazole (final concentration 15mM). Any particles were removed by centrifugation at 13000 × g. The supernatant was loaded onto a 5ml HisTrap Excel column (GE Healthcare) at 4°C with a flow rate of 2ml / min and spent the night. The column was washed with 6 column volumes (CV) of IMAC buffer A (50mM Tris-HCl, 500mM NaCl; pH 7.5) containing 15mM imidazole. The column was washed again with 10CV of IMAC buffer A containing 25mM imidazole. The bound protein was eluted with a 20CV linear gradient to 100% IMAC buffer B (50mMTris-HCl, 500mM NaCl, 0.5M imidazole, pH 7.5). The eluted protein was concentrated, centrifuged and run on a HiLoad 26 / 600 Superdex 200 preparative gel filtration column (GE Healthcare) pre-equilibrated with SEC buffer (50 mM HEPES, 500 mM NaCl, 2 mM EDTA; pH 7). Fractions from the target peak (1E6-1F8) were combined and concentrated. The total yield was 6.2 mg, and the sample purity was approximately 85%.

[0854] BSSL:Fab' complex formation and purification

[0855] Prior to complex generation, BSSL was evaluated by SEC on a HiLoad 26 / 600 Superdex 200 prep-grade column to confirm the absence of concentration / storage-induced oligomerization. The complexes were mixed at a molar ratio of 1:1.3 BSSL:Fab' and incubated on ice for 1 hour. The incubated complexes were run on the same SEC column using the same buffer as BSSL. Fractions of interest (B3-C4) were pooled and the buffer exchanged to 50 mM HEPES, 250 mM NaCl, 2 mM EDTA; pH 7. The buffer-exchanged sample was concentrated to 17 mg / ml, flash-frozen in liquid nitrogen, and stored at -80°C until crystallization. The purity of the sample was estimated to be >90% based on SDS-PAGE analysis.

[0856] Formation and purification of BSSL:Alk-Fab′ complex

[0857] The complex was mixed at a 1.1:1 molar ratio of BSSL:Alk-Fab' and incubated on ice for 1 hour. The incubated complex was run on the same SEC column using the same buffer as the BSSL:Fab' complex. Fractions of interest were pooled and the buffer exchanged to 50 mM HEPES, 250 mM NaCl, 2 mM EDTA, pH 7. The buffer-exchanged sample was concentrated to 14.5 mg / ml, flash-frozen in liquid nitrogen, and stored at -80°C until crystallization.

[0858] Crystallization and cryoprotection of Fab-BSSL complexes

[0859] The best diffracting crystals were grown at 20°C from 14.5 mg / ml Fab-BSSL complex in buffer (50 mM HEPES, 250 mM NaCl, 2 mM EDTA, pH 7.0) and mixed with reservoir solution (16% (w / v) PEG 4000, 0.1 M sodium citrate pH 5 and 6% (v / v) ethanol) and seed solution as follows:

[0860] 150 nl complex + 37 nl seeds (crushed crystals in 0.1 M sodium citrate pH 5.0, 20% (w / v) PEG4000 and 0.2 M ammonium sulfate) + 113 nl reservoir solution

[0861] The settled drop was pipetted onto an MRC plate with 40 μl of reservoir solution. Crystals appeared within a few days and froze in a freezing solution containing 20% ​​(v / v) glycerol, 16% (w / v) PEG4000, 0.1 M sodium citrate pH 5.0 and 3% (v / v) ethanol.

[0862] Data Collection

[0863] The results were obtained at 100 K on a BioMAX station equipped with an Eiger 16M hybrid pixel detector, MAX IV, Lund, The data set was collected using an exposure time of 0.011 s and an oscillation of 0.1° / image, for a total of 360°. The data were processed using the autoPROC pipeline into space group P21.

[0864] result

[0865] Structure determination

[0866] The catalytic domain of human bile salt-activated lipase BSSL was characterized by using Phaser software. Structure (from PDB:1F6W) and homology The Fab structure (from PDB: 3NFP) was used as a template for molecular replacement to determine the Fab-BSSL structure. A complex was found in the asymmetric unit. The structure was refined in Refmac5, then in Buster, and modeled in Coot. The final model included three protein chains (chain A) with BSSL and the heavy (H) and light (L) chains of S-SL048-116 Fab ( Figure 22 ). The final model includes amino acids 1-531 in chain A, except for the flexible loop (amino acids 117-123) that is not seen in the electron density. The model includes amino acids 1-227 in chain H and amino acids 1-211 in chain L. The first amino acid Gln1 in chain H has been modeled as PCA, with pyroglutamic acid to best fit the electron density. In chain H, two free cysteines (Cys133 and Cys225) have been modeled as non-alkylated cysteines, although the alkylation of S-SL048-116 Fab is essential for crystallization. There is no additional density to fit the methylamide group presumably connected to the sulfur atom of cysteine. It is possible that the cysteine ​​residues are at least partially alkylated, but the atoms connected are so flexible that they cannot be clearly seen in the electron density map. In addition, 87 water molecules were modeled.

[0867] Epitope analysis

[0868] Epitope analysis was performed using the coordinates of the Fab-BSSL complex. Analysis was performed using CONTACT software from the CCP4 program suite. S-SL048-116-Fab binds to BSSL via both the heavy and light chains. In the heavy chain, all three CDR loops (CDR1, CDR2, and CDR3) are involved, whereas in the light chain, only CDR1 and CDR3 make contact with BSSL ( Figure 23, Table 26). Ile 2 near the N-terminus of the light chain also undergoes hydrophobic van der Waals interactions with BSSL. The hydrogen bond network stabilizes the interactions between the heavy chain and BSSL (a total of 7 hydrogen bonds) and between BSSL and the light chain (a total of 5 hydrogen bonds) calculated using PISA (QT) analysis. PISA (QT) analysis shows that The solvent accessible region of is buried in the interface between BSSL and the heavy chain, and Embedded in the interface between BSSL and the light chain.

[0869] Table 26-Between S-SL048-116-Fab and BSSL Summary of all interacting residues between

[0870]

[0871]

[0872] in conclusion

[0873] The results for S-SCL048-116 obtained in this example are consistent with the results obtained from the HDX-MS studies in Example 14.

[0874] Example 23 - Efficacy Verification of S-SL048-116 in Rheumatoid Arthritis Mouse Model

[0875] In this example, the effects of S-SL048-116 (heavy chain SEQ ID NO: 125 and light chain SEQ ID NO: 126) were investigated in an in vivo mouse model of rheumatoid arthritis (RA), collagen antibody-induced arthritis (CAIA). The CAIA model is described in Example 18. This study was approved by the local animal ethics committee in Malmö / Lund, Sweden (02896-20).

[0876] Materials and methods

[0877] Disease induction

[0878] DBA / 1 mice (male, 8 weeks) were injected intravenously with 2 mg / mouse of a monoclonal anti-CII antibody cocktail (CIA-MAB-50, MD Bioproducts) on day 0. On day 5, mice were injected intraperitoneally with LPS (50 μg / mouse) to aggravate the disease.

[0879] Experimental groups and administration of test items

[0880] S-SL048-116 was delivered at a concentration of 5 mg / ml and further diluted in vehicle (25 mM histidine, 150 mM NaCl, 0.02% P80, pH 6.0). The test items (antibody and vehicle) were administered intraperitoneally every 4 days starting one day before disease induction (day -1) and then on days 3, 7, 11, and 15. S-SL048-116 was administered at three different doses, 10, 30, and 90 mg / kg, based on the average body weight of the animals on day -2. The relatively high dose was chosen to compensate for the differences in the size of the human BSSL (K D =0.5 nM) compared to the low affinity of S-SL048-116 for mouse BSSL (K D =40 nM), as described in Example 11. The experimental groups are summarized in Table 27. The first administration on Day -1 was a bolus dose, i.e., the test item was given as a double dose divided into two injections, the first injection was given in the morning and the second injection was given in the afternoon. The following administrations (Days 3, 7, 11 and 15) were given as single doses. Animals were weighed before each administration, and the dose volume of 20 ml / kg was based on the individual weight of the animal.

[0881] Table 27 - Experimental Groups

[0882]

[0883] Disease evaluation

[0884] Disease was assessed daily in a blinded manner starting on day 3 using a macroscopic limb scoring system ranging from 0 to 15 (1 point for each swollen or red toe, 1 point for a swollen or red middle toe or knuckle, and 5 points for a swollen ankle), resulting in a maximum total score for each mouse of 60. Animals with a score of more than 45 were removed from the experiment due to ethical restrictions.

[0885] result

[0886] Severity of arthritis

[0887] CAIA-induced and vehicle-treated animals developed moderate to severe disease with a 100% morbidity. The efficacy of S-SL048-116 (SOL-116) administered intraperitoneally every 4 days from day -1 to termination was evaluated at three doses (10, 30, and 90 mg / kg). S-SL048-116 administered at 90 mg / kg showed an ameliorative effect on disease severity compared to vehicle ( Figure 24 and 25 ).

[0888] For ethical reasons (high score), two animals were removed before termination, one in the vehicle group (day 15) and one in the 90 mg / kg group (day 12). These animals were included in the maximum score, but not in the mean, AUC and % inhibition ( Figure 25 , Table 28). One mouse in the vehicle group did not recover from the LPS boost, i.e., was hypothermic, had a kyphotic posture, and very low body weight. This mouse was removed before the onset of disease (day 7) and was therefore excluded from all results.

[0889] Disease parameter statistics are summarized in Table 28.

[0890] Table 28 - Statistics of CAIA severity parameters

[0891]

[0892] 1 Cumulative incidence: Mice were considered to have developed disease if the score was 1 or higher on 2 consecutive scoring days. 2 The mean CAIA score across all scoring time points. 3 Area under the curve. 4 The percent change in overall disease burden for each animal relative to the vehicle control treated group was calculated by determining the difference between the mean AUC of the vehicle control group and the AUC of each individual animal, dividing by the vehicle control group AUC and multiplying by 100*(-1).

[0893] Health Assessment

[0894] General health assessments were performed daily from day 3 until termination, in conjunction with disease assessments. Mice were weighed twice weekly as part of general health assessments. No side effects of treatment were observed in the animals.

[0895] in conclusion

[0896] CAIA-induced vehicle-treated mice developed moderate to severe disease with a 100% morbidity. The efficacy of S-SL048-116 was evaluated at three different doses: 10, 30, and 90 mg / kg intraperitoneal doses, with treatment beginning on day -1. An improvement in disease severity was observed with the 90 mg / kg dose of S-SL048-116 compared to vehicle. No adverse effects were observed with treatment with S-SL048-116 or vehicle alone.

[0897] Example 24 - Comparison of cytoarchitecture analysis in blood, spleen, and mesenteric lymph nodes of BSSL knockout (KO) and wild-type (WT) mice

[0898] In this example, the numbers of different leukocyte subsets were investigated by fluorescence-activated cell sorting (FACS) in blood, spleen, and mesenteric lymph nodes (MLN) collected from BSSL-deficient knockout (KO) mice and wild-type littermates.

[0899] Materials and methods

[0900] Leukocytes were isolated from the blood, spleen, and MLN of 10 BSSL KO mice and 10 wild-type littermates (15-19 weeks) for analysis. Total leukocyte counts in the spleen and MLN were determined using manual counting in a Burcher chamber. Cells isolated from blood, spleen, and MLN were incubated with FC-Block (CD16 / C032 clone 2.4G2) and subsequently incubated with two different antibody cocktails, Stain A and Stain B.

[0901] Stain A

[0902] Antibodies / clones / fluorochromes used for staining A were: CD19 (ID3 / BB515), γδ TCR (GL3 / PE), CD8a (53-6.7 / PerCP-Cy5.5), CD45.2 (104 / PE-Cy7), NK1.1 (PK136 / APC), CD4 (GK1.5 / APC-H7), TCRβ (H57-597 / BV421), and fixable viability dye (FVD) (Horizon 510).

[0903] The cell populations identified by staining A are:

[0904] αβ T cells: CD45.2 + , TCRβ +

[0905] CD4αβ T cells: CD45.2 + , TCRβ + 、CD4 +

[0906] CD8αβ T cells: CD45.2 + , TCRβ + 、CD8 +

[0907] γδ T cells: CD45.2 + , TCRγδ +

[0908] NKT cells: CD45.2 + , TCRβ + NK1.1 +

[0909] B cells: CD45.2 + 、CD19 +

[0910] NK cells: CD45.2 + , TCRβ - , TCRγδ - NK1.1 +

[0911] Staining B

[0912] The antibodies / clones / fluorochromes used for staining B were: MHCII (MS / 114.15.2 / Alexa488), FcεRlα (MAR-1 / PE), SiglecF (E50-2440 / PE-CF594), Ly6G (1A8 / PerCP-Cy5.5), CD45.2 (104 / PE-Cy7), Ly6C (AL-21 / APC), CD11b (M1 / 70 / APC-Cy7), CD117 (2B8 / BV421) and fixable viability dye (FVD) (Horizon 510).

[0913] The cell populations identified by staining B are:

[0914] Bone marrow cells: CD45.2 + 、CD11b +

[0915] Neutrophils: CD45.2 + 、CD11b + 、Ly6G +

[0916] Eosinophils: CD45.2 + 、CD11b + 、Ly6G - 、Siglec F + 、SSC 高

[0917] Monocytes: CD45.2 + 、CD11b + 、Ly6G - 、Siglec F - 、SSC 低 、Ly6C + , MHCII -

[0918] Macrophages: CD45.2 + 、CD11b + 、Ly6G - 、Siglec F- 、SSC 低 、Ly6C - , MHCII +

[0919] Mast cells: CD45.2 + 、CD11b + , FcεRlα + 、CD117 +

[0920] Basophils: CD45.2 + 、CD11b + , FcεRlα + 、CD117 -

[0921] After incubation (20 min on ice), cells were washed, resuspended in FACS buffer, and different cell subsets were identified by FACS.

[0922] result

[0923] Total white blood cell count in spleen and MLN

[0924] The number of leukocytes in the spleen of KO mice was found to be significantly reduced to about 50% of that in WT mice. No significant difference in the number of leukocytes was observed in the MLN between KO and WT mice ( Figure 26 ).

[0925] αβ T cells

[0926] The total number of αβ T cells in the spleen of KO mice was found to be reduced compared with that of WT mice. + Similar results were observed for leukocytes. No significant differences in the number of αβ T cells were observed in MLN. αβ T cells were found to account for the majority of total CD45 + In the spleen, the percentage of CD4 + / CD8 + The ratio was higher than that in WT mice.

[0927] γδ T cells

[0928] The total number of γδ T cells in the spleen of KO mice was found to be reduced compared with that of WT mice. + Similar results were observed for leukocytes. In MLN, this reduction was even more pronounced than in spleen. It was also found that γδ T cells accounted for more CD45 in MLN in KO mice. + The proportion of cells was lower than that in WT mice. No such difference was observed in the spleen and blood.

[0929] NKT cells

[0930] The total number of NKT cells in the spleen of KO mice was found to be reduced compared with that of WT mice. + Similar results were observed for leukocytes. No significant difference in the number of NKT cells was observed in MLN. NKT cells accounted for the majority of CD45 + The proportion of cells was lower than that in WT mice. No such difference was observed in the spleen and MLN.

[0931] B cells

[0932] The total number of B cells in the spleen of KO mice was found to be reduced compared with WT mice. + Similar results were observed for leukocytes. No significant difference in the number of B cells was observed in MLN. B cells were found to account for the majority of CD45 + The proportion of cells was lower than that in WT mice. No such difference was observed in the MLN.

[0933] NK cells

[0934] The total number of NK cells in the spleen of KO mice was found to be reduced compared with that of WT mice. + The difference in NK cell numbers was more pronounced in leukocytes and other lymphoid subsets. No significant difference in NK cell numbers was observed in MLN. NK cells were found to account for the majority of CD45 + The proportion of cells was lower than that of WT mice. No such difference was observed in MLN ( Figure 27 ).

[0935] bone marrow cells

[0936] The total number of myeloid cells in the spleen and MLN of KO mice was reduced compared with that of WT mice. + The proportion of cells was lower than that in WT mice. No such difference was observed in the blood.

[0937] Neutrophils

[0938] No significant differences in neutrophil numbers were observed in the spleen or MLN between KO and WT mice. KO mice had a higher proportion of neutrophils in the blood, but no significant differences were observed in the spleen or MLN.

[0939] eosinophils

[0940] No significant differences in the number of eosinophils were observed in the spleen or MLN between KO and WT mice. A higher proportion of eosinophils was found in the blood of KO mice, but no significant differences were found in the spleen or MLN.

[0941] Monocytes

[0942] The total number of monocytes was found to be lower in the spleen or MLN of KO mice compared to WT mice. No significant differences in the proportion of monocytes were observed between KO and WT mice in any organ studied.

[0943] macrophages

[0944] The total number of macrophages was found to be lower in the spleen or MLN of KO mice compared to WT mice. No significant differences in the proportion of macrophages were observed between KO and WT mice in any organ studied.

[0945] Mast cells

[0946] No significant differences in the number of mast cells in the spleen or MLN were observed between KO and WT mice. A higher proportion of mast cells was found in the spleen of KO mice compared to WT mice. No differences were observed in the blood or MLN.

[0947] basophils

[0948] Compared with WT mice, the total number of basophils in the spleen of KO mice was found to be lower. No difference in basophil numbers was observed in the MLNs. Compared with WT mice, the proportion of basophils in the blood of KO mice was higher. No such differences were observed in the spleen or MLNs.

[0949] in conclusion

[0950] In addition to the global effects on CD45+ leukocytes, the data suggest an additional specific effect on NK cells, namely that CD45 in the blood and spleen of native BSSL-deficient mice was significantly increased compared to wild-type littermates. + These data support the finding that treatment with AS20 hIgG1 LALA-PG (90 mg / kg dose) significantly reduced the number of CD45 + The total number of NK cells and the ratio of T cells, NKT cells and NK cells in the cells (Example 19).

[0951] Table 29 - List of sequences used herein and SEQ ID NOs:

[0952]

[0953]

[0954]

[0955]

[0956]

[0957]

[0958]

[0959]

[0960]

[0961]

[0962]

[0963]

[0964]

[0965]

[0966]

[0967]

[0968]

[0969]

[0970]

[0971]

[0972]

[0973]

[0974]

[0975] ...

Claims

1. An isolated antibody or antigen-binding fragment thereof, comprising: the three complementarity determining regions HCDR of the heavy chain variable region HCVR; and The light chain variable region LCVR has three complementary determining regions LCDR, among which The first HCDR consists of the amino acid sequence according to SEQ ID NO: 7; The second HCDR consists of an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:18 and SEQ ID NO:19; The third HCDR consists of the amino acid sequence according to SEQ ID NO: 9; The first LCDR consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 20; the second LCDR consists of an amino acid sequence selected from the group consisting of ATS and AAS, and The third LCDR consists of an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:21 and SEQ ID NO:

22.

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The first HCDR consists of the amino acid sequence according to SEQ ID NO: 7; the second HCDR consists of the amino acid sequence according to SEQ ID NO: 8; the third HCDR consisting of the amino acid sequence according to SEQ ID NO: 9; The first LCDR consists of the amino acid sequence according to SEQ ID NO: 10; The second LCDR consists of the amino acid sequence ATS; and The third LCDR consists of the amino acid sequence according to SEQ ID NO:

11.

3. The isolated antibody or antigen-binding fragment thereof according to claim 2, wherein the isolated antibody or antigen-binding fragment thereof comprises: an extended second HCDR consisting of the amino acid sequence according to SEQ ID NO: 12; an extended first LCDR consisting of the amino acid sequence according to SEQ ID NO: 14; and An extended second LCDR consisting of the amino acid sequence according to SEQ ID NO:

15.

4. The isolated antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein the HCVR comprises the amino acid sequence according to SEQ ID NO: 36; and / or The LCVR comprises the amino acid sequence according to SEQ ID NO:

37.

5. The isolated antibody or antigen-binding fragment thereof according to claim 4, wherein The HCVR consists of the amino acid sequence according to SEQ ID NO: 36; and / or The LCVR consists of the amino acid sequence according to SEQ ID NO:

37.

6. The isolated antibody or antigen-binding fragment thereof according to claim 2, wherein the isolated antibody or antigen-binding fragment thereof comprises: an extended second HCDR consisting of the amino acid sequence according to SEQ ID NO: 12; an extended first LCDR consisting of the amino acid sequence according to SEQ ID NO: 16; and An extended second LCDR consisting of the amino acid sequence according to SEQ ID NO:

17.

7. The isolated antibody or antigen-binding fragment thereof according to claim 2 or 6, wherein The HCVR comprises the amino acid sequence according to SEQ ID NO: 36; and / or The LCVR comprises the amino acid sequence according to SEQ ID NO:

38.

8. The isolated antibody or antigen-binding fragment thereof according to claim 7, wherein The HCVR consists of the amino acid sequence according to SEQ ID NO: 36; and / or The LCVR consists of the amino acid sequence according to SEQ ID NO:

38.

9. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The first HCDR consists of the amino acid sequence according to SEQ ID NO: 7; the second HCDR consists of the amino acid sequence according to SEQ ID NO: 18; the third HCDR consisting of the amino acid sequence according to SEQ ID NO: 9; The first LCDR consists of the amino acid sequence according to SEQ ID NO: 10; The second LCDR consists of the amino acid sequence ATS; and The third LCDR consists of the amino acid sequence according to SEQ ID NO:

21.

10. The isolated antibody or antigen-binding fragment thereof according to claim 9, wherein the isolated antibody or antigen-binding fragment thereof comprises: an extended second HCDR consisting of the amino acid sequence according to SEQ ID NO: 23; an extended first LCDR consisting of the amino acid sequence according to SEQ ID NO: 16; and An extended second LCDR consisting of the amino acid sequence according to SEQ ID NO:

15.

11. The isolated antibody or antigen-binding fragment thereof according to claim 9 or 10, wherein the HCVR comprises the amino acid sequence according to SEQ ID NO: 30; and / or The LCVR comprises the amino acid sequence according to SEQ ID NO:

31.

12. The isolated antibody or antigen-binding fragment thereof according to claim 11, wherein The HCVR consists of the amino acid sequence according to SEQ ID NO: 30; and / or The LCVR consists of the amino acid sequence according to SEQ ID NO:

31.

13. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The first HCDR consists of the amino acid sequence according to SEQ ID NO: 7; the second HCDR consists of the amino acid sequence according to SEQ ID NO: 8; the third HCDR consisting of the amino acid sequence according to SEQ ID NO: 9; The first LCDR consists of the amino acid sequence according to SEQ ID NO: 20; The second LCDR consists of the amino acid sequence AAS; and The third LCDR consists of the amino acid sequence according to SEQ ID NO:

11.

14. The isolated antibody or antigen-binding fragment thereof according to claim 13, wherein the isolated antibody or antigen-binding fragment thereof comprises: an extended second HCDR consisting of the amino acid sequence according to SEQ ID NO: 24; an extended first LCDR consisting of the amino acid sequence according to SEQ ID NO: 27; and An extended second LCDR consisting of the amino acid sequence according to SEQ ID NO:

29.

15. The isolated antibody or antigen-binding fragment thereof according to claim 12 or 13, wherein The HCVR comprises the amino acid sequence according to SEQ ID NO: 32; and / or The LCVR comprises the amino acid sequence according to SEQ ID NO:

33.

16. The isolated antibody or antigen-binding fragment thereof according to claim 15, wherein The HCVR consists of the amino acid sequence according to SEQ ID NO: 32; and / or The LCVR consists of the amino acid sequence according to SEQ ID NO:

33.

17. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The first HCDR consists of the amino acid sequence according to SEQ ID NO: 7; the second HCDR consists of the amino acid sequence according to SEQ ID NO: 19; the third HCDR consisting of the amino acid sequence according to SEQ ID NO: 9; The first LCDR consists of the amino acid sequence according to SEQ ID NO: 20; The second LCDR consists of the amino acid sequence ATS; and The third LCDR consists of the amino acid sequence according to SEQ ID NO:

22.

18. The isolated antibody or antigen-binding fragment thereof according to claim 17, wherein the isolated antibody or antigen-binding fragment thereof comprises: an extended second HCDR consisting of the amino acid sequence according to SEQ ID NO: 25; an extended first LCDR consisting of the amino acid sequence according to SEQ ID NO: 26; and An extended second LCDR consisting of the amino acid sequence according to SEQ ID NO:

28.

19. The isolated antibody or antigen-binding fragment thereof according to claim 17 or 18, wherein The HCVR comprises the amino acid sequence according to SEQ ID NO: 34; and / or The LCVR comprises the amino acid sequence according to SEQ ID NO:

35.

20. The isolated antibody or antigen-binding fragment thereof according to claim 19, wherein The HCVR consists of the amino acid sequence according to SEQ ID NO: 34; and / or The LCVR consists of the amino acid sequence according to SEQ ID NO:

35.

21. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:

36.

22. The isolated antibody or antigen-binding fragment thereof of claim 21, wherein the HCVR consists of an amino acid sequence selected from the group consisting of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and SEQ ID NO:

36.

23. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:

38.

24. The isolated antibody or antigen-binding fragment thereof of claim 23, wherein the LCVR consists of an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:

38.

25. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the isolated antibody or antigen-binding fragment thereof specifically binds bile salt-stimulated lipase (BSSL).

26. The isolated antibody or antigen-binding fragment thereof according to claim 25, wherein the isolated antibody or antigen-binding fragment thereof specifically binds to human BSSL (hBSSL).

27. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, and a chimeric antibody or antigen-binding fragment thereof.

28. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is a single chain variable fragment scFV.

29. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

30. The isolated antibody or antigen-binding fragment thereof of claim 1, having an isotype class selected from the group consisting of IgG, IgA, IgM, IgD and IgE.

31. The isolated antibody or antigen-binding fragment thereof of claim 30, which has an isotype class of IgG.

32. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the isolated antibody or antigen-binding fragment thereof comprises at least one Fc silent mutation that inhibits interaction with an Fc receptor (FcγR).

33. The isolated antibody or antigen-binding fragment thereof according to claim 32, wherein the isolated antibody or antigen-binding fragment thereof belongs to the IgG isotype class and the at least one Fc silent mutation is selected from the group consisting of L234A, L235A and P329G.

34. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the isolated antibody or antigen-binding fragment thereof comprises at least one stabilizing mutation that prevents or reduces Fab arm exchange in vivo.

35. The isolated antibody or antigen-binding fragment thereof of claim 34, wherein the isolated antibody or antigen-binding fragment thereof is of IgG4 isotype subclass and the at least one stabilizing mutation is S228P.

36. A pharmaceutical composition comprising the isolated antibody and / or antigen-binding fragment thereof according to any one of claims 1 to 35 and a pharmaceutically acceptable carrier or excipient.

37. Use of the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, or the pharmaceutical composition according to claim 36, in the preparation of a medicament for treating and / or preventing rheumatoid arthritis (RA).

38. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 35.

39. An expression vector comprising the polynucleotide according to claim 38.

40. A cell comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, the polynucleotide according to claim 38 and / or the expression vector according to claim 39.

41. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising: The cell according to claim 40 comprising the expression vector according to claim 39 is cultured under conditions where the antibody or antigen-binding fragment thereof is expressed by the cell.

42. The method of claim 41, further comprising: The antibody or antigen-binding fragment thereof is separated from the cell or the medium in which the cell is cultured.

43. A method for detecting the presence of bile salt stimulated lipase (BSSL) in a sample and / or quantifying the amount of BSSL in a sample, the method comprising: contacting the sample with an isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 35; as well as The presence or absence of BSSL in the sample is detected and / or the amount of BSSL in the sample is quantified based on the amount of isolated antibody or antigen-binding fragment thereof that binds to BSSL.

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