Novel BSSL antibodies

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 in the N-terminal portion of hBSSL, identified as comprising amino acid residues 7 to 12 and amino acid residues 42 to 55. The present invention also relates to medical uses of the antibodies and / or antigen-binding fragments thereof, particularly in the treatment of inflammatory conditions, and related pharmaceutical compositions.

Description

[Technical field]

[0001] The present invention relates to novel isolated antibodies and antigen-binding fragments thereof that bind to previously uncharacterized epitopes of bile salt stimulated lipase (BSSL) located in the N-terminal portion of the BSSL protein. The present disclosure also relates to medical uses of the antibodies and antigen-binding fragments thereof, particularly in the treatment of inflammatory conditions, and related pharmaceutical compositions. The present disclosure also discloses the use of the antibodies or antigen-binding fragments thereof as molecular tools in the detection of BSSL and / or for the diagnosis of BSSL-related diseases. [Background technology]

[0002] background Inflammatory conditions, including autoimmune and autoinflammatory diseases, continue to pose a significant threat to human health. Despite advances in the treatment of inflammatory conditions, improved therapies remain needed.

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

[0004] Some inflammatory conditions are chronic. Chronic inflammation occurs when the inflammatory response persists and puts the body on constant alert. Examples of inflammatory diseases and conditions that involve chronic inflammation include 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 treatments for RA include nonsteroidal anti-inflammatory drugs (NSAIDs) for pain treatment, disease-modifying antirheumatic drugs (DMARDs), and biologic agents that target specific inflammatory 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 begins before age 16, and the causes of JIA are largely unknown. The main focus of treatment for JIA is to help children regain normal levels of physical and social activity. Most children are treated with NSAIDs and intra-articular corticosteroid injections. The DMARD methotrexate is a powerful drug that helps suppress joint inflammation in the majority of JIA patients with polyarthritis, but has been reported to be less useful in systemic arthritis, and many children are given TNF-alpha inhibitors such as etanercept.

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

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

[0009] Bile salt-stimulated lipase (BSSL), also known as bile salt-dependent lipase (BSDL), carboxylester lipase (CEL) or bile salt-activated lipase (BAL), encoded by the CEL gene, is a lipolytic enzyme expressed in the exocrine pancreas and secreted into the intestinal lumen in all species investigated so far, where it aids in lipid digestion.

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

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

[0012] DNA encoding BSSL may 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 purification of BSSL from different tissues and transfected cell lines are known in the art [1].

[0014] Document [2] describes antigen-binding compounds that bind to BSSL or Feto-Acinar Pancreatic Protein (FAPP). The compounds are disclosed to recognize the C-terminal peptide (J28 epitope) of BSSL. 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 BSSL or FAPP polypeptides. Document [2] describes compounds that can directly target tumor cells, particularly BSSL or FAPP-expressing pancreatic tumor cells, and cause their death via apoptosis and / or stop their proliferation.

[0015] References [3, 4] describe the discovery that BSSL plays a role in inflammatory processes and that inhibiting or eliminating BSSL in animal models protects against the development of chronic arthritis. References [3, 4] disclose that BSSL protein is present in inflammatory cells and tissues and that BSSL-deficient mice are protected from the development of inflammatory diseases exemplified by 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 such as antibodies, most regimens and drugs have in common that they aim to suppress the immune system, as is the case for example with all TNFα inhibitors and corticosteroids, which in turn increases the risk of secondary infections and complications. Summary of the Invention [Problem to be solved by the invention]

[0017] As a result, there remains a significant clinical need for new selective, preferably biological, drugs for the treatment, prophylaxis and prevention of inflammatory diseases that are directed to different and / or new targets involved in inflammatory signaling and processes and that do not act primarily by suppressing the immune system and are therefore expected to have fewer and / or less severe adverse effects. [Means for solving the problem]

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

[0019] This and other objectives are met by the embodiments disclosed herein.

[0020] The invention is defined in the independent claims. Further embodiments of the invention are defined in the independent claims. Effect of the Invention

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

[0022] Another aspect of the present invention is 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 115In one embodiment, ZH1, ZH2, ZH3, and ZH4 each independently represent zero, one, or several independently selected amino acid residues, and X is an integer from 1 to 4; 53 is selected from I and M, and X 57 is selected from N and Y, and X 64 is selected from A and S, and X 68 is selected from A and N, and X 72 is selected from K and Q. Furthermore, each of ZL1, ZL2, ZL3 and ZL4 independently represents zero, one or several independently selected amino acid residues; 24 is selected from S and R, and X 27 is selected from S and P; X 39 is selected from M and L; X 57 is selected from A and T, and X 66 is selected from K and S, and X 68 is selected from A and P; X 115 is selected from S, T and Y.

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

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

[0025] A further aspect of the present invention relates to an isolated antibody or an antigen-binding fragment thereof as defined above for use as a medicament and for use in the treatment and / or prevention of an inflammatory disease, or a pharmaceutical composition as defined above.

[0026] A related aspect of the invention defines the use of the isolated antibody or antigen-binding fragment thereof as defined above, or the pharmaceutical composition as defined 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 defines a method for the treatment and / or amelioration and / or preventing and / or prophylaxis of an inflammatory disease, comprising administering to a subject in need thereof a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof as described above, or the pharmaceutical composition as described above.

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

[0029] Another aspect of the invention relates to a method for detecting the presence or absence of BSSL and / or quantifying the amount of BSSL in a sample, the method comprising contacting a sample with the isolated antibody or antigen-binding fragment thereof described above, and detecting the presence or absence 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 bound to the BSSL.

[0030] A further aspect of the present invention relates to a method for diagnosing a BSSL-associated disorder, which comprises contacting a sample from a subject with the above-described isolated antibody or antigen-binding fragment thereof, and detecting the presence or absence of BSSL and / or quantifying the amount of BSSL in the sample based on the amount of the isolated antibody or antigen-binding fragment thereof bound to the BSSL.The method also comprises concluding whether the subject suffers from a BSSL-associated disorder based on the results from the detection and / or quantification.

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

[0032] The antibodies and antigen-binding fragments thereof of the present invention bind to a previously uncharacterized epitope in hBSSL that is distinct from the active site of the enzyme. The antibodies and antigen-binding fragments thereof are therefore capable of binding to hBSSL without competing with the enzymatic activity of hBSSL. The antibodies and antigen-binding fragments thereof of the present invention are useful in the treatment and / or prevention of inflammatory conditions, and alleviate the above and other shortcomings of current therapies.

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

[0034] [Figure 1] Figure 1 shows the interaction of hBSSL with immobilized AS20 mIgG1. The sensorgrams were fitted to a 1:1 binding model. The fit is so good that the sensorgrams and the fitted line are indistinguishable. [Diagram 2] Figure 2 shows steady-state analysis of the interaction of mBSSL with immobilized AS20 mIgG1. KD is from half-maximal response automatically corrected for high bulk effects with an offset of -173 RU (from above). Bulk effects are not removed in the figure. [Diagram 3] Figure 3a shows a graph showing AS20 scFv ELISA binding to non-biotinylated and biotinylated human BSSL. The absorbance values ​​displayed (y-axis) are the average of the two. Figure 3b shows a graph showing AS20 scFv ELISA binding to mouse and human BSSL, and to an unrelated protein. The absorbance values ​​displayed (y-axis) are the average of the two. [Figure 4] 4 shows a bar graph of the results of a multiplex bead assay (LUMINEX®) analyzing the ability of AS20 scFv (left bar) to bind to human BSSL and 30 different unrelated proteins. A positive control scFv, unrelated scFv_1 (right bar), predicted to bind to bunrelated protein_1, was also included in the assay. [Diagram 5] FIG. 5 shows the results of an HTRF-based competition assay analyzing the binding of AS20 scFv to mouse and human BSSL. [Figure 6] Figure 6 is a sequence comparison of AS20, AS20 CDR graft and AS20 humanized library scaffold. * indicates an in-frame stop introduced in LCDR3 to ensure that only clones mutated in this region are displayed on phage. X indicates the position mutated in the AS20 humanized library. CDR boundaries are as defined by Kabat and residue numbering is as defined by IMGT nomenclature [5]. eHCDR2, eLCDR1 and eLCDR2 indicate extended HCDR2, LCDR1 and LCDR2 regions that include amino acid positions outside the respective CDR regions according to IMGT. [Figure 7] FIG. 7 is a graph showing size exclusion chromatography data trends at A) +40° C. and B) +4° C. as described in Example 12. [Figure 8] Figure 8 is a graph showing the mean Tm1 (circles) and Tm2 (squares) values ​​plotted for each candidate. Bars indicate standard deviation; a) +4°C b) +40°C. [Figure 9] Figure 9 shows the results of DLS analysis showing the intensity versus size of the candidates. Samples were analyzed after 30 days of storage at -80°C (hatched arrow), +4°C (dotted arrow) and +40°C (solid arrow), respectively. [Figure 10]FIG. 10 is a diagram of the structure of a BSSL with the indicated epitope regions of the pre-designated antibody candidates (aa7-12 for S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116, S-SL048-118; aa42-55 for S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116, S-SL048-118; aa84-101 for S-SL048-46; aa174-180 for S-SL048-116; aa283-295 for S-SL048-11). [Figure 11] FIG. 11 is a diagram of the S-SL048-106 svFv highlighting possible post-translational impediments. [Figure 12] Figure 12 is a schematic diagram of the t-hBSSL showing an "oven globe" view and the epitopes colored in light grey within a dashed circle around the back of the globe. Strands are indicated by arrows and helices are indicated by spirals. [Figure 13] Figure 13 shows in the left panel t-hBSSL in a surface representation in dark grey with the sequence interacting with AS20-Fab in light grey. The variable regions of the heavy and light chains are shown in ribbon representation (light chain in grey and heavy chain in black). The right panel shows the same view, but t-hBSSL is represented as "sticks" and the active site triad is highlighted in black. In the left panel the active site is hidden below the surface. [Figure 14] FIG. 14 is a table showing the amino acid sequence differences among the 38 candidate scFvs. [Figure 15] 15A and 15B summarize the design of the combinatorial scFv library for heavy chain variable regions described in Example 5. [Figure 16] 16A and 16B summarize the design of the combinatorial scFv library for the light chain variable region described in Example 5. [Figure 17]17 is a graph showing the BSSL activity assay according to Example 20. A) and B) show the results of the triglyceride hydrolysis assay, and C) and D) show the results of the cholesterol ester hydrolysis assay. Chimeric AS20 is shown in the figure as AS20. [Figure 18] Figure 18 shows the severity of arthritis. After CIA-MAB-50 injection, (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 -1 day 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 AS20 hIgG1 LALA-PG 10 mg / kg. Onset of CAIA. Two animals, one in the AS20 hIgG1 LALA-PG 10 mg / kg group and one in the isotype control group, were removed before termination (day 12) for ethical reasons (high score). These animals are included in the results up to the day of removal. Data are presented as mean ± SEM. *p<0.05, **p<0.01. [Figure 19] Figure 19 shows disease parameters graphs. CAIA disease parameters including animals iP-treated with isotype control anti-NP hIgG1 LALA-PG (90 mg / kg), AS20 hIgG1 LALA-PG (90, 30 and 10 mg / kg) every 4 days from -1 to day 15. (A) Mean CAIA score (sum of scores during the experiment divided by number of days scored). (B) Maximum CAIA score. (C) Total disease burden (AUC). (D) Percentage of inhibition. Two animals, one in the AS20 hIgG1 LALA-PG 10 mg / kg group and one in the isotype control group, were removed before termination (day 12) for ethical reasons (high score). These animals are included only in the maximum score. Data are presented as mean ± SEM. *p<0.05, **p<0.01. [Figure 20] Figure 20 shows a graph of cell subsets in total numbers. Data are presented as mean ± SD. [Figure 21] Figure 21 shows a graph of cell subsets in percentages. Data are presented as mean values ​​± SD. [Figure 22] Figure 22 shows the structure of a depicted Fab-BSSL complex. A dimeric complex of S-SL048-116Fab with light and heavy chains and BSSL. The same complex rotated 180° to the right. [Figure 23] Figure 23. Interaction of S-SL048-116 Fab with BSSL. Variable Ig domain of Fab with light and heavy chain interacting with BSSL. Two important amino acids for epitopes Arg 176 and Gln 52 are depicted as circles and sticks. [Figure 24] Figure 24 shows the severity of arthritis. Development of CAIA in mice treated with (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 after CIA-MAB-50 injection. For ethical reasons, three animals were removed before termination: two from the vehicle group (days 7 and 15) and one from the 90 mg / kg group (day 12). These animals are included in the results until the day of removal, and the animal removed on day 7 is completely excluded from the data. Data are presented as mean ± SEM. [Diagram 25] Figure 25 shows CAIA disease parameters including animals treated ip with vehicle and different doses of S-SL048-116 (SOL-116) (90, 30 and 10 mg / kg). (A) Mean CAIA score (sum of scores during the experiment divided by number of days scored). (B) Maximum CAIA score. (C) Total disease burden (AUC). (D) Percentage of inhibition. For ethical reasons, three animals were removed before termination: two from the vehicle group (days 7 and 15) and one from the 90 mg / kg group (day 12). These animals are included only in the maximum score. Data are presented as mean ± SEM. [Figure 26]Figure 26 shows the total number of leukocytes in (A) the spleen and (B) the mesenteric lymph nodes. Data are presented as mean ± SEM. **p<0.01. [Figure 27] Figure 27 shows the percentage of NK cells among CD45+ cells in (A) spleen, (B) blood and (C) mesenteric lymph nodes. Data are presented as mean ± SEM. ****p<0.001 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Detailed Description definition The term "isolated" as used in relation to an antibody, such as in the phrase "isolated antibody", means that the antibody has been removed from its original environment. An isolated antibody, as used herein, refers to an antibody that is substantially free of other antibodies having different antigen specificities, and is intended to refer to, for example, an isolated antibody that specifically binds to BSSL, particularly human BSSL (hBSSL), and 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 to 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 an antigen-binding fragment thereof may be purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), capillary electrophoresis, or chromatography, such as ion exchange chromatography or reverse-phase high performance liquid chromatography (HPLC). Those of skill in the art will understand that whenever the term "antibody or antigen-binding fragment thereof" or the like is used, an isolated antibody or antigen-binding fragment thereof is referred to herein, even if the term "isolated" is not explicitly stated.

[0036] The term "isolated humanized antibody" and the like, as used herein, refers to an isolated antibody that has been humanized.

[0037] The term "antigen-binding fragment" is intended in the present context to mean 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 substantial portion of the antigen-binding properties of the corresponding full-length antibody. An antigen-binding fragment may comprise one or more complementarity determining region (CDR) sequences of an antibody or a portion of these CDR sequences, a portion or all of the heavy chain variable region (HCVR), a portion or all of the light chain variable region (LCVR), or a combination thereof. In one embodiment, an antigen-binding fragment of an antibody may consist of a contiguous amino acid sequence of an antibody, or may be obtained or consist of different portions of the amino acid sequence of an antibody, with or without association with a linker(s). Examples of antigen-binding fragments include single chain variable fragments (scFv), Fab fragments, F(ab')2 fragments, F(ab')3 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, isolated complementarity determining regions (CDRs) and nanobodies.

[0038] "Single chain variable fragments" or "single chain variable fragments" ("scFv") are fusion proteins of the variable regions of immunoglobulin heavy and light chains linked with a short linker peptide, typically of about 10-25 amino acids. scFvs of the same or different types (having affinity for the same or different epitopes) can be combined in different ways known to those skilled in the art. Non-limiting examples of such combinations include tandem di-scFvs, diabodies, tandem tri-scFvs or tri(a)bodies.

[0039] The term "epitope" refers to a part of an antigen that is recognized by the immune system, such as by an antibody. An epitope is also called an antigenic determinant.

[0040] The term "paratope" refers to the part of an antibody that binds to an epitope.

[0041] As used herein, the terms "binds to," "has 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 to assess the binding ability of an antibody or antigen-binding fragment to a target molecule include, for example, enzyme immunoassays (EIAs), such as enzyme-linked immunosorbent assays (ELISAs), Western blots, radioimmunoassays (RIAs), surface plasmon resonance (SPR), LUMINEX® multiplex assays, and flow cytometry analysis. As exemplified in the experimental section, the binding kinetics, e.g., binding affinity, of an antibody can also be assessed by standard assays known in the art, such as by BIACORE® system analysis.

[0042] "Specifically binds," "specifically binding," and the like, mean that the molecule in question, such as an antibody or antigen-binding fragment thereof, specifically binds to a target antigen without significant binding to other molecules. The specificity of an antibody or antigen-binding fragment thereof can be determined based on affinity and / or avidity. The equilibrium constant for dissociation of an antibody or antigen-binding fragment thereof with an antigen (K D Affinity, expressed by K), is a measure of the strength of binding between an antigenic determinant, i.e., an epitope, and an antigen-binding site on an antibody or antigen-binding fragment thereof. D The lower the value of K, the stronger the binding strength between the antigenic determinant and the antibody or antigen-binding fragment thereof. Alternatively, affinity can be expressed as 1 / K D The affinity constant (K A As will be appreciated by those skilled in the art, depending on the specific antigen of interest, affinity can be determined by methods known per se.

[0043] Typically, the antibody or antigen-binding fragment thereof is -5 ~10 -12 moles / liter (M) or less, and preferably 10 -7 ~10 -12 M or less, and more preferably 10 -8 ~10 -12 The equilibrium dissociation constant of M (K D ), i.e., 10 5 ~1012 M -1 More than 10, preferably 7 ~10 12 M -1 More preferably, 10 8 ~10 12 M -1 The affinity constant (K A ) to bind to their antigens. -4 K is bigger than M D value (or 10 4 M -1 Lower K A Values ​​below 0.1 are considered to indicate non-specific binding. Preferably, the antibodies or antigen-binding fragments thereof of the embodiments bind to BSSL with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, less than 5 nM, etc.

[0044] The terms "detection," "detecting," and the like include any means of detection, including direct and indirect detection.

[0045] Herein, all amino acids within the variable regions, including the CDRs described herein, are consequently numbered according to the IMGT specific numbering system defined by Marie-Paule Lefranc [5].

[0046] The term "Kabat numbering" and the like refers to a scheme for numbering amino acid residues in an antibody based on the variable region.

[0047] As used herein, the terms "monoclonal antibody", "monoclonal antibodies" refer to an antibody / antibodies with monovalent affinity, meaning that each antibody molecule in a sample of monoclonal antibodies binds to the same epitope on the antigen. Monoclonal antibodies are made by the same immune cell that is a clone of a unique parent cell, e.g., a hybridoma cell line.

[0048] As used herein, the term "polyclonal antibody" refers to a population of antibodies reactive against a particular antigen, although in the collection there may be, for example, different antibody molecules that recognize different epitopes on the antigen. Polyclonal antibodies are typically produced by inoculation of a suitable mammal and purified from the mammal's serum.

[0049] The term "human antibody derivatives" refers to any modified form of a human antibody, for example, a conjugate of the antibody and another agent or antibody.

[0050] The term "full antibody" as used herein 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.

[0051] The term "chimeric antibody" as used herein refers to a recombinant or genetically engineered antibody, such as a murine monoclonal antibody, that contains a polypeptide or domain from a different species, e.g., human, that is introduced to reduce the immunogenicity of the antibody.

[0052] As used herein, the term "at least one" should be interpreted as one or more.

[0053] As will be appreciated by those of skill in the art, reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter per se. For example, a reference to "about X" includes a description of "X."

[0054] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymer 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.

[0055] As used herein, a "host cell" includes an individual cell or cell culture that can be or has been the recipient of any of the vectors herein. A host cell includes the progeny of a single host cell, which progeny may not necessarily be completely identical in morphology or total DNA complement to the original parent cell due to natural, accidental, or deliberate mutations and / or changes. A host cell includes cells that have been transfected or infected with a vector containing a nucleic acid herein. A host cell can be a prokaryotic or eukaryotic cell.

[0056] The term "isolated" as used in reference to polynucleotides, polypeptides, and the like, means that the molecule or polypeptide has been removed from its original environment.

[0057] As used herein, the term "% identity" or "% identical" may be determined using methods well known in the art. For example, % identity is calculated as follows: A query sequence is aligned to a target sequence using the CLUSTAL W algorithm [6]. A comparison is made over a window corresponding to the shortest of the aligned sequences. In some cases, the shortest of the aligned sequences may be the target sequence. In other cases, the query sequence may constitute the shortest of the aligned sequences. The amino acid residues or nucleotides at each position are compared, and the percentage of positions in the query sequence that have identical counterparts in the target sequence is reported as % identity.

[0058] 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.

[0059] Aspects and embodiments described herein include aspects and embodiments "consisting of" and / or "consisting essentially of." As used herein, the singular forms "a," "an," and "the" include plural references unless specifically indicated.

[0060] "hIgG1 LALA-PG" described in [7] "hIgG4 S228P, hIgG4 S241 P" described in [8] "G-SP140-8", SP140-binding clone, negative control "expiHEK293 cells", human cells derived from the 293 cell line, and the core component of the Expi293™ Expression System

[0061] It is an object of the present invention to provide antibodies and / or antigen-binding fragments thereof that are useful in the treatment and / or prevention of inflammatory conditions and that alleviate the above-mentioned and other shortcomings of current therapies. It is a further object of the present disclosure to provide agents for use in the diagnosis of inflammatory conditions and in the study of the Bile Salt Stimulated Lipase (BSSL) protein.

[0062] More particularly, 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 specification also relates to medical uses of the antibodies and antigen-binding fragments thereof, particularly in the treatment of inflammatory conditions, and related pharmaceutical compositions. The present specification also discloses the use of the antibodies or antigen-binding fragments thereof as molecular tools for the detection of BSSL and / or the diagnosis of BSSL-related diseases.

[0063] In one embodiment, whenever BSSL is referred to, it also includes human BSSL (hBSSL), unless the context makes clear that hBSSL is not intended to be included.

[0064] The present disclosure describes a novel group of antibodies against BSSL, including antigen-binding fragments thereof, that bind to previously unrecognized epitopes on hBSSL. The antibodies may be humanized or have their CDR sequences grafted onto a non-human scaffold. The antibodies or antigen-binding fragments thereof may also bind to mouse or murine BSSL (mBSSL), but the affinity for hBSSL and mBSSL may differ due to an amino acid difference(s) in one of the epitopes to which the antibody and / or antigen-binding fragment thereof binds.

[0065] As is well known, an antibody is an immunoglobulin molecule that can specifically bind to a target (antigen), such as a carbohydrate, polynucleotide, lipid, polypeptide, or other, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. An antibody is a glycoprotein that comprises 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 antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to various cells of the immune system, such as effector cells, and to host tissues or factors, including the first component of the classical complement system, i.e., complement component 1q (C1q).

[0066] The antibodies or antigen-binding fragments thereof disclosed herein can be used to inhibit or reduce at least some biological activity of the BSSL protein when bound thereto. This binding can, for example, significantly or completely inhibit some biological activity of the BSSL protein. These effects of the antibodies or antigen-binding fragments thereof of the present invention were very surprising, given that the antibodies or antigen-binding fragments thereof do not bind to the active site of BSSL. Therefore, it is preferred that the antibodies or antigen-binding fragments thereof of the present invention do not significantly inhibit or reduce the enzymatic activity of BSSL, e.g., do not significantly inhibit or reduce the ability of BSSL to hydrolyze cholesterol esters (EC 3.1.1.13).

[0067] The antibody or antigen-binding fragment thereof can be used to reduce the pro-inflammatory effects of BSSL in a subject in need thereof. Therefore, the antibody or antigen-binding fragment thereof can be further used in the treatment and / or prevention of various inflammatory diseases as described herein. These medical uses of the antibody or antigen-binding fragment thereof of the present invention can be achieved without blocking the enzymatic activity of BSSL. Thus, the antibody or antigen-binding fragment thereof of the present invention does not contribute to the negative effects caused by the inhibition of the enzymatic activity of BSSL that other anti-BSSL antibodies that bind to or associate with the active site of BSSL may have.

[0068] The antibodies or antigen-binding fragments thereof disclosed herein may also be used for diagnostic purposes to diagnose BSSL-associated conditions, such as BSSL-associated inflammatory conditions.

[0069] As demonstrated in the experimental section, several approaches were used in attempts to develop humanized BSSL antibodies or antigen-binding fragments thereof, but despite an initial number of scFv candidates of about 1,000,000, a surprisingly small number were found to show 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 finally to 5 candidates that were 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 thereof has been a major challenge, which has been overcome as described herein to achieve humanized antibodies or antigen-binding fragments thereof that still show sufficient binding affinity to hBSSL. These antibodies or antigen-binding fragments thereof of the present invention share common structural and functional characteristics described elsewhere herein.

[0070] Generation of antibodies and antigen-binding fragments The antibodies and antigen-binding fragments thereof of the present invention were produced in a multi-step process with the aim of finding antibodies and antigen-binding fragments thereof that have sufficiently good binding affinity to hBSSL. Another aim was 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.

[0071] Antibodies and antigen-binding fragments thereof were generated based on the sequence of a non-human monoclonal mBSSL antibody: DNA encoding the heavy and light immunoglobulin chains was obtained from a non-human hybridoma expressing this antibody and engineered to contain non-mouse, e.g., human, immunoglobulin sequences using standard molecular biology techniques.

[0072] However, it was a surprising discovery that the CDR grafts constructed as disclosed in Example 6 did not have as high a binding affinity as would be 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, further modifications to the antibody framework (FW) had to be made by introducing mutations in both the CDR and adjacent FW regions, as disclosed in Example 5. This was achieved by using phage display to construct a humanized library that was used for the selection and isolation of scFv fragments that bind to mouse and human BSSL. Such phage display methods for isolating human antibodies are established in the art, see, e.g., U.S. Patent Nos. 5,223,409; 5,403,484; 5,571,698; 5,427,908; 5,580,717; 5,969,108; 6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6,593,081.

[0073] The resulting isolated antibodies and antigen-binding fragments thereof had a minimal animal-derived CDR content, allowing only essential non-human germline residues. The remaining CDRs were converted to human v-gene sequences, except for some novel mutations due to the introduction of species-neutral essential de novo residues such as 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.

[0074] In humanized antibodies, the constant and variable regions, except for the CDR sequences, have frameworks 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, which are grafted onto human framework sequences. Such humanized antibodies may also refer to CDR grafting in the context of the present invention. The advantage of using humanized antibodies is that they reduce the risk of immunogenic reactions that may occur when the antibody is injected into a human subject, when a framework from another species is used. This allows them to be used for medical applications in humans. The antibodies or antigen-binding fragments thereof disclosed herein are useful for diagnostic applications and for the detection of BSSL proteins, such as hBSSL, in different types of samples, as disclosed in more detail elsewhere herein.

[0075] The present specification therefore discloses a method for producing an isolated antibody or antigen-binding fragment thereof according to the invention. The method comprises culturing a host cell expressing 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 under conditions permissive for expression of the antibody or antigen-binding fragment thereof. The method also comprises isolating the antibody or antigen-binding fragment thereof from the host cell or from the medium in which the host cell is cultured.

[0076] Epitope binding of antibodies or antigen-binding fragments thereof The isolated antibody or antigen-binding fragment thereof has been found to bind to a previously unrecognized epitope of human BSSL protein located in the N-terminal portion of the BSSL protein, which epitope may form a conformational epitope of BSSL.

[0077] It was a surprising discovery that the antibody and its antigen-binding fragments made according to the present invention bind to a previously unrecognized epitope of hBSSL protein.More surprisingly, it was found that the epitope is not located near the active site of BSSL for lipid metabolism, but rather in the N-terminal part of BSSL.This has several advantages.One is that the antibody or its antigen-binding fragment does not significantly affect the enzyme lipase activity of BSSL, so it is less likely to cause negative side effects.Another advantage is that the antibody or its antigen-binding fragment is suitable for studying BSSL protein and its lipase activity, since the BSSL protein is not significantly affected by the antibody or its antigen-binding fragment of the present invention.

[0078] The BSSL structure has been described as having a large core region consisting of 11 stranded beta sheets twisted and surrounded by alpha helices and connecting loops ([9], Figure 12). At the N-terminus there are three smaller stranded beta sheets. The structure has been likened to a left oven glove with the palm containing the active site triad near the "thumb". Similarly, a small N-terminal beta sheet is located at 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 beta sheet and the C-terminal part of the alpha C, the third alpha helix in the structure. See Figure 13. In other words, the binding region of the antibody is not near the active site of the BSSL but on the opposite side of the BSSL.

[0079] The currently identified epitope region includes residues 7-12 (strands 1 and 2) and 42-55 (loop region leading to strand 3 of the sheet). The epitope is rather flat, with only a few characteristic residues protruding, namely Tyr7, Phe12 and Gln52 (major interactions listed in Table 25). The loop region 47-54 forms a well-defined and uniform surface. Proline 47 is important for stacking interactions with Tyr31 of the antibody, but overall the surface is flat. In a preferred embodiment, the epitope region also includes residues 174-180 (C-terminal of alpha C).

[0080] An embodiment of the 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 or defined by an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence having at least 80%, such as at least 83%, identity with SEQ ID NO: 1. The epitope also comprises a second surface comprising or defined by an amino acid sequence according to SEQ ID NO: 2 or an amino acid sequence having at least 80%, such as at least 85% or at least 92% identity with SEQ ID NO: 2.

[0081] A first peptide comprising the amino acid sequence of SEQ ID NO:1 defines a first surface of an epitope within the BSSL, and a second peptide comprising the amino acid sequence of SEQ ID NO:2 defines a second surface of an epitope in the BSSL. Thus, the first surface comprises, or is rather defined by, the amino acid sequence of SEQ ID NO:1, and the second surface comprises, or is rather defined by, the amino acid sequence of SEQ ID NO:2.

[0082] In one embodiment, the first peptide comprises an amino acid sequence according to SEQ ID NO: 3 or an amino acid sequence having at least 80%, preferably at least 83%, and more preferably at least 91% identity with SEQ ID NO: 3. SEQ ID NO: 3 is a longer amino acid sequence which includes, in its part, the amino acid sequence according to SEQ ID NO: 1.

[0083] The isolated antibody or antigen-binding fragment thereof may further specifically bind to another peptide and surface of a BSSL, such as hBSSL, i.e., a third peptide and a third surface. In one embodiment, the third peptide comprises an amino acid sequence according to SEQ ID NO:5 or an amino acid sequence having at least 80%, preferably at least 85%, identity with SEQ ID NO:5. In another embodiment, the third peptide comprises an amino acid sequence according to SEQ ID NO:4 or an amino acid sequence having at least 80%, preferably at least 83%, more preferably at least 88%, such as at least 94%, identity with SEQ ID NO:4. In a further embodiment, the third peptide comprises an amino acid sequence according to SEQ ID NO:6 or an amino acid sequence having at least 80%, preferably at least 84%, more preferably at least 92% identity with SEQ ID NO:6.

[0084] In one embodiment, an isolated antibody or antigen-binding fragment thereof may specifically bind to a first peptide comprising, e.g., consisting of, SEQ ID NO:1, a second peptide comprising, e.g., consisting of, SEQ ID NO:2, and a third peptide comprising, e.g., consisting of, SEQ ID NO:4, or an amino acid sequence having a respective identity thereto as defined herein. Alternatively, instead of binding to a shorter amino acid sequence according to SEQ ID NO:1, such an antibody or antigen-binding fragment thereof may bind to a longer amino acid sequence according to SEQ ID NO:3, or an amino acid sequence having an identity thereto as specified herein.

[0085] In another embodiment, the isolated antibody or antigen-binding fragment thereof specifically binds to an amino acid sequence having a first peptide comprising, e.g., consisting of, SEQ ID NO:1, a second peptide comprising, e.g., consisting of, SEQ ID NO:2, and a third peptide comprising, e.g., consisting of, SEQ ID NO:5, or their respective identities as defined herein. Alternatively, instead of binding to a shorter amino acid sequence according to SEQ ID NO:1, such an antibody or antigen-binding fragment thereof may specifically bind to a longer amino acid sequence according to SEQ ID NO:3, or an amino acid sequence having an identity thereto as specified herein.

[0086] In further embodiments, the isolated antibody or antigen-binding fragment thereof specifically binds to an amino acid sequence comprising, e.g., a first peptide consisting of, e.g., SEQ ID NO:1, a second peptide consisting of, e.g., SEQ ID NO:2, and a third peptide consisting of, e.g., SEQ ID NO:6, or a respective specified identity thereto as defined herein. Alternatively, instead of binding to a shorter amino acid sequence according to SEQ ID NO:1, such an antibody or antigen-binding fragment thereof may bind to a longer amino acid sequence according to SEQ ID NO:3, or an amino acid sequence having a specified identity thereto as defined herein.

[0087] Another aspect of the invention is directed to a BSSL epitope, such as 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 having at least 80%, preferably at least 83%, identity to SEQ ID NO: 1. BSSL epitopes also include a second peptide comprising, e.g., consisting of, an amino acid sequence according to SEQ ID NO: 2, or an amino acid sequence having at least 80%, preferably at least 85% or at least 92% identity to SEQ ID NO: 2.

[0088] 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 identity thereto as defined herein.

[0089] The epitope may further comprise an amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence having identity thereto as defined herein, an amino acid sequence according to SEQ ID NO: 5, or an amino acid sequence having identity thereto as defined herein, or an amino acid sequence according to SEQ ID NO: 6, or an amino acid sequence having identity thereto as defined herein.

[0090] Such epitopes may be useful, for example, for the development of antibodies or antigen-binding fragments thereof that bind to BSSL proteins, such as hBSSL, for use in the treatment and / or prevention of BSSL-associated pathologies and / or for use as molecular tools to study BSSL proteins. The present invention is therefore also directed to the use of such epitopes for the development of antibodies or antigen-binding fragments thereof. As these epitopes are not located in the lipase catalytic center of the BSSL protein, it is an attractive goal to develop anti-BSSL antibodies or antigen-binding fragments thereto.

[0091] An isolated antibody or antigen-binding fragment thereof according to the invention may specifically bind to an epitope(s) defined herein.

[0092] Antigen-binding portions of antibodies and antigen-binding fragments thereof A full-length antibody comprises two heavy chains and two light chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (HVCR) and first, second and third constant regions (C H 1. C H 2 and C H 3). In this disclosure, the term V H Each light chain comprises a light chain variable region (LVCR) and a light chain constant region (CCR). L In this disclosure, the term V L , VL and LCVR are used interchangeably.

[0093] The HCVR and LCVR regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FR or FW). Each HCVR and LCVR is composed of three CDRs and four FR / FWs arranged from N-terminus to C-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4.

[0094] Extended CDR (eCDR) as used herein relates to an amino acid sequence which comprises at least one additional amino acid residue beyond that of the CDR defined according to the IMGT nomenclature.

[0095] 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 Fv region of an antibody and contains parts of the antibody's heavy and light chains. Each arm of the Y-shape of the antibody monomer is terminated by a paratope, a set of six CDRs. The paratope consists of three light chain CDRs (LCDRs) and three heavy chain CDRs (HCDRs) that extend from an antiparallel beta sheet fold.

[0096] In the following sections, the isolated antibody or antigen-binding fragment thereof of the present invention is defined by the structural features of its CDRs, in other words, the amino acid sequences of its HCDRs and / or LCDRs, or the amino acid structures of the regions containing HCDRs and / or LCDRs. A person skilled in the art will understand that minor changes such as substitutions of one, two, three, four or more amino acid residues in the amino acid sequence may occur without affecting the functional properties of the isolated antibody or antigen-binding fragment thereof, such as its binding ability or binding affinity to a BSSL, such as hBSSL. It is understood that the first HCDR, second HCDR, third HCDR, first LCDR, second LCDR and third LCDR may be independently selected from the recited amino acid sequences.

[0097] One aspect of the invention thus relates to an isolated antibody or antigen-binding fragment thereof comprising the three CDRs of HCVR (HCDR) and the three CDRs of LCHV (LCDR), in which the first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7 or an amino acid sequence having at least 87%, such as at least 87.5%, identity with 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 having at least 75% identity with 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 having at least 83%, such as at least 91.6%, identity with SEQ ID NO: 9. Further, in this embodiment, the first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 10 or an amino acid sequence having at least 80% identity with SEQ ID NO: 10, the second LCDR comprises, preferably consists of, an amino acid sequence ATS such as AAS or an amino acid sequence having at least 66% identity with the amino acid sequence ATS, and the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 11 or an amino acid sequence having at least 87% identity with SEQ ID NO: 11.

[0098] The experimental data shown in Example 22 indicates that the second LCDR may not be as important for the interaction with BSSL.Thus, in embodiments, an 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 having at least 87%, such as at least 87.5%, identity with 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 having at least 75% identity with 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 having at least 83%, such as at least 91.6%, identity with 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 having at least 80% identity with 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 having at least 87% identity with SEQ ID NO:11.

[0099] 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 one 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.

[0100] 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 according to SEQ ID NO: 8 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 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, an amino acid sequence according to SEQ ID NO: 11.

[0101] In a particular embodiment, the isolated antibody or antigen-binding fragment thereof comprises an extended second HCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 12, an extended first LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 14, and an extended second LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 15.

[0102] In another specific embodiment, the isolated antibody or antigen-binding fragment thereof comprises an extended second HCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 12, an extended first LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 16, and an extended second LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 17.

[0103] 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 according to SEQ ID NO: 18 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 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, an amino acid sequence according to SEQ ID NO: 21.

[0104] In a particular embodiment, the isolated antibody or antigen-binding fragment thereof comprises an extended second HCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 23, an extended first LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 16, and an extended second LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 15.

[0105] 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 according to SEQ ID NO: 8 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 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, an amino acid sequence according to SEQ ID NO: 11.

[0106] In a particular embodiment, the isolated antibody or antigen-binding fragment thereof comprises an extended second HCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 24, an extended first LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 27, and an extended second LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 29.

[0107] 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 according to 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 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, an amino acid sequence according to SEQ ID NO: 22.

[0108] In a particular embodiment, the isolated antibody or antigen-binding fragment thereof comprises an extended second HCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 25, an extended first LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 26, and an extended second LCDR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO: 28.

[0109] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises a first HCDR which comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:7, a second HCDR which comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs:12, 23, 24 and 25, and a third HCDR which comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:9. In this embodiment, the isolated antibody or antigen-binding fragment thereof, an extended first LCDR which comprises, preferably consists 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 which comprises, preferably consists 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 which 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.

[0110] In one embodiment, when the first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:7, it also comprises an amino acid sequence which is at least 87.5% identical to SEQ ID NO:7.

[0111] In one embodiment, when the second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:8, it also comprises an amino acid sequence which is at least 75%, such as at least 87%, or at least 87.5% identical to SEQ ID NO:8.

[0112] In one embodiment, when the third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:9, it also comprises an amino acid sequence which is at least 83%, such as at least 91.6%, identical to SEQ ID NO:9.

[0113] In one embodiment, when the first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:10, it also comprises an amino acid sequence which is at least 80% identical to SEQ ID NO:10.

[0114] In one embodiment, when the second LCDR comprises, preferably consists of, the amino acid sequence ATS or AAS, it also comprises an amino acid sequence which is at least 66% identical to any one of the amino acid sequences ATS and AAS.

[0115] In one embodiment, when the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:11, it also comprises an amino acid sequence which is at least 87.5% identical to SEQ ID NO:11.

[0116] In one embodiment, where the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:21, it also comprises an amino acid sequence which is at least 75%, such as at least 87.5%, identical to SEQ ID NO:21.

[0117] In one embodiment, where the third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:22, it also comprises an amino acid sequence which is at least 75%, such as at least 87.5%, identical to SEQ ID NO:22.

[0118] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 12, it also comprises an amino acid sequence which is at least 77.8%, such as at least 83%, for example at least 83.3%, such as at least 88%, for example at least 88.9%, such as at least 94%, for example at least 94.4% identical to SEQ ID NO: 12.

[0119] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 18, it also comprises an amino acid sequence which is at least 75%, such as 87.5%, identical to SEQ ID NO: 18.

[0120] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 19, it also comprises an amino acid sequence which is at least 75%, such as 87.5%, identical to SEQ ID NO: 19.

[0121] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 23, it also comprises an amino acid sequence which is at least 77.8%, such as at least 83%, for example at least 83.3%, such as at least 88%, for example at least 88.9%, such as at least 94%, for example at least 94.4% identical to SEQ ID NO: 23.

[0122] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:24, it also comprises an amino acid sequence which is at least 77.8%, such as at least 83%, for example at least 83.3%, such as at least 88%, for example at least 88.9%, such as at least 94%, for example at least 94.4% identical to SEQ ID NO:24.

[0123] In one embodiment, when the extended second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:25, it also comprises an amino acid sequence which is at least 77.8%, such as at least 83%, for example at least 83.3%, such as at least 88%, for example at least 88.9%, such as at least 94%, for example at least 94.4% identical to SEQ ID NO:25.

[0124] In one embodiment, when the extended first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:14, it also comprises an amino acid sequence that is at least 80%, for example at least 90%, identical to SEQ ID NO:14.

[0125] In one embodiment, when the extended first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:16, it also comprises an amino acid sequence that is at least 80%, for example at least 90%, identical to SEQ ID NO:16.

[0126] In one embodiment, when the extended first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:20, it also comprises an amino acid sequence which is at least 80% identical to SEQ ID NO:20.

[0127] In one embodiment, when the extended first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:26, it also comprises an amino acid sequence that is at least 80%, for example at least 90%, identical to SEQ ID NO:26.

[0128] In one embodiment, when the extended first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:27, it also comprises an amino acid sequence that is at least 80%, such as at least 90%, identical to SEQ ID NO:27.

[0129] In one embodiment, when the extended second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 15, it also comprises an amino acid sequence that is at least 66.7%, such as at least 83%, for example at least 83.3% identical to SEQ ID NO: 15.

[0130] In one embodiment, when the extended second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 17, it also comprises an amino acid sequence that is at least 66.7%, such as at least 83%, for example at least 83.3% identical to SEQ ID NO: 17.

[0131] In one embodiment, when the extended second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:28, it also comprises an amino acid sequence that is at least 66.7%, such as at least 83%, for example at least 83.3% identical to SEQ ID NO:28.

[0132] In one embodiment, when the extended second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:29, it also comprises an amino acid sequence that is at least 66.7%, such as at least 83%, for example at least 83.3% identical to SEQ ID NO:29.

[0133] The isolated antibodies or antigen-binding fragments thereof disclosed herein may also, or alternatively, be structurally described by the amino acid sequences of their HCVRs and / or LCVRs. A person skilled in the art will understand that the HCVRs and LCVRs may be independently selected from the recited amino acid sequences. As mentioned above, a person skilled in the art will understand that minor changes, such as substitutions including amino acid deletions or additions of one, two, three, four or more amino acid residues in the amino acid sequence, may occur without affecting the functional properties of the isolated antibodies or antigen-binding fragments thereof, such as their ability to bind to hBSSL. The changes may occur in the amino acid sequences of the CDRs, in the amino acid sequences outside the CDR regions, referred to herein as framework regions, or both in the amino acid sequences of the CDRs and in the amino acid sequences outside the CDR regions of the HCVRs or LCVRs.

[0134] Thus, in one embodiment, the antibody or antigen-binding fragment thereof comprises a HCVR comprising, preferably consisting of, an amino acid sequence consisting of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and SEQ ID NO:36, and an amino acid sequence having at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to any one of SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and SEQ ID NO:36.

[0135] In certain embodiments, the amino acid sequence of the 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 particular embodiment, the amino acid sequence of the 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, the HCVR may comprise an 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.

[0136] In one embodiment, the antibody or antigen-binding fragment thereof comprises an 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.

[0137] In certain embodiments, the amino acid sequence of the 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 particular embodiment, the amino acid sequence of the 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, 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, an 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.

[0138] In one embodiment, the antibody or antigen-binding fragment thereof comprises an 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 also comprises an 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.

[0139] In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO:36, and a LCVR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO:37.

[0140] In another specific embodiment, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO:36, and a LCVR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO:38.

[0141] In a further embodiment, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO:30, and a LCVR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO:31.

[0142] In yet another embodiment, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO:32, and a LCVR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO:33.

[0143] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO:34, and a LCVR comprising, preferably consisting of, an amino acid sequence according to SEQ ID NO:35.

[0144] An embodiment of the present invention is 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), for example, an amino acid sequence having 93% or more, such as 94% or more, for example 95% or more, such as 96% or more, for example 97% or more, such as 98% or more, for example 99% or more identity to the sequence defined in i), and 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 having at least 87% identity to the sequence defined in ii), for example 88% or more, such as 89% or more, for example 90% or more, such as 91% or more, for example 92% or more, such as 93% or more, for example 94% or more, such as 95% or more, for example 96% or more, such as 97% or more, for example 98% or more, such as 99% or more identity to the sequence defined in ii).

[0145] In this embodiment, 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 is selected from I and M, and X 57 is selected from N and Y, and X 64 is selected from A and S, and X 68 is selected from A and N, and X 72 is selected from K and Q, and X 24 is selected from S and R, and X 27 is selected from S and P, and X 39 is selected from M and L, and X 57 is selected from A and T, and X 66 is selected from K and S, and X 68 is selected from A and P, and X 115is selected from S, T and Y. The numbering of amino acid residues is according to the IMTG numbering standard, i.e., at position "X n ", n is an integer indicating the position of amino acid residue X according to the IMGT numbering system.

[0146] GYTFTSYN is represented by SEQ ID NO: 7; 53 GVIX 57 PGDGX 64 TSYX 68 QKFX 72 is represented by SEQ ID NO: 169, ARDYYGSSPLGY is represented by SEQ ID NO: 9, and X 24 ASX 27 SISYX 39 N is represented by SEQ ID NO: 170, and AX 57 SX 66 LX 68 is represented by SEQ ID NO: 171 and HQRSSX 115 PT is shown in SEQ ID NO:172.

[0147] Provided herein is an antibody or antigen-binding fragment thereof, comprising in sequence i) X n are independently selected from the group of possible residues listed in List A below. n may be selected from any one of the listed groups of possible residues, and this selection may be selected from any one of X, where n is not m. m It will be appreciated that the present invention is independent of the choice of amino acid at position X. n Any of the possible residues listed in can be combined independently with any of the possible residues listed at any other various positions according to List A.

[0148] List A: A list of possible amino acid residues in sequence i) X 53 can be I; X 53 may be M; X 57 can be N; X 57 can be Y; X59 can be G; X 59 can be S; X 64 can be A; X 64 can be S; X 68 may be selected from A and T; X 68 may be selected from A and N; X 68 may be selected from T and N; X 68 can be A; X 68 can be N; X 68 can be T; X 72 can be K; and X 72 can also be Q.

[0149] In a similar manner, there is provided herein an antibody or antigen-binding fragment, comprising in sequence ii) X k are independently selected from the group of possible residues according to the following list of List B. k may be selected from any one of the enumerated groups of possible residues, and this selection is l It will be appreciated that the present invention is independent of the choice of amino acid in position X of list A. k Any of the possible residues listed in can be combined independently with any of the possible residues listed at any other various positions according to List B. List B: A list of possible amino acid residues in sequence ii) X 24 can be S; X 24 may be R; X 27 can be S; X 27 can be P; X 39 may be M; X 39 can be L; X40 can be H; X 40 can be N; X 57 can be A; X 57 can be T; X 66 may be selected from K and S; X 66 may be selected from R and S; X 66 may be selected from R and K; X 66 can be K; X 66 may be R; X 66 can be S; X 68 may be selected from A and P; X 68 may be selected from A and Q; X 68 may be selected from P and Q; X 68 can be A; X 68 can be P; X 68 can be Q; X 105 can be H; X 105 can be Q; X 115 may be selected from S and T; X 115 may be selected from S and Y; X 115 may be selected from T and Y; X 115 can be S; X 115 can be Y; and X 115 can also be T.

[0150] For clarity, the selection of an amino acid residue at an amino acid position in sequence i) from List A is independent of the selection of an amino acid residue at an 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 in accordance with the present disclosure, and the listed examples may be freely combined.

[0151] As defined above, each of ZH1, ZH2, ZH3 and ZH4 may represent zero, one or several independently selected amino acid residues. The identity and number of amino acid residues in each of ZH1, ZH2, ZH3 and ZH4 may be independently selected.

[0152] Similarly, each of ZL1, ZL2, ZL3 and ZL4 may 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 may be independently selected. Furthermore, ZL1 may be linked to ZH1 or ZH4 via an amino acid linker or other linker. Also, ZL4 may 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) may be part of one amino acid sequence, in other words, part of one polypeptide.

[0153] 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%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99% identical to SEQ ID NO: 39.

[0154] In one embodiment, ZH2 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:40, or an amino acid sequence that is at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99% identical to SEQ ID NO:40.

[0155] 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%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99% identical to SEQ ID NO: 41.

[0156] In one embodiment, ZH4 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 42, or an amino acid sequence that is at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99% identical to SEQ ID NO: 42.

[0157] 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%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99% identical to SEQ ID NO: 43.

[0158] 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%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99% identical to SEQ ID NO: 44.

[0159] 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%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99% identical to SEQ ID NO:45.

[0160] 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%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99% identical to SEQ ID NO:46.

[0161] Those skilled in the art will understand that the percent identity of each of ZH1, ZH2, ZH3, ZH4, ZL1, ZL2, ZL3 and ZL4 with the amino acid sequence according to their respective SEQ ID NOs listed above is independent of any other percent identity of ZH1, ZH2, ZH3, ZH4, ZL1, ZL2, ZL3 and ZL4 with its respective SEQ ID NOs. Thus, for example, ZH1 may exhibit 95% identity with SEQ ID NO:39 and ZH2 may exhibit 99% identity with SEQ ID NO:40.

[0162] During the humanization process resulting in the novel anti-BSSL antibodies or antigen-binding fragments thereof presented herein, several mutations have been introduced in both the CDRs and the adjacent FW regions. For the purposes of this disclosure, each antibody HCVR and LCVR is composed of three CDRs, one, two or three of which may be an extended CDRS (eCDRS), and four FR / FWs, arranged from N-terminus to C-terminus in the following order as defined in Table 1:

[0163] Table 1 - IMGT Numbering [Table 1]

[0164] The three CDRs of HCRV are flanked by framework regions which may be the regions ZH1, ZH2, ZH3 and ZH4 described above. The three CDRs of LCRV are flanked by framework regions which may be the regions ZL1, ZL2, ZL3 and ZL4 described above.

[0165] The HVCR of the antibody or antigen-binding fragment thereof constructed herein is recited in the amino acid sequence within the group consisting of SEQ ID NOs: 30, 32, 34, 36, and 47 to 84. Correspondingly, the LVCR of the antibody or antigen-binding fragment thereof constructed herein is recited in the amino acid sequence within the group consisting of SEQ ID NOs: 31, 33, 35, 37, 38, and 86 to 123. Specific examples of these antibodies or antigen-binding fragments thereof include the following combinations of 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; The sequences include, or preferably consist of, sequence numbers 61 and 95; SEQ ID NOs: 62 and 96; SEQ ID NOs: 63 and 97; SEQ ID NOs: 64 and 98; SEQ ID NOs: 65 and 99; SEQ ID NOs: 66 and 100; SEQ ID NOs: 67 and 101; SEQ ID NOs: 68 and 102; SEQ ID NOs: 69 and 103; SEQ ID NOs: 70 and 104; SEQ ID NOs: 71 and 105; SEQ ID NOs: 72 and 106; SEQ ID NOs: 73 and 107; SEQ ID NOs: 74 and 108; SEQ ID NOs: 85 and 109; SEQ ID NOs: 86 and 110; SEQ ID NOs: 77 and 111; SEQ ID NOs: 78 and 112; SEQ ID NOs: 79 and 113 or SEQ ID NOs: 80 and SEQ ID NO: 114.

[0166] Those skilled in the art will appreciate that various modifications and / or additions can be made to the antibodies or antigen-binding fragments thereof disclosed herein to tailor the antibodies or antigen-binding fragments thereof to specific applications without departing from the scope of the present disclosure. For example, the antibodies or antigen-binding fragments thereof can have an amino acid sequence that is extended with and / or includes additional amino acids at the C-terminus and / or N-terminus, e.g., at the C-terminus and / or N-terminus of the heavy or light chain. Thus, the antibodies or antigen-binding fragments thereof may include any suitable number of additional amino acid residues, e.g., at least one additional amino acid residue. Each additional amino acid residue may be added individually or collectively, for example, to improve and / or simplify the production, purification, in vivo or in vitro stabilization, coupling, or detection of the polypeptide. Such additional amino acid residues may include one or more amino acid residues added for chemical coupling purposes. One example is the addition of a cysteine ​​residue. The additional amino acid residues may also provide a "tag" for purification or detection of the antibody or antigen-binding fragment thereof, such as a His6 tag, a (HisGlu)3 tag, a "myc" (c-myc) tag, or a FLAG tag.

[0167] The isolated antibodies or antigen-binding fragments thereof of the present invention may be selected from, but are not limited to, full length antibodies, combinations of CDR sequences, single chain variable fragments, Fab fragments, F(ab')2 fragments, F(ab')3 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, isolated complementarity determining regions (CDRs), and nanobodies.

[0168] 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.

[0169] For example, it may be desirable to reduce or eliminate effector functions by an antibody or its antigen-binding fragment, to prevent target cell death or undesired cytokine secretion. This may be particularly appropriate when the antibody or its antigen-binding fragment is intended to engage a cell surface receptor to prevent receptor-ligand interaction, i.e., an antagonist. Other examples where reduced effector functions may be warranted include preventing an antibody-drug conjugate from interacting with Fc receptors (FcγR), which results in off-target cytotoxicity.

[0170] Thus, in one embodiment, the isolated antibody or antigen-binding fragment thereof comprises at least one Fc silencing 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, more preferably all three of the Fc silencing mutations L234A, L235A and P329G.

[0171] Also, antibodies of IgG4 isotype or antigen-binding fragments thereof are considered as potential candidates for immunotherapy when reduced effector function is desired. IgG4 antibodies are known to be dynamic molecules that can undergo a process known as Fab arm exchange (FAE), and without being bound by theory, it is believed that this results in functionally monovalent bispecific antibodies (bsAbs) with unknown specificity, thus potentially reducing therapeutic efficacy. This may result in undesirable pharmacodynamic unpredictability for human immunotherapy.

[0172] 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, the art suggests that a single amino acid mutation (S228P) in the IgG4 core hinge region is sufficient to prevent FAE in vivo [8]. In a particular embodiment, the isolated antibody or antigen-binding fragment thereof is of the IgG4 isotype subclass, and the at least one stabilizing mutation is S228P.

[0173] 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 particular embodiment, the isotype class is IgG. For example, the isolated antibody or antigen-binding fragment thereof may be selected from the group consisting of isotype subclasses IgG1 and IgG4.

[0174] 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.

[0175] Presently 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).

[0176] The stability of these five candidates was assessed by size exclusion chromatography (SEC), SDS-PAGE, nano-differential scanning fluorimetry (nano-DSF), and differential light scattering. Combining these data into an overall ranking, we concluded that the most stable candidates in the hIgG4 S228P format were candidates 106, 118, and 116.

[0177] binding affinity In one embodiment, the isolated antibody or antigen-binding fragment thereof according to the present invention has a K D 1×10 -7 M or less, preferably K D 1×10 -8For example, the isolated antibody or antigen-binding fragment thereof may have an affinity for hBSSL of less than or equal to K D K below 3 nM D It may have an affinity for hBSSL of 5 nM or less.

[0178] As shown in the experimental section, the isolated antibody or antigen-binding fragment thereof has a K D K of 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7 or less, or 0.6 or less D In one example, an isolated antibody or antigen-binding fragment thereof that binds to hBSSL according to the present disclosure may have an affinity for hBSSL of K D K of 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 D It has an affinity for hBSSL of 0.6 to 1.7 nM.

[0179] Pharmaceutical Compositions The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. The pharmaceutical composition herein comprises an antibody, as defined herein, and / or an antigen-binding fragment thereof, such as an scFv, and a pharma- ceutical acceptable carrier or excipient.

[0180] The antibodies defined herein, or antigen-binding fragments thereof, such as scFvs, may be formulated by means known in the art, for example in the form of tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols for inhalation, sterile aqueous or oily solutions or suspensions or sterile emulsions for parenteral use (including intravenous, subcutaneous or intramuscular injection).

[0181] Therefore, provided herein is a composition comprising the isolated antibody or antigen-binding fragment thereof described herein and at least one pharma- ceutically 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, at least three additional active agents, etc.A non-limiting example of an additional active agent that may prove useful in such combination is an immune response modifier.

[0182] A "pharmaceutical acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0183] As used herein, pharma- ceutically acceptable carriers include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Preferably, the carrier is suitable for oral, as well as intravenous, intramuscular, subcutaneous, spinal or epidermal administration (e.g., by injection or infusion).

[0184] The pharmaceutical compositions disclosed herein may contain pharma- ceutical acceptable antioxidants. Examples of pharma-ceutical acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium sulfite, 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 chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0185] Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0186] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by the inclusion of both sterilization procedures and various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. in the compositions. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0187] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutical active substances is known in the art.

[0188] Also provided herein is a kit of parts comprising an antibody or antigen-binding fragment thereof, or pharmaceutical composition according to the invention, a means for administering the antibody or antigen-binding fragment thereof, or pharmaceutical composition, and, optionally, a package insert comprising instructions for use. The means for administering the antibody or antigen-binding fragment thereof, or pharmaceutical composition may, for example, be a syringe. The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, including information regarding indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings regarding the use of such therapeutic products.

[0189] Medical Uses of Isolated Antibodies or Antigen-Binding Fragments Thereof The antibodies or antigen-binding fragments thereof according to the invention may be used to effectively reduce the pro-inflammatory effects of BSSL in a subject, such as a human.

[0190] The advantage of using the antibodies and antigen-binding fragments thereof of the present invention is that they do not bind to the active site on the BSSL protein responsible for the lipase activity of BSSL, as demonstrated and further detailed in the experimental section, and therefore the risk of negative side effects is reduced as lipase activity is not significantly affected.

[0191] The present invention therefore relates to an isolated antibody, or an antigen-binding fragment thereof, such as an scFv, as defined herein, or a pharmaceutical composition for use as a medicament.

[0192] The present specification is also directed to an isolated antibody or antigen-binding fragment thereof, or a pharmaceutical composition as defined herein, for use in the treatment and / or prevention of an inflammatory disease. The present specification is also directed to the use of an isolated antibody or antigen-binding fragment thereof, such as an scFv, or a pharmaceutical composition as defined herein, for the manufacture of a medicament for the treatment and / or prevention of an inflammatory disease. The present specification is also directed to a method for the treatment and / or amelioration and / or prevention and / or prophylaxis of an inflammatory disease. The method comprises administering to a subject in need thereof a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof, such as an scFv, or a pharmaceutical composition.

[0193] Different in vivo models can be used to predict the effect of antibodies and antigen-binding fragments in the treatment and / or prevention of inflammatory diseases. Typically, mice are used in these models and different substances are injected to induce an immune response. The effect of antibodies or their antigen-binding fragments on such immune response can then be examined after administration of the antibodies / antigen-binding fragments.

[0194] One model that can be used is the so-called "collagen-induced arthritis" (CIA) model. In this model, collagen type II (CII) in complete Freund's adjuvant (CFA) is injected, typically on day 21, with a boost of CII in incomplete Freund's adjuvant (IFA). This model induces autoimmune arthritis. Arthritis typically appears 21-28 days after the first injection with CII. This model is B-cell and T-cell dependent (adaptive immunity).

[0195] Another model is "Collagen Antibody-Induced Arthritis" (CAIA). In this model, a cocktail of CII antibodies is injected, typically with a boost of lipopolysaccharide (LPS) on day 5. This model is B-cell and T-cell independent (innate immunity). Protocols for testing the in vivo efficacy of the antibodies and antigen-binding fragments thereof herein in treating and / or preventing inflammatory diseases using the CAIA model are disclosed in the Experimental Section herein.

[0196] Yet another model is the "glucose-6-phosphate isomerase-induced arthritis" model, in which a peptide corresponding to a sequence in glucose-6-phosphate isomerase is injected to elicit an immune response. This model is T cell dependent.

[0197] Another model is the "pristane-induced arthritis" (PIA) model in which pristane is injected. This model is T cell dependent.

[0198] Alternatively, a "dextran sulfate sodium induced colitis" model can be used in which dextran sulfate sodium (DSS) is included in the drinking water.

[0199] All of the above methods are well known to those skilled in the art and can be used to test the in vivo efficacy of the antibodies or antigen-binding fragments thereof herein.

[0200] The inflammatory disease to be treated and / or prevented herein may be, for example, a chronic inflammatory disease. The inflammatory disease may be a local or systemic inflammatory disease.

[0201] The inflammatory disease may be, for example, an autoimmune disease or an autoinflammatory disease. Another type of inflammatory disease is 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), Sjogren's syndrome and inflammatory bowel disease (IBD).

[0202] 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 liver steatosis, and hyperinflammation.

[0203] In certain embodiments, 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-gamma-inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1-alpha (MIP-1α), and TNF-α.

[0204] RA primarily affects the joints, but can also be a systemic inflammatory disease that can cause extra-articular manifestations in several organs. Thus, RA can be considered a systemic inflammatory disease.

[0205] Furthermore, the isolated antibodies or antigen-binding fragments thereof, such as scFv fragments, or pharmaceutical compositions disclosed herein may replace current biological therapies for patients who do not respond or respond temporarily to current tumor necrosis factor alpha (TNFα) inhibitors reducing the need to administer corticosteroids and / or immunosuppressants and / or pharmaceuticals. Thus, the use of the isolated antibodies or antigen-binding fragments thereof, such as scFv fragments, disclosed herein may halt and / or reduce the adverse effects and / or side effects of alternative treatment regimens in patients, which is an important issue in quality care in general, and particularly in young patients and children, as well as immunosuppressed and / or elderly patients.

[0206] Treatment and / or prophylaxis using the isolated antibodies and / or antigen-binding fragments thereof or pharmaceutical compositions disclosed herein is typically passive immunotherapy, in which the antibodies or antigen-binding fragments thereof, or pharmaceutical compositions comprising such antibodies and / or antigen-binding fragments thereof, are administered to a subject in need thereof. However, instead of administering the antibodies or antigen-binding fragments thereof directly, other types of immunotherapy may also be employed, such as gene therapy, in which a genetic construct capable of expressing such antibodies or antigen-binding fragments thereof is administered to the subject.

[0207] The subject according to the present disclosure may 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, cows, chickens, amphibians, reptiles, etc. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). The term "subject" may be used interchangeably with the term "patient" herein. The subject may be a human.

[0208] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that seeks to alter the natural course of the disease in the individual being treated, and can be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms (improved quality of life), reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, remission or alleviation of the disease state, and remission or improved prognosis. An antibody or antigen-binding fragment thereof according to the invention may be used to delay the onset of the disease or slow the progression of the disease.

[0209] "Reduction" or "inhibition" refers to the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more overall. Reduction or inhibition can refer to the symptoms of the disorder being treated. Reduction or inhibition also encompasses delaying the onset of a disease, particularly an inflammatory disease.

[0210] Mode of administration An isolated antibody, or antigen-binding fragment thereof, such as scFv, or pharmaceutical composition according to the invention may be administered in standard manner for the condition it is desired to treat and / or prevent, for example by oral, topical, parenteral, intravenous, subcutaneous, buccal, nasal, or rectal administration, or by inhalation. For example, an antibody, or antigen-binding fragment thereof, such as scFv, or pharmaceutical composition for use as described herein may be formulated for parenteral administration, such as intravenous or subcutaneous administration, in particular subcutaneous administration.

[0211] Typically, the isolated antibody, or antigen-binding fragment thereof, such as an scFv, or pharmaceutical composition as defined herein is administered systemically. The mode of administration may be parenteral, for example by intravenous or subcutaneous administration, in particular subcutaneous administration.

[0212] Although dosing regimens can be adjusted for the particular disease and subject being treated, typically an isolated antibody, or antigen-binding fragment thereof, such as an scFv, or pharmaceutical composition defined herein is administered 1-3 times per week, for example 1-2 times per week, for example once per week, although other dosing regimens are possible.

[0213] The antibodies, antigen-binding fragments thereof, and / or pharmaceutical compositions of the present invention may also be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy may include an antibody according to the present invention, or an antigen-binding fragment thereof, such as an scFv, in combination with at least one other anti-inflammatory or immunosuppressant agent. It is understood that combination therapy encompasses sequential and simultaneous administration. The term "concurrent" is used herein to refer to the administration of two or more therapeutic agents, where at least a portion of the administration overlaps in time. Thus, simultaneous administration includes a dosing regimen in which administration of one or more agent(s) continues after administration of one or more other agent(s) has ceased.

[0214] Dosage regimens can be adjusted to provide the optimum desired response, e.g., a therapeutic response. For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can 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.

[0215] The therapeutically effective amount of an antibody or an antigen-binding fragment thereof, such as an scFv, may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antigen-binding fragment thereof, such as an scFv, to induce a desired response in the subject. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the administered substance. Although antibodies or antigen-binding fragments thereof, such as scFv, are typically used for the prevention of disease states (prophylactic purposes), the prophylactically effective amount is not necessarily less than the therapeutically effective amount, since a prophylactic dose is used in subjects prior to or at an early stage of disease.

[0216] A medicamentously effective amount, i.e., dose, of an antibody or antigen-binding fragment thereof such as an scFv according to the invention will typically be in the range of about 0.0001-100 mg / kg of host body weight, more usually 0.01-5 mg / kg, however the exact dose must be adjusted depending, for example, on the condition to be treated or prevented, the age and / or sex of the subject, and whether a pathology is intended to be treated or prevented.

[0217] Expression system The present invention also relates to a polynucleotide, such as an isolated polynucleotide, that encodes an antibody or antigen-binding fragment thereof according to the invention.

[0218] Exemplary polynucleotides according to embodiments include the HCs and LCs 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: 180; 191; 200) and LC (SEQ ID NOs: 182; 193; 202) and AS20 HC (SEQ ID NO: 183) and LC (SEQ ID NO: 184), as well as CDR grafts HC (SEQ ID NO: 194) and LC (SEQ ID NO: 195), are shown in SEQ ID NOs: 174 to 202, which show DNA sequences encoding the HC (SEQ ID NO: 183) and LC (SEQ ID NO: 184) and CDR grafts HC (SEQ ID NO

[0219] Thus, in one embodiment, the polynucleotide is selected from the group consisting of SEQ ID NOs: 174-202, and any combination and / or variant thereof. As used herein, a variant of any of SEQ ID NOs: 174-202 includes a polynucleotide encoding the same antibody or antigen-binding fragment thereof as a polynucleotide defined in any of SEQ ID NOs: 174-202, but may have at least one synonymous substitution, i.e., a substitution of at least one base with another base, such that the resulting amino acid sequence is not altered. Thus, such a synonymous substitution changes at least one base of a codon in the polynucleotide, both of which code for an amino acid residue, to another codon. For example, a polynucleotide according to any of SEQ ID NOs: 174-202, or a combination thereof, can be codon-optimized for expression in a particular host cell.

[0220] The polynucleotide encoding the antibody or antigen-binding fragment thereof disclosed herein may be introduced into an expression vector. The expression vector allows the propagation of the polynucleotide introduced therein. The vector may be a self-replicating nucleic acid structure that is integrated into the genome of the host cell into which it is introduced. The present invention is therefore also directed to such expression vectors comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof.

[0221] 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 sequence of DNA to which a protein binds and initiates 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 to which an activator can bind to increase the likelihood that transcription of a particular gene will occur.

[0222] Examples of expression vectors include DNA molecules, RNA molecules, plasmids, episomal plasmids, and viral vectors. Non-limiting exemplary examples of viral vectors include lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, Semliki Forest virus, poliovirus, and hybrid vectors.

[0223] The expression vector may be introduced into a host cell for expression and / or propagation of the vector containing the polynucleotide, in particular the expression vector is for use in treating and / or preventing an inflammatory disease by expression in a subject, thereby producing an antibody or antigen-binding fragment thereof in the subject.

[0224] Thus, host cells comprising the expression vectors are also provided herein. The host cells used can be any type of host cell, including both eukaryotic and prokaryotic host cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of transfers.

[0225] The present invention also relates to a cell comprising an antibody or an antigen-binding fragment thereof according to the invention, a polynucleotide according to the invention and / or an expression vector according to the invention.

[0226] The cell may be an isolated cell, including a cell of a cell line. The cell may be selected from a eukaryotic cell, such as a bacterial cell, a yeast cell, a mammalian cell, a human cell, or a non-human cell.

[0227] The antibodies or antigen-binding fragments thereof are produced by introducing their sequences into an expression vector and allowing the expression vector to express the antibodies or antigen-binding fragments thereof in a host cell, after which the produced antibodies or antigen-binding fragments thereof are isolated / purified before use, e.g., for medical treatment purposes or diagnostic purposes as disclosed elsewhere herein. The vector itself may also be introduced into a subject for direct expression of the antibodies or antigen-binding fragments thereof in the subject to be treated. The expression vector then preferably comprises a promoter-controlled expression of a polynucleotide encoding the antibody or antigen-binding fragment thereof.

[0228] The present invention therefore also relates to a method for producing an antibody or antigen-binding fragment thereof, the method comprising culturing a cell according to the invention comprising an expression vector according to the 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 medium in which the cell is cultured.

[0229] Diagnostic Uses of Isolated Antibodies or Antigen-Binding Fragments Thereof The antibodies or antigen-binding fragments thereof of the present invention can also be used to detect BSSL, such as hBSSL, in a sample using standard techniques, including but not limited to, ELISA, Western blot, RIA, surface plasmon resonance (SPR) and flow cytometry analysis.

[0230] The 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 lipase activity of the protein.Therefore, as demonstrated in the experimental section, it is possible to use the antibodies or antigen-binding fragments thereof to study the BSSL protein without significantly affecting the lipase activity.The antibodies or antigen-binding fragments thereof are therefore useful as molecular tools in studying the BSSL protein and / or its enzymatic activity in vitro / ex vivo and / or in vivo.

[0231] The present invention therefore discloses a method for detecting the presence or absence of a BSSL, such as hBSSL, in a sample and / or for quantifying the amount of BSSL. The method comprises contacting the sample with an isolated antibody or antigen-binding fragment thereof according to the present invention. The method also comprises detecting the presence or absence of BSSL and quantifying the amount of BSSL in the sample based on the amount of the isolated antibody or antigen-binding fragment thereof bound to the BSSL. Detection or quantification may be performed, for example, using ELISA, Western blot, RIA, surface plasmon resonance (SPR), proximity ligation assay (PLA) or flow cytometry analysis. One or more of the antibodies or antigen-binding fragments thereof of the present invention, i.e. at least two of them, may be used for such detection.

[0232] The above method may be in the form of an ex vivo or in vitro method, in which case the method comprises contacting the sample with an isolated antibody or antigen-binding fragment thereof according to the invention ex vivo or in vitro.

[0233] In one embodiment, the method also includes providing a sample potentially containing a BSSL.

[0234] The present invention also discloses a method for diagnosing a BSSL-associated 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 or absence of BSSL and / or quantifying the amount of BSSL in the sample based on the amount of the isolated antibody or antigen-binding fragment thereof bound to the BSSL. The detection or quantification may be performed, for example, using ELISA, Western blot, RIA, SPR, PLA or flow cytometry analysis. The method also comprises c) concluding based on the result of step b) whether the subject is diagnosed with a BSSL-associated disorder.

[0235] In one embodiment, the method also includes providing a sample from a subject suspected of suffering from a BSSL-associated disorder.

[0236] In certain embodiments, the method comprises comparing the quantified amount of BSSL in the sample with a threshold value.In such certain embodiments, step c) comprises not based on comparing the quantified amount of BSSL in the sample with a threshold value, but rather concludes that the subject is diagnosed with or has a BSSL-related disorder.For example, if the amount of BSSL in the BSSL exceeds a threshold value, the subject is diagnosed and concludes that the subject is diagnosed with or has a BSSL-related disorder.

[0237] The threshold value depends on the particular BSSL-related disorder and can be defined by quantifying the amount of BSSL in a sample taken from a subject already diagnosed with a particular BSSL-related disorder and / or by quantifying the amount of BSSL in a sample taken from a healthy subject not suffering from a particular BSSL-related disorder. The threshold value can be determined based on these quantified amounts of BSSL from subjects suffering from a particular BSSL-related disorder, preferably based on the quantified amount of BSSL from healthy subjects.

[0238] The BSSL-associated disorder is typically an inflammatory condition as disclosed elsewhere herein. The inflammatory condition may be, for example, a chronic or systemic inflammatory disease, such as an inflammatory disease, an autoinflammatory disease and / or an autoimmune disease. The inflammatory condition may be, for example, rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, atherogenesis, Crohn's disease, or ulcerative colitis.

[0239] The sample potentially containing BSSL may be any type of sample, such as a sample obtained from a subject. Thus, in one embodiment, the sample is a biological sample. One 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 may be a natural sample or an in vitro sample potentially containing BSSL. Methods for detecting BSSL and / or diagnosing a BSSL-associated condition include both in vitro and in vivo methods, such as in situ hybridization.

[0240] As mentioned elsewhere herein, the antibodies or antigen-binding fragments thereof may be humanized or have their CDR sequences (or parts thereof) grafted onto a non-human scaffold. The latter may be advantageous when the antibodies or antigen-binding fragments thereof are used as molecular tools to study BSSL proteins in species other than human, for example to reduce negative immunogenic responses against the antibodies and / or antigen-binding fragments.

[0241] Exemplary embodiments One embodiment relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to bile salt stimulated lipase (BSSL), such as human BSSL (hBSSL), wherein the antibody or antigen-binding fragment thereof binds to at least one of an identified first and second epitope on BSSL. The first epitope comprises, and preferably consists of, an amino acid sequence according to SEQ ID NO:1 or an amino acid sequence having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:1, and the second epitope comprises an amino acid sequence according to SEQ ID NO:2 or an amino acid sequence having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:2.

[0242] 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 having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3.

[0243] In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to both the first epitope and the second epitope.

[0244] 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 having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4.

[0245] 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 having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0246] 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 having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0247] 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 that is at least 87% identical to SEQ ID NO:7, the second HCDR comprises or 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 or consists of an amino acid sequence according to SEQ ID NO:9 or an amino acid sequence that is at least 90% identical to SEQ ID NO:9. 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 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, the second LCDR comprises or consists of an 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 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.

[0248] 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.

[0249] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, a second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 18, and a third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of an LCVR: a first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 10, a second LCDR comprises, preferably consists of, the amino acid sequence ATS, and a third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 21.

[0250] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, a second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 8, and a third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of an LCVR: a first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 10, a second LCDR comprises, preferably consists of, the amino acid sequence ATS, and a third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 11.

[0251] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, a second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 19, and a third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of an LCVR: a first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 20, a second LCDR comprises, preferably consists of, the amino acid sequence ATS, and a third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 22.

[0252] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, a second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 8, and a third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of an LCVR: a first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 20, a second LCDR comprises, preferably consists of, the amino acid sequence AAS, and a third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 11.

[0253] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a 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, a 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 a 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 an LCVR: a 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, a 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 a 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.

[0254] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDRs) of the 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 the light chain variable region (LCVR). The first LCDR comprises, and 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, and 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, and 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.

[0255] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a 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, a 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 a 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 an LCVR: a 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, a 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 a 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.

[0256] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a 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, a 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 a 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 an LCVR: a 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, a 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 a 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.

[0257] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a 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, a 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 a 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 an LCVR: a 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, a 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 a 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.

[0258] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of the HCVR: a first HCDR comprises, and preferably consists of, an amino acid sequence according to SEQ ID NO: 7, a second HCDR comprises, and preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 23, 24 and 25, and a third HCDR comprises, and preferably consists of, an 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: a first LCDR comprises, and preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 16, 26 and 27, a second LCDR comprises, and preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 17, 28 and 29, and a third LCDR comprises, and preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 21 and 22.

[0259] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, a second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 23, and a third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of an LCVR: a first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 16, a second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 15, and a third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 21.

[0260] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, a second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 24, and a third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of an LCVR: a first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 27, a second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 29, and a third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 11.

[0261] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, a second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 25, and a third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of an LCVR: a first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 26, a second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 28, and a third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 22.

[0262] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, a second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 12, and a third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of an LCVR: a first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 14, a second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 15, and a third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 11.

[0263] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs of an HCVR: a first HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 7, a second HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 12, and a third HCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 9. In this embodiment, the isolated antibody or antigen-binding fragment thereof comprises three LCDRs of an LCVR: a first LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 16, a second LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 17, and a third LCDR comprises, preferably consists of, an amino acid sequence according to SEQ ID NO: 11.

[0264] In one embodiment, the HCVR comprises, preferably consists of, one amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 32, 34, and 36 or amino acid sequences that are at least 98% identical thereto; for example, the group consisting of SEQ ID NOs: 30, 34 and 36 or amino acid sequences that are at least 96% identical thereto; for example, the group consisting of SEQ ID NOs: 34 and 36 or amino acid sequences that are at least 96% identical thereto.

[0265] 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.

[0266] In one embodiment, the LCVR comprises, preferably consists of, one amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 33, 35, 37 and 38 or amino acid sequences that are at least 96% identical thereto; for example, the group consisting of SEQ ID NOs: 31, 35, 37 and 38 or amino acid sequences that are at least 96% identical thereto; for example, the group consisting of SEQ ID NOs: 35, 37 and 38 or amino acid sequences that are at least 96% identical thereto; for example, the group consisting of SEQ ID NOs: 37 and 38 or amino acid sequences that are at least 96% identical thereto.

[0267] In one embodiment, the LCVR comprises or consists of an amino acid sequence according to SEQ ID NO: 37, or an amino acid sequence which is at least 96% identical thereto.

[0268] 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 amino acid sequences that are at least 96% identical 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 amino acid sequences that are at least 96% identical thereto. In a particular 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 amino acid sequences that are at least 96% identical 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 amino acid sequences that are at least 96% identical 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 amino acid sequences that are at least 96% identical 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 amino acid sequences that are at least 96% identical thereto. In a further specific embodiment, the HCVR comprises, preferably consists of, an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 36, or an amino acid sequence that is at least 96% identical 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 amino acid sequences that are at least 96% identical thereto.

[0269] In one embodiment, the isolated antibody or antigen-binding fragment thereof 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.

[0270] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises an HCVR and an LCVR, the HCVR and LCVR being a pair of amino acid sequences selected from the group consisting of the amino acid sequence pair SEQ ID NOs: 30 and 31; the amino acid sequence pair SEQ ID NOs: 32 and 33; the amino acid sequence pair SEQ ID NOs: 34 and 35; the amino acid sequence pair SEQ ID NOs: 36 and 37; and the amino acid sequence pair SEQ ID NOs: 36 and 38; for example, a pair of amino acid sequences that are at least 96% identical to a pair of amino acid sequences selected from the group consisting of the amino acid sequence pair SEQ ID NOs: 36 and 37; and the amino acid sequence pair SEQ ID NOs: 36 and 38.

[0271] 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, where ZH1, ZH2, ZH3 and ZH4 each represent zero, one or several independently selected amino acid residues. In one embodiment, the heavy chain variable region comprises i) ZH1-[GYTFTSYN]-ZH2-[X 53 GVIX 57 PGDGX 64 TSYX 68 QKFX 72 ]-ZH3-[ARDYYGSSPLGY]-ZH4, wherein, independently of each other, X 53 is selected from I and M; X 57 is selected from N and Y; X 64 is selected from A and S; X 68 is selected from A and N; X 72 is 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 an amino acid sequence comprising ZL1-[eCDR-L1]-ZL2-[eCDR-L2]-ZL3-[CDR-L3]-ZL4, where each of ZL1, ZL2, ZL3 and ZL4 represents zero, one or several independently selected amino acid residues. In one embodiment, the light chain variable region comprises iii) ZL1-[X 24 ASX 27 SISYX 39N]-ZL2-[AX 57 SX 66 LX 68 ]-ZL2-[HQRSSX 115 PT]-ZL4, wherein, independently of each other, X 24 is selected from S and R; X 27 is selected from S and P; X 39 is selected from M and L; X 57 is selected from A and T; X 66 is selected from K and S; X 68 is selected from A and P; and X 115 is selected from S, T and Y, and iv) an amino acid sequence selected from an amino acid sequence having at least 87% identity with the sequence defined in iii).

[0272] In one embodiment, ZH1 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:39 or an amino acid sequence that is at least 90% identical to SEQ ID NO:39.

[0273] In one embodiment, ZH2 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:40 or an amino acid sequence that is at least 90% identical to SEQ ID NO:40.

[0274] In one embodiment, ZH3 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:41 or an amino acid sequence that is at least 90% identical to SEQ ID NO:41.

[0275] In one embodiment, ZH4 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:42 or an amino acid sequence that is at least 90% identical to SEQ ID NO:42.

[0276] 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% identical to SEQ ID NO:43.

[0277] In one embodiment, ZL2 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:44 or an amino acid sequence that is at least 90% identical to SEQ ID NO:44.

[0278] In one embodiment, ZL3 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:45 or an amino acid sequence that is at least 90% identical to SEQ ID NO:45.

[0279] In one embodiment, ZL4 comprises, preferably consists of, an amino acid sequence according to SEQ ID NO:46 or an amino acid sequence that is at least 90% identical to SEQ ID NO:46.

[0280] In one embodiment, the antibody is a full-length antibody.

[0281] In one embodiment, the antibody is selected from the group consisting of a human antibody, a humanized antibody, and a chimeric antibody.

[0282] 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 particular embodiment, the antigen-binding fragment is an scFv fragment.

[0283] In one embodiment, the isolated antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof. In a particular embodiment, the monoclonal antibody or antigen-binding fragment thereof is a humanized monoclonal antibody or antigen-binding fragment thereof.

[0284] In one embodiment, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of isotype classes IgG, such as IgG, IgA, IgM, IgD and IgE. In a particular embodiment, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of isotype subclasses IgG1 and IgG4.

[0285] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises one or more Fc silencing mutations. In a particular embodiment, the IgG1 comprises the Fc silencing mutations L234A, L235A and P329G.

[0286] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises one or more stabilizing mutations that prevent or reduce in vivo Fab arm exchange, hi a particular embodiment, the IgG4 comprises the stabilizing mutation S228P.

[0287] In one embodiment, the isolated antibody or antigen-binding fragment thereof specifically binds to hBSSL and is a single chain variable fragment (scFv) comprising an 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 an 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, the amino acid sequence ATS, and SEQ ID NO:11, respectively.

[0288] In one embodiment, the first HCDR, the second HCDR and the third HCDR consist of the amino acid sequences 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 sequences according to SEQ ID NO:10, the amino acid sequence ATS and SEQ ID NO:11, respectively.

[0289] In one embodiment, the antibody is a humanized antibody.

[0290] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K D It has an affinity for hBSSL of 1.7 nM or less.

[0291] In one embodiment, the isolated antibody or antigen-binding fragment thereof is directed against monocytes, preferably CD14 + The binding of hBSSL to monocytes can be displaced.

[0292] An embodiment relates to a pharmaceutical composition comprising an isolated antibody and / or antigen-binding fragment thereof according to the invention and a pharma- ceutically acceptable carrier or excipient.

[0293] An 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.

[0294] An embodiment relates to an isolated antibody and / or antigen-binding fragment thereof, or a pharmaceutical composition according to the invention for use in the treatment and / or prevention of an inflammatory disease.

[0295] An embodiment relates to the use of an isolated antibody and / or antigen-binding fragment thereof, or a pharmaceutical composition according to the invention for the manufacture of a pharmaceutical composition for the treatment and / or prevention of an inflammatory disease.

[0296] An embodiment relates to a method for the treatment and / or amelioration and / or prevention and / or prophylaxis of an inflammatory disease, in which a therapeutically effective amount of an isolated antibody and / or antigen-binding fragment thereof, or a pharmaceutical composition according to the invention is administered to a subject in need thereof.

[0297] In one embodiment, the inflammatory disease is a chronic inflammatory disease.

[0298] In one embodiment, the inflammatory disease is a systemic inflammatory disease.

[0299] In one embodiment, the inflammatory disease is an autoimmune disease. In a particular embodiment, the autoimmune disease is rheumatoid arthritis or juvenile rheumatoid arthritis. In another particular embodiment, the autoimmune disease is an inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis.

[0300] In one embodiment, the inflammatory disease is an autoinflammatory disease, hi a particular embodiment, the autoinflammatory disease is psoriatic arthritis.

[0301] In one embodiment, the inflammatory disease is fatty liver.

[0302] In one embodiment, the isolated antibody and / or antigen-binding fragment thereof, or the pharmaceutical composition is administered systemically.

[0303] In one embodiment, the isolated antibody and / or antigen-binding fragment thereof, or the pharmaceutical composition is administered parenterally, e.g., subcutaneously. Thus, in certain embodiments, the isolated antibody and / or antigen-binding fragment thereof, or the pharmaceutical composition is formulated for parenteral administration, e.g., subcutaneous administration.

[0304] In one embodiment, the isolated antibody and / or antigen-binding fragment thereof or the pharmaceutical composition is administered 1-3 times per week, such as 1-2 times per week, for example, once per week.

[0305] In one embodiment, the treatment and / or prevention is by passive immunotherapy.

[0306] Embodiments relate to polynucleotides encoding an isolated antibody or antigen-binding fragment thereof defined according to the invention, expression vectors comprising a polynucleotide according to the invention and host cells comprising an expression vector according to the invention.

[0307] An embodiment relates to a method of producing an isolated antibody or antigen-binding fragment thereof according to the invention, the method comprising culturing a host cell according to the invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and isolation of the antibody or antigen-binding fragment thereof.

[0308] An embodiment relates to a method of detecting the presence or absence of a BSSL in a sample and / or a method of quantifying the amount of a BSSL, the method comprising the steps of a) providing a sample potentially containing a 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 a BSSL in said sample and / or quantifying the amount of a BSSL.

[0309] An embodiment relates to a method for diagnosing a BSSL-related disorder, comprising the steps of: a) providing a sample from a subject suspected of suffering from a BSSL-related disorder, b) contacting said sample with an isolated antibody or antigen-binding fragment thereof according to the invention, c) detecting the presence or absence of BSSL in the sample and / or quantifying the amount of BSSL, and d) concluding based on the result of step c) whether the subject is diagnosed with a BSSL-related disorder.

[0310] 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 fatty liver.

[0311] An embodiment relates to a method for determining the enzymatic activity of a BSSL, the method comprising the steps of a) providing a sample comprising a BSSL, b) contacting the sample with an isolated antibody or antigen-binding fragment thereof according to the invention, and c) determining the enzymatic activity of the BSSL in the sample.

[0312] An 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 having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1. The second epitope comprises a second surface comprising or consisting of an amino acid sequence according to SEQ ID NO: 2 or an amino acid sequence having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 2.

[0313] 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 having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0314] In one embodiment, the epitope further comprises an amino acid sequence according to SEQ ID NO: 4, or an amino acid sequence having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0315] In one embodiment, the epitope further comprises an amino acid sequence according to SEQ ID NO: 5, or an amino acid sequence having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0316] In one embodiment, the epitope further comprises an amino acid sequence according to SEQ ID NO: 6, or an amino acid sequence having at least 80%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0317] Although the present invention has been described with reference to various exemplary aspects and embodiments, those skilled in the art will recognize that various changes may be made and equivalents may be substituted for those elements without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from its essential scope. Therefore, the present invention is not limited to any particular embodiment contemplated, but is intended to include all embodiments falling within the scope of the appended claims.

[0318] Working Example Table 2 - BSSL Materials Used in the Examples [Table 2]

[0319] Example 1 - Binding of AS20 IgG to human and mouse BSSL (SPR) In this example, the binding of antibody AS20 Mouse-IgG1 (AS20 mIgG) to both 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 full-length BSSL protein (SEQ ID NO: 138) purified from human milk. Reactivity against mouse BSSL was unknown.

[0320] Materials and Methods hBSSL and mBSSL (Table 2) were used together with AS20 mouse IgG1 (AS20 mIgG1) (generated in the laboratory, HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114) in the experiments described in this example.

[0321] SPR measurements were performed on a BIACORE® T200 instrument (GE Healthcare). To minimize avidity effects, antibodies were immobilized on the sensor surface and BSSL was injected as analyte. Immobilization of AS20 mIgG1 was performed by amine coupling to a BIACORE® 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 min. Antibodies were diluted to 14-50 μg / ml in 10 mM acetate-HCl pH 5.0 (set 1) or pH 6.0 (set 2) and injected for 0.2-2.8 min until a final immobilization level of 560-830 RU was reached. The remaining activated carboxyl groups on the sensor surface were inactivated by injecting 1 M ethanolamine for 7 min.

[0322] 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 Tris HCl, pH 7.5, 150 mM NaCl. 146 mM H3PO4 was used as the standard regeneration solution. Kinetic studies and nonlinear regression analysis were performed according to the Single Cycle Kinetics (SCK) method of the BIACORE® T200 instrument and evaluation software. The interaction of mBSSL with AS20 mIgG1 was analyzed using a steady-state affinity model.

[0323] In experimental set 1, three SCK experiments were performed at the highest hBSSL concentration within the concentration series, which were 300, 100 and 50 nM, respectively. The concentration series was made at 1:3, 1:3.16 (half log) and 1:2 dilutions.

[0324] In the second set of experiments, hBSSL was diluted in running buffer to a starting concentration of 20 nM, followed by a 1:1 serial dilution in the same buffer to give five concentrations ranging from 20 nM to 1.25 nM. mBSSL was diluted in running buffer to a starting concentration of 2000 nM, followed by a 1:1 serial dilution in the same buffer to give five concentrations ranging from 2000 nM to 125 nM.

[0325] result AS20 mIgG1 was found to bind to both human and mouse BSSL. The affinity to 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 equilibrium dissociation constants from nonlinear regression analysis of the SCK experiments are shown in Table 3. In the first set of experiments, measurements were performed in triplicate, therefore, the means and standard deviations are presented.

[0326] Mouse BSSL was also found to interact with immobilized AS20 mIgG1, but with a weaker affinity approximately 100-fold. To determine affinity, steady-state analyses were performed (Figure 2 and Table 3).

[0327] Table 3 - Kinetic parameters of the interaction between AS20 mIgG1 and hBSSL and AS20 mIgG1 and mBSSL [Table 3] * Single cycle kinetics ** Steady-State Analysis ND Not decided 1 First set of experiments 2 Second set of experiments

[0328] Example 2 - Generation of AS20 scFv and binding properties of AS20 scFv to human and mouse BSSL (ELISA, SPR and LUMINEX®) In this example, a single chain variable fragment (scFv) version based on AS20 mIgG1 was generated, designated AS20 scFv, which retains binding to human BSSL as assessed by enzyme-linked immunosorbent assay (ELISA), SPR and LUMINEX® (comprising HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114).

[0329] Materials and Methods Small scale production and purification 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 a secretion signal for the scFv together with a C-terminal triple FLAG tag and a hexahistidine (His) tag. The construct was then transformed into TOP10 E. coli. The bacterial supernatant of 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 by a BCA (bicinchoninic acid) assay kit (Pierce).

[0330] ELISA Non-biotinylated human BSSL (hBSSL) and biotinylated human BSSL (b-hBSSL) were either directly coated or coated via streptavidin in 384-well ELISA plates at two different concentrations, 1 μg / ml and 0.5 μg / ml, in PBS overnight at 4 °C. Purified AS20 scFv was serially diluted 3-fold in blocking buffer (phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA) and 0.05% Tween 20) at concentrations ranging from 1 μg / ml to 4 ng / ml. Detection of binding was enabled via horseradish peroxidase (HRP)-conjugated α-FLAG M2 antibody (Sigma-Aldrich) followed by incubation with the chromogenic substrate Ultra 3,3',5,5'-tetramethylbenzidine (TMB) ELISA. Signal development was stopped by the addition of 1 M sulfuric acid and absorbance was measured at 450 nM.

[0331] In the second ELISA assay, 1 μg / ml of human and mouse BSSL, as well as a negative control protein, were directly coated onto 384-well ELISA plates and incubated overnight at 4° C. Purified AS20 scFv and negative control scFv were added at two different concentrations, 1 μg / ml and 0.2 μg / ml. Detection of binding signals was performed as described above. All samples were assayed in duplicate in both ELISA settings. hBSSL, b-hBSSL and mBSSL (Table 2) were used together with AS20 scFv (produced in the lab, HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114) in the experiments performed in this example.

[0332] SPR measurement Affinity evaluation of the scFv clones was performed by SPR using a BIACORE® T200 (GE Healthcare). α-FLAG M2 antibody was immobilized on a CM5 S chip via primary amine coupling using NHS-EDC chemistry, allowing capture of AS20 scFv via its 3×FLAG tag. A 3-fold dilution series consisting of five different concentrations of hBSSL and b-hBSSL, from 200 nM to 2 nM, was sequentially injected over the flow cell, allowing binding to the captured AS20 scFv. Regeneration of the surface was achieved under acidic conditions using 10 mM glycine-HCl at pH 2.2. The obtained single cycle kinetic data was fitted to a 1:1 Langmuir binding model, giving the kinetic parameters K a (1 / Ms), k d (1 / s) and K D (M) was acquired using the software BIAevaluation.

[0333] LUMINEX® Analysis Biotinylated hBSSL was incubated with neutravidin-conjugated LUMINEXEX® beads and mixed with 30 different bead IDs, each conjugated with 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). One positive scFv control was also included, i.e., an scFv expected to bind to beads coated with one of the unrelated proteins. Binding of scFv clones to specific protein-conjugated beads was validated via R-PE-conjugated anti-FLAG M2 antibody and subsequently analyzed on a FlexMAP 3D instrument.

[0334] result Small scale production and purification Gel electrophoresis of the bacterially expressed and purified AS20 scFv demonstrated high purity with one major protein band corresponding closely to the predicted molecular weight of the scFv (data not shown).

[0335] ELISA AS20 scFv showed concentration-dependent binding to both non-biotinylated and biotinylated human BSSL (Figure 3a).Then, the signal intensity at a certain scFv concentration was much higher for biotinylated BSSL than for non-biotinylated BSSL, which may be due to the difference in coating conditions.

[0336] Although AS20 scFv also showed binding to mouse BSSL, the signal intensity was much weaker than that of human BSSL (FIG. 3b), suggesting that AS20 scFv may have a weaker affinity for mouse BSSL. No binding of AS20 scFv to the negative control was detected.

[0337] SPR measurement A single cycle kinetic approach was used to determine the affinity of AS20 scFv to non-biotinylated and biotinylated human BSSL. AS20 scFv was found to bind to non-biotinylated human BSSL (KD = 0.6 nM) and biotinylated human BSSL (K D = 0.8 nM) (Table 4).

[0338] Table 4 - Kinetic parameters of the interaction between AS20 scFv and native and biotinylated hBSSL [Table 4]

[0339] Luminex analysis To determine whether AS20 scFv is prone to nonspecific interactions with unrelated proteins, a Luminex assay was performed in which AS20 scFv was analyzed with 30 different unrelated proteins and their cognate targets. Only AS20 scFv showed binding to human BSSL, with no or very low binding to all other proteins included in the assay (Figure 4).

[0340] conclusion The AS20 scFv exhibits similar affinities in the subnanomolar range (similar K D The K value obtained for non-biotinylated BSSL was D The values ​​are in good agreement with those reported in Example 1 for full-length IgG antibodies. AS20 scFv also showed low off-target binding to 30 unrelated proteins when assayed with a Luminex-based approach. As shown by the ELISA results, AS20 scFv showed binding to mouse BSSL, which was shown for full-length IgG in Example 1.

[0341] Example 3 - Binding characteristics of AS20 scFv to mouse BSSL by HTRF Homogeneous time-resolved fluorescence (HTRF) is a spectrophotometric method based on the phenomenon of fluorescence resonance energy transfer (FRET) between two different molecules, known as the 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 of AS20 scFv with murine BSSL.

[0342] Materials and Methods A competition assay was developed to study the interaction of AS20 scFv with native mouse BSSL. Detection of binding was made possible via the donor molecule terbium-conjugated α-FLAG antibody (Cisbio #611FG2TL), which interacts with the FLAG tag located at the C-terminus of the scFv, and the acceptor molecule streptavidin-conjugated XL665 (Cisbio #610SAXL), which interacts with the victor moiety on human BSSL. Experiments were performed using a range of concentrations of non-biotinylated proteins; 0-500 nM for mBSSL and 0-80 nM for hBSSL. hBSSL, b-hBSSL and mBSSL (Table 2) were used together with AS20 scFv (generated in the lab, HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114) in the experiments performed in this example.

[0343] 2.5 nM of AS20 scFv was pre-incubated with mouse or human orthologues of BSSL for 2 h, followed by the addition of 5 nM of b-hBSSL and FRET donor and acceptor molecules. Finally, the mixture was incubated at room temperature for 16 h, and the binding signal (665 nm) and background / noise signal (615 nm) were measured using EnVision (PerkinElmer). The experimental output, delta R, was calculated for each point using four replicates and two blanks.

[0344] result 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 observation was seen for human native BSSL. A 50% reduction in delta R was achieved at 150-200 nM mouse BSSL and approximately 2 nM human BSSL, suggesting that AS20 scFv has approximately 100-fold lower affinity for mouse BSSL compared to the human orthologue.

[0345] conclusion The data obtained showed that the AS20 scFv has approximately 100-fold lower affinity for mouse BSSL compared to the human orthologue, which was in close agreement with the affinities obtained using SPR for the same clonal full-length antibody formats; AS20 mIgG1 (Example 1) and chimeric AS20 (Example 4).

[0346] Example 4 - Generation of chimeric AS20 In this example, a chimeric AS20 is generated. More specifically, a chimera of the human IgG4 subclass was constructed.

[0347] Materials and Methods The production of the material was outsourced to GenScript (Piscataway, NJ, USA). The sequences of the variable domains of the heavy (VH) and light (VL) chains of the murine 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 into Freestyle 293-F cells and transiently expressed. The expressed antibodies were then purified by affinity chromatography using Protein A, followed by preparative size-exclusion chromatography (SEC), purity was determined by high-performance liquid chromatography (HPLC), and concentration was measured spectrophotometrically at 280 nM.

[0348] result Purity was determined to be >98% by HPLC. The sequences of the chimeric AS20 were determined to be SEQ ID NO: 139 for the heavy chain and SEQ ID NO: 140 for the light chain.

[0349] Chimeric AS20 retained the same binding affinity for mouse and human BSSL as the AS20 mouse IgG1 antibody of Example 1 (data not shown).

[0350] Example 5 - Design and construction of AS20 CDR-grafted antibodies and AS20 humanized libraries AS20 is a murine antibody, AS20 mIgG, see Example 1. Non-human antibodies have been shown to induce human immune responses, which can result in neutralization of the administered antibody, thus limiting the efficacy of the antibody in treating disease. To overcome this potential problem, antibody humanization was performed. In this example, two strategies for humanization of AS20 are described: complementarity determining region (CDR) grafting and library-based approaches. The resulting CDR-grafted antibody is referred to herein as AS20 CDR graft or CDR graft, and the generated library is referred to herein as AS20 humanized library. The library was then used for the selection and isolation of AS20-binding scFv fragments using phage display (see Example 6).

[0351] Materials and Methods 15A and 15B show an overview of the design of a combinatorial scFv library of heavy chain variable regions, and FIG. 16A and 16B show an overview of the design of a combinatorial scFv library of light chain variable regions.

[0352] The scFv format was chosen as a scaffold for both CDR grafting and humanized libraries. Data presented in Example 2 demonstrated that the AS20 scFv fully retained the binding ability of its full-length parental IgG counterpart, suggesting that the scFv gene represents a good scaffold format for both CDR grafting and combinatorial library construction.

[0353] The human immunoglobulin heavy chain variable region germline gene (IHGV) was selected as the IHGV framework, IGHV1-46, which has the highest sequence homology to the AS20 heavy chain variable region, with 73.5% (residues 1-104) homology according to IMGT / DomainGapAlign (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi). For the selection of the light chain sequence, homology was considered, but also the pairing of heavy and light chains with good biophysical properties was taken into account

[10] . Taken together, this result led to the selection of the human germline gene IGKV1-39. Based on the IMGT / DomainGapAlign domain search, IGHJ4 and IKVJ2 were also selected as the joining fragments to obtain the complete variable heavy and light chain domains, respectively.

[0354] The AS20 CDR graft was obtained by grafting six mouse CDR loops into a human germline gene. For the heavy chain, the following regions were grafted into the IGHV1-46 framework: heavy chain complementarity 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 a HCVR according to SEQ ID NO: 144. For the light chain, the following regions extended light chain complementarity 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 a LCVR according to SEQ ID NO: 145. The gene encoding the corresponding scFv construct was 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 end of the LCVR to include a BsiWI restriction site. Synthesis and subcloning of scFv genes were outsourced to GenScript (Piscataway, NJ, USA). After synthesis, the scFv genes were cloned into a laboratory phagemid using the restriction enzymes SfiI and BsiWII.

[0355] The scaffold for the AS20 humanized library was constructed using the same HCVR and LCVR frameworks used for CDR grafting (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. We determined that this loop is also most likely important for AS20-BSSL interactions and was therefore kept constant. Instead, for diversity, we selected 22 positions that differ between AS20 and the AS20 humanized scaffold in the other five CDR regions (Figure 6). Herein, we primarily attempted double diversity; that is, we allowed residues found in AS20 and in the human germline genes constructing the humanized scaffold at certain positions. However, not all pairs of amino acids could be established by NNS oligos without introducing additional amino acids, and therefore additional chemical diversity was added at six positions (three in VH: 62, 64, 68 and three in VL: 27, 66, 68). For LCDR3, an alternative strategy was taken. Herein, we considered the diversity found in rearranged functional antibodies encoded by germline genes IGVK1-39 / IKV1D-39 as found in the IMGT database (http: / / www.imgt.org / ligmdb / ). More specifically, the sequence of an antibody with an LCDR3 of 8 amino acids in length, the length of the LCDR3 in AS20, was used as a guide for the diversity to be introduced. The resulting consensus sequence was QQSYSTPT (aa105-117, 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 by restriction of NNS oligos. As a result of the VJ gene joining process, most of the diversity in LCDR3 is found at position 116. In an attempt to mimic this variability, we allowed six amino acids here (P, H, L, Y, S and F), although they are also restricted by the use of NNS codons. This strategy allows capturing more than 50% of the diversity found among antibodies at this position. Overall, the above procedure theoretically allows for approximately 1.2 × 10 9 This results in combinatorial diversity of different mutants.

[0356] Table 5 - Positions targeted for mutagenesis in the AS20 humanized library. The HCVR and LCVR positions are listed at the top and bottom of the table, respectively. Amino acids marked in bold are those found in AS20, while underlined amino acids are the corresponding diversity found in the human germline gene. Numbering is as defined by the IMGT nomenclature, and for codon definitions, the IUPAC nucleotide code is used. Diversity was introduced using one primer each for the five target regions (HCDR1, HCDR2, LCDR1, LCDR2 and LCDR3) (Table 6). [Table 5]

[0357] Diversity was introduced into the library scaffold gene using an optimized Kunkel mutagenesis method essentially as described in

[11] , utilizing the AS20 humanized library scaffold gene (Figure 6) together with five mutagenic oligonucleotides (Table 6). To assess whether the intended diversity was incorporated, TOP10 E. coli cells were chemically transformed with a small aliquot of DNA generated by Kunkel mutagenesis, and 96 clones were selected and sent for sequencing (GATC, Germany). The remaining DNA was then electroporated into SS320 cells (Lucigen, Middleton, WI, USA), yielding approximately 1.7 × 10 10A highly diverse library containing clones was obtained. Transformed SS320 cells were harvested and stored at -80°C in 15% glycerol. Bacterial glycerol stocks were used to inoculate a total of 600 ml of 2xYT with antibiotics selective for both the phagemid and the F' episome. Bacteria were grown to log phase and then infected with M13KO7 helper phage (New England Biolabs, Ipswich, MA, USA) using a multiple of infection of 5. Cultures were grown overnight and scFv-displaying phages were 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.

[0358] Table 6 - Oligonucleotide primers used in the construction of the AS20 humanized library. Sequences are formatted using the IUPAC nucleotide code. [Table 6]

[0359] result Genes encoding the AS20 CDR graft and AS20 humanized library scaffolds were synthesized and cloned into the pHAT4 phagemid vector. The AS20 humanized library was constructed by using an optimized Kunkel procedure and contained 1.7 × 10 10 Transformants were obtained. Sequencing of 96 randomly selected clones confirmed the intended introduction of diversity (data not shown).

[0360] conclusion AS20 CDR-grafted and AS20 humanized libraries were successfully constructed. In both cases, IGHV1-46 and IGKV1-39 were used as human framework scaffold genes. Binding of AS20 CDR-grafted to BSSL was evaluated in both 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). Some of the selected clones showed binding with affinity and specificity comparable to the parental IgG to the cognate target (human BSSL) and even better affinity to the mouse orthologue (Example 9). When analyzing the sequences of the selected clones (Example 11), it is clear that certain positions are enriched for specific residues. Interestingly, at some positions (e.g., VH:62, 64 and VL:115), there is preferential selection of amino acids beyond the double diversity, indicating that the strategy of introducing additional diversity was successful.

[0361] Example 6 - Phage display selection on human and mouse BSSL followed by screening and sequencing In this example, phage display selection was performed, allowing the isolation of scFv fragments specific for human and mouse BSSL.

[0362] Materials and Methods antigen During phage display selection, mouse BSSL and non-biotinylated and biotinylated human BSSL were used as target antigens. More specifically, two variants with different degrees of biotinylation and conjugation chemistry were generated. These were BSSL-b amine and BSSL-b glycan. hBSSL, b-hBSSL, hBSSL-b amine and mBSSL (Table 2) were used as targets for phage display selection in this example.

[0363] Phage display selection For all antigens, phage display was performed using four rounds of enrichment employing two human synthetic scFv phage libraries constructed in the laboratory, SciLifeLib2 and the AS20 humanized library (see Example 5). SciLifeLib2 is a natural human synthetic scFv library similar in design and structure to the one previously reported

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

[0364] Table 7 - Phage display tracks [Table 7] 1- Selection performed on biotinylated BSSL (amine-coupled, 10x) coupled to magnetic SAV beads (M280) 2- Selection performed on biotinylated BSSL (carbohydrate-based, BH8520) coupled to magnetic SAV beads (M280) 3- Selection performed on native antigens coated on the surface of immunotubes 4- Phage elution using competition with BSSL of another species (mouse or human) (trypsin is used for elution in all other cases)

[0365] Re-cloning Phagemid DNA was isolated from the third and fourth rounds of each selection track to allow the generation of soluble scFvs. In the pools, genes encoding the scFv fragments were subcloned into a screening vector to provide a secretion signal for the scFvs with a C-terminal triple FLAG tag and a hexahistidine (His) tag. The constructs were then transformed into TOP10 E. coli.

[0366] Primary ELISA and sequencing A total of 89-222 colonies from rounds 3 and 4 for each selection track were picked and cultured in 96-well plates and grown overnight. The expressed scFvs (supernatants) were screened by ELISA for binding to the native form of the respective selection target. Experiments in this screening process included the AS20 scFv previously constructed, generated, and characterized for binding to both human and mouse BSSL (see Example 2). Also included was the AS20 CDR graft as a scFv (see Example 5). Clones deemed positive in ELISA were subjected to DNA sequencing (GATC Biotech, Cologne, Germany).

[0367] Secondary ELISA and HTRF All sequence-specific clones identified for each selection track were further analyzed in a secondary screen by ELISA. Herein, 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 fluorometry).

[0368] result A total of 14 phage selection tracks were performed in parallel on four forms of BSSL using SciLifeLib2 and the AS20 humanized library. 89 222 clones were picked and analyzed from each of the 14 tracks. ELISA and HTRF binding screening and sequencing yielded a total of 68 unique scFv clones capable of binding to the orthologues of the BSSLs in which they were selected. Unexpectedly, no binding of the AS20 CDR-grafted scFvs could be detected.

[0369] conclusion Primary screening of a total of 2365 clones by ELISA yielded a total of 467 scFv fragments with potential binding affinity to human and mouse BSSL, which have been sent for sequencing. Secondary ELISA screening followed by HTRF and resequencing yielded a total of 68 sequence-unique scFv clones. These binding data suggest that their relative binding to human and mouse BSSL falls into three groups with the characteristic of recognizing one or both of these orthologues.

[0370] Sixty-four isolate scFvs were derived from the AS20 humanized library, of which only four were derived from SciLifeLib2, indicating that BSSL is a challenging target for phage display selection using naive antibody libraries.

[0371] Example 7 - ELISA and affinity ranking by SPR of 68 anti-BSSL scFvs In this example, the 68 unique scFvs generated in Example 6 were analyzed by ELISA and further ranked based on affinity using SPR. Together with previous binding data (ELISA and HTRF), these results were used as decision points to select candidates for further development.

[0372] Materials and Methods hBSSL and mBSSL (Table 2) were used as the BSSL reagents in this example.

[0373] ELISA Human and mouse BSSL were coated onto 384-ELISA well plates at 1 μg / ml in PBS overnight at 4° C. 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% Tween 20) and allowed to bind to the coated proteins. All samples were assayed in duplicate. Detection of binding was enabled via HRP-conjugated α-FLAG M2 antibody (Sigma-Aldrich #A8592) followed by incubation with TMB ELISA substrate (ThermoFisher Scientific #34029). Colorimetric signal development was stopped by adding 1 M sulfuric acid and plates were analyzed at 450 nm.

[0374] SPR Kinetic screening was performed on a BIACORE® T200 biosensor instrument (GE Healthcare). The α-FLAG M2 antibody (Sigma-Aldrich #F1804), which serves as a capture ligand, was immobilized on all four surfaces of a CM5-S amine sensor chip according to the manufacturer's recommendations.

[0375] FLAG-tagged scFv clones present in bacterial supernatants were injected and captured on the chip surface, followed by injection of either human or mouse BSSL at 50 nM and 200 nM, respectively. Surfaces were 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).

[0376] result ELISA Binding of the 68 scFv clones to directly coated human and mouse BSSL was confirmed for the majority of the clones. As observed in Example 6, the BSSL-binding clones can be divided into three groups with the characteristics of recognizing either human BSSL, mouse BSSL, or both human and mouse BSSL. The majority of the clones show preferential binding to human BSSL (data not shown).

[0377] SPR Analysis of the data was performed by visual inspection of the sensorgrams (not shown). In these studies, the majority of clones showed significantly higher affinity, lower K DIt was clear that the humanized clones had affinity in the same range as observed for AS20 scFv. Inspection of the sensorgrams also showed that many of the humanized clones exhibited affinities in the same range as observed for 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 mouse BSSL for these particular clones was also similar to that seen for AS20.

[0378] Several clones from the phage display selection track panned against mouse BSSL showed preferential binding to mouse BSSL over human BSSL, exemplified by clone S-SL048-66 (containing HCVR SEQ ID NO:61 and LCVR SEQ ID NO:95).

[0379] conclusion In this example, the binding of 68 previously identified scFv clones to BSSL was confirmed by ELISA. Kinetic screening was also performed on all 68 clones using a single concentration of human and mouse BSSL. As seen for AS20, a much larger proportion of clones had a dissociation constant K 100 for human BSSL than for mouse BSSL. D The predicted affinities for human BSSL were in the low nanomolar to nanomolar range, with the benchmark AS20 scFv having a K of 2 nM. D A small set of scFv clones showed higher affinity for mouse BSSL than for human BSSL, with the highest affinity being a K of 43 nM.D Corresponded to the value.

[0380] From all collected data, 38 clones (including 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 the 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 humanized library and not from SciLifeLib.

[0381] Example 8 - Conversion of 38 humanized BSSL-specific antibodies into hIgG4 S228P format and small-scale transient expression In this experiment, the 38 most promising humanized scFv clones (including 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 (parent clone) (including HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114) and the AS20 CDR graft (including HCVR SEQ ID NO: 144 and LCVR SEQ ID NO: 145) were also converted to IgG4 S228P.

[0382] Briefly, human IgG4 is considered to be the most Fc-silent natural IgG subclass in humans, i.e., it does not mediate major effector functions through the Fc portion of the antibody. Similar to IgG1, IgG4 has a serum half-life of 21 days. However, IgG4 tends to spontaneously dissociate in vivo into half IgG4 molecules, which can then combine with other circulating IgG4 molecules. This half-molecule exchange can be avoided by the introduction of a stabilizing mutation in the hinge region, namely S228P (Eu numbering; this is identical to Kabat numbering S241P)

[13] .

[0383] Genes encoding the VH and VL of 38 scFv clones AS20 and AS20 CDR-grafted were successfully transferred into a vector encoding the human IgG4 S228P subclass. ExpiHEK293 cells were transiently transfected and antibodies were expressed at small scale (4 ml) and purified with Protein A. Purity and integrity / monomer content were analyzed by SDS-PAGE and analytical size-exclusion chromatography (SEC).

[0384] Materials and Methods Sequence analysis The amino acid sequences of the HCVR and LCVR of the scFvs selected for IgG conversion are presented for clarity in the sequence listing in Table 8, along with an anti-hapten (4-hydroxy-3-nitrophenylacetyl, NP) antibody (anti-NP).

[0385] Table 8 - Antibodies used in this example [Table 8]

[0386] Figure 14 shows the sequence differences between the 38 humanized clones converted to hIgG4 S228P. In the AS20 humanized library, a total of 20 positions were targeted for diversification in CDR1 and CDR2 of the heavy chain and CDR1, CDR2 and CDR3 of the light chain. For comparison, AS20 and the CDR-grafted constructs are included in the figure.

[0387] In-fusion cloning Plasmid DNA of 38BSSL-specific scFv and AS20 were purified from bacterial cultures by standard miniprep procedures. Genes of AS20 CDR grafts were synthesized by Genscript. VH and VL regions were PCR amplified and inserted into the laboratory constructed vector pHAT-hIgG4-S241P using the Infusion HD Plus Cloning Kit (Clontech #638909). A representative example of the resulting full-length IgG sequence is that of S-SL048-11 hIgG4 S228P heavy chain (VH-CH1-hinge-CH2-CH3) corresponding to SEQ ID NO: 119 and S-SL048-11 hIgG4 S228P light chain (VL-CL) corresponding to SEQ ID NO: 120.

[0388] Transfection into HEK293, expression and purification Transfection of plasmid DNA into expiHEK293 cells in 4 ml cultures in 24 deep-well plates was performed using the ExpiFectamineTM 293 Transfection Kit (ThermoFisher Scientific #A14525). After 5 days of culture at 37°C, 6% CO2, 80% rH and 400 rpm, culture supernatants were mixed with Protein A-conjugated magnetic beads and purified on a KingFisher Flex apparatus. Immediately after elution with 0.1 M glycine pH 2.7, neutralization was performed by addition of 1 M Tris-HCl, pH 8.8, and buffer exchange into PBS was performed using a 96-well spin 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).

[0389] result Infusion cloning Thirty-eight unique scFvS, AS20 and AS20 CDR grafts were successfully converted into full-length human IgG4 antibodies as confirmed by sequencing.

[0390] HEK293 transfection, expression and purification The antibody was expressed in expiHEK293 cells and purified from the supernatant by protein A purification. The purity and integrity of the purified IgG was confirmed by SDS-PAGE (data not shown).

[0391] conclusion 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 apparatus, and all showed acceptable levels of purity as assessed by SDS-PAGE.

[0392] Example 9 - Binding of 38 hIgG4 S228P clones to human and mouse BSSL Described in this example is target binding analysis of 38 hIgG4 S228P clones by surface plasmon resonance (SPR) to confirm that binding to human and mouse BSSL is retained after conversion from scFv to IgG format (Example 8, Table 8).

[0393] Materials and Methods Kinetic parameters of the hIgG4 S228P clone were determined by SPR using a BIACORE® T200 (GE Healthcare). Single cycle kinetics were used to measure the affinity of purified hIgG4 molecules to human and mouse BSSL. Anti-Fab antibody (GE Healthcare, #28958325) was immobilized on a CM5 S sensor chip by primary amine coupling using NHS-EDC chemistry. hIgG4 was captured by the anti-Fab antibody, after which five different concentrations of hBSSL (1:5 dilution starting from 50 nM) or mBSSL (1:5 dilution starting from 500 nM) were injected over the surface. The sensor chip surface was regenerated with 10 mM glycine-HCl pH 2.1. The BSSL reagents 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 obtained using the software BIAevaluation. For mouse BSSL, steady-state analysis was performed by plotting the response level at equilibrium against each concentration, and the K was calculated using the BIAevaluation software. D The value was obtained.

[0394] result A single cycle kinetic approach was used to determine the affinity of the converted antibodies to non-biotinylated human and mouse BSSL. The resulting equilibrium dissociation constants (K D ) are listed in Table 9. For hBSSL, the data generally fit well to a 1:1 binding model. For mouse BSSL, the data did not always fit very well to a 1:1 binding model. Instead, the data were analyzed using steady-state analysis. The K determined for binding to human and mouse BSSL D Values ​​are K determined for the same clone in scFv format. D The values ​​were in the same range as those for the AS20 CDR-grafted clone (see Example 7). A low degree of binding to human BSSL was observed for the AS20 CDR-grafted clone (data not shown). However, the model fit was not considered accurate, so the K D No value was obtained.

[0395] Table 9 - Measured equilibrium dissociation constants (K D In some cases, the data were of such low quality that they were considered unreliable and are indicated here as "nd" (not determined). [Table 9]

[0396] conclusion Overall, the results show that the binding affinity of the antibodies to human and mouse BSSL is not affected by recloning into hIgG4 format. However, the AS20 CDR graft behaved differently. As shown in Example 6, the AS20 CDR graft showed no binding to BSSL when expressed in scFv format. However, in IgG format, a binding signal to human BSSL was observed.

[0397] Example 10 - Functional testing of 28 hIgG4 S228P clones using a flow cytometric displacement assay In this example, the 28 hIgG4 S228P antibodies (including 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) that have the highest binding affinity to human and / or mouse BSSL in Example 9 were analyzed using a flow cytometry-based displacement assay to identify CD14 of human BSSL. + The ability to block binding to monocytes was tested. Five antibodies were included as benchmarks; AS20 mIgG1 (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).

[0398] Materials and Methods Preparation of buffy coat Human blood was collected from a single healthy donor in vacutainer tubes supplemented with citrate anticoagulant (BD Vacutainer). The buffy coat, consisting of leukocytes and platelets, was isolated after centrifugation at 1300 × g for 10 min at room temperature in a swing-out bucket rotor.

[0399] Flow cytometry displacement assay Twenty-eight BSSL-specific hIgG4 antibodies and five reference antibodies at different concentrations (ranging from 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 mixtures were incubated at +4°C for 30 min to promote antibody binding to the BSSL. Buffy coat (50 μl) was then added to each antibody / b-hBSSL mixture, and incubation was continued at +4°C for another 30 min. Afterwards, 2 ml of FACS lysis solution (BD Biosciences) was added, and the cells were incubated at room temperature for 10 min to lyse the red blood cells and fix the white blood cells. The cells were then centrifuged at 200×g for 5 min 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.

[0400] 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 for 30 min at +4° C. and protected from light. Cells were then washed twice with FACS buffer, resuspended in 500 μl FACS buffer, and run on a BD LSRII flow cytometer (BD Biosciences). Finally, data were analyzed using FlowJo software (BD Biosciences).

[0401] result To investigate the monocyte population, CD14 + Cells were first gated out. Then, gated on CD14 + Binding of b-hBSSL to monocytes was detected with BB515-conjugated streptavidin and quantified as median fluorescence intensity (MFI) in the BB515 channel. + The ability of BSSL-specific hIgG4 and reference antibodies to displace b-hBSSL binding to monocytes was quantified as the decrease in BB515 MFI in monocytes after incubation with increasing concentrations of BSSL-specific hIgG4 or reference antibody.

[0402] Table 10 - Human CD14 + Ability of 28 BSSL-specific hIgG4 S228P and reference antibodies to block BSSL binding to monocytes. Note that not all antibodies were tested at all concentrations. [Table 10]

[0403] conclusion The molecular mass 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 corresponded to an approximately 1:1 molar ratio. Using the highest antibody concentration (3 μg per reaction), 9 out of 28 BSSL-specific hIgG4 S228P antibodies were shown to inhibit (displace) the binding of BSSL (1 μg per reaction) to monocytes by at least 60%. The most effective antibody for displacing binding was AS20 mIgG1, whereas AS20 CDR-grafted hIgG4, anti-NP hIgG4 and anti-α-synuclein mIgG1 had no effect on binding.

[0404] Example 11 - Generation of five pre-specified hIgG4 S228P antibodies and controls Based on the results obtained from the binding assays performed in Example 9 and the in vitro functional tests in Example 10, as well as the sequence content, five humanized hIgG4 S228P clones, namely S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116 and S-SL048-118, were selected for larger scale production (10 mg).

[0405] Two controls were also included; AS20 and an AS20 CDR-grafted clone. In addition, 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 chosen, hIgG4 S228P, is the same as that used previously in Example 8 for the evaluation of 38 BSSL-specific clones.

[0406] Materials and Methods Generation of antibodies Production of material was outsourced to Absolute Antibody (Oxford, UK). VH and VL sequence information of seven clones was sent to the company, where genes were synthesized and cloned into a vector encoding the human IgG4-S228P subclass. Antibodies were transiently expressed in mammalian HEK293 cells and subsequently purified by affinity chromatography with Protein A. Purity and integrity were assessed by SDS-PAGE and endotoxin levels determined by LAL chromogenic endotoxin assay.

[0407] The amino acid sequences of the eight antibodies correspond to the SEQ ID NOs shown in Table 11.

[0408] Table 11 - Antibodies used in this example [Table 11]

[0409] Surface Plasmon Resonance (SPR) Kinetic parameters of the hIgG4 S228P clone were determined by SPR using a BIACORE® T200 (GE Healthcare). Anti-Fab antibody (GE #28958325) was immobilized on a CM5 S sensor chip by primary amine coupling using NHS-EDC chemistry. hIgG4 antibody was captured by anti-Fab antibody, after which five different concentrations of hBSSL (1:5 dilution, 0.08-50 nM) or mBSSL (1:2 dilution, 50-800 nM) were injected over the surface. The sensor chip surface was regenerated with 10 mM glycine-HCl pH 2.1. In the case of binding to human BSSL, single cycle kinetic data were fitted to a 1:1 binding model and kinetic parameters were obtained using the software BIAevaluation. In the case of mouse BSSL, steady state analysis was performed by plotting the response level at equilibrium against each concentration and the K was calculated by the BIAevalution software. D The value was obtained.

[0410] result Generation of antibodies Ten milligrams of eight antibodies at concentrations of 4.6-5 mg / ml in 25 mM histidine, 150 mM NaCl, 0.02% P80 (pH 6.0) were received from Absolute Antibody. Purity was determined to be greater than 98% by SDS-PAGE and endotoxin levels were determined to be less than 0.05 EU / mg by LAL chromogenic endotoxin assay. Monomeric content of each clone was determined to be greater than 98% by analytical size exclusion chromatography.

[0411] SPR SPR was used to determine the affinity of the antibodies for human and mouse BSSL. Table 12 shows the different K values ​​of pre-specified clones for hBSSL and mBSSL binding. D For comparison, the K values ​​determined for the same clones generated in the laboratory are summarized. D Values ​​are also included (these experiments are described in Example 9).

[0412] Table 12 - Summary of SPR analysis results for antibodies in hIgG4 S228P format. * 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 data was of such low quality that it was deemed unreliable and is indicated here as "nd" (not determined). [Table 12]

[0413] conclusion Five pre-specified candidates and three controls generated by Absolute Antibody were analyzed for BSSL binding by ELISA (data not shown) and SPR. The results showed that the binding to BSSL was generally very similar to that observed for each of the lab-generated clones in the same IgG format (see Example 9). The results also showed that the isotype control anti-NP hIgG4 S228P did not bind to BSSL and should 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). Very similar results were obtained for the different clones reported in Example 10 (data not shown).

[0414] In this example, it was possible to measure the affinity of the CDR grafts for the first time. Here, AS20 CDR grafted hIgG4 S228P (comprising HC SEQ ID NO: 131 and LC SEQ ID NO: 132) showed clear binding to hBSSL at higher concentrations, but binding was significantly reduced compared to AS20 hsIgG S228P (comprising HC SEQ ID NO: 129 and LC SEQ ID NO: 130). The reduction in affinity measured here was about 100-fold for hBSSL, and for mBSSL, the affinity was too low to be determined. Thus, when the CDRs were grafted into the new framework, the affinity for the target was significantly reduced. The observed stronger BSSL binding of AS20 CDR grafted hIgG4 S228P compared to scFv AS20 CDR grafted (comprising HCVR SEQ ID NO: 144 and LCVR SEQ ID NO: 145, see Example 6) may be due to the different antibody formats.

[0415] Example 12 - Stability testing of five candidate antibodies In this example, stability studies of S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116 and S-SL048-118 in hIgG4 S228P format are described to investigate the biophysical stability of the antibodies. For comparison, both AS20 as hIgG4 S228P and hIgG1 LALA-PG (see Example 17) were included. AS20 CDR-grafted hIgG4 S228P and anti-NP hIgG1 LALA-PG isotype controls were also included. Analysis was performed by SDS-PAGE, analytical SEC, nanoDSF, and DLS.

[0416] Materials and Methods The nine different antibodies and their corresponding SEQ ID NOs included in the stability study are listed in Table 13. Antibodies were dispensed into vials and incubated at 5 mg / ml in 25 mM histidine, 150 mM NaCl, 0.02% P80, pH 6.0 at -80° C., +4° C., and +40° C. Samples were removed and analyzed according to the schedule in Table 14, starting on day 0.

[0417] Table 13 - Antibodies used in this example [Table 13]

[0418] Table 14 - Sample analysis schedule [Table 14]

[0419] The elution was performed using a BioSEC column (300A, 7.8×300 mm; πP.N. 5190-2511, Agilent) coupled to an Agilent 1100 system and eluting with 0.15 M sodium phosphate (Na x H y Analytical size exclusion chromatography (SEC) was performed using a running buffer of pH 6.8 (PO4). 280 and A 220 Proteins were detected by measuring absorbance at 37 °C. 20 μg of each sample was loaded onto the column. SDS-PAGE was performed using NuPAGE 4-12% Bis-Tris gels (InVitrogen NP0321BOX) with NuPAGE MES SDS running buffer (InVItrogen NP000202) according to the manufacturer's instructions. Samples were run under reducing or non-reducing conditions. Bands were visualized using SimplyBlue stain (Invitrogen LC6065) and quantified using a densitometric scanner (Oddyssey, Li-cor). 5 μg of each sample was loaded per well.

[0420] NanoDSF (differential scanning fluorometry) was performed using a Prometheus instrument (NanoTemper) applying a temperature gradient from 20 to 95 °C at a ramp of 1 degree / min. Fluorescence emission at 300 nm and 350 nm was recorded as well as the backscattering signal. 10 μL of sample at a concentration of 5 mg / ml was loaded for each sample. Data was analyzed using PR Stability Analysis v1.02 from Nanotemper.

[0421] Dynamic light scattering was performed using a Zetasizer Pro (Malvern). Scattered light was recorded and analyzed using ZS Explorer software v 1.0.0.436 and built-in algorithms. Samples were analyzed at 5 mg / ml.

[0422] result Size Exclusion Chromatography (SEC) Chromatograms of each sample were obtained according to the schedule shown in Table 14. Note that additional peaks appearing over time are of lower molecular weight, suggesting degradation of material. A plot of the total integrated area over time indicated that no material was lost in the prefilter or on the column.

[0423] Size exclusion chromatography data are shown in Figure 7. Looking at the percentage of the total area made up of the main peak, only small changes were observed at +4° C. At +40° C., a more pronounced effect was seen.

[0424] The highest reduction was observed for S-SL048-46 hIgG4 S228P and anti-NP hIgG1 LALA-PG. S-SL048-118 hIgG4 S228P, AS20 in hIgG4 S228P and AS20 hIgG1 LALA-PG showed moderate reduction, while CDR grafts showed virtually no reduction.

[0425] SDS-PAGE Samples were analyzed under reducing and non-reducing conditions as outlined in Table 14. The -80°C sample showed no additional bands, with the exception of S-SL048-46 hIgG4 S228P and anti-NP hIgG1 LALA-PG, which showed one weak band that constituted less than 1% each under reducing conditions. This was nearly identical to that observed for the day 0 sample. In fact, the day 0 sample showed an additional band that was not seen at -80°C analyzed at day 30, reflecting analytical variability. The non-reduced sample was nearly identical to the day 0 sample. At +4°C, no additional significant bands were seen at day 9 or day 30 under reducing or non-reducing conditions. At +40°C, all samples showed additional bands that appeared at days 9 and 30. Under reducing conditions, all candidates showed low molecular weight bands, i.e., below 25 kD, while AS20 hIgG4 S228P, AS20 hIgG1 LALA-PG and anti-NP hIgG1 LALA-PG all showed higher Mw bands, i.e., above 50 kDa on days 9 and 30. In both cases, these bands became more prominent over time. Under non-reducing conditions, additional bands appeared in all samples, with clear differences. This result suggests that candidates S-SL048-46 and S-SL048-106 may be more susceptible to degradation.

[0426] Nano DSF The samples were analyzed according to the schedule in Table 14 and the results are shown in Figure 8. The main observations that can be made from the nanoDSF data are the main melting temperatures T m A shift was visible upon storage at +40° C. for the AS20 hIgG4 S228P, AS20 hIgG1 LALA-PG formats, and the changes occurring with anti-NP hIgG1 LALA-PG.

[0427] For the candidate, the main observation was a second T that may reflect a major contribution from the Fab portion of the antibody. mThe amplitude of the changes was decreased (data not shown). 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. These differences are considered minor for all candidates. The CDR graft appeared to be the most stable of the samples tested.

[0428] Dynamic Light Scattering (DLS) The samples were analyzed by DLS on day 30. The results for each sample at three different temperatures are shown in Figure 9. At -80°C and +4°C, all samples show one peak with an apparent diameter of about 10-11 nm, in line with the expected result for a monomeric antibody. No larger particles could be observed in any of the samples, i.e. only protein molecules could be detected.

[0429] At +40°C, larger particles are evident to varying degrees. Using the mean Z-average and polydispersity values ​​as indices of size homogeneity (data not shown), S-SL048-106 showed the lowest amount of higher Mw particles. In contrast, AS20 hIgG1 LALA-PG and anti-NP hIgG1 LALA-PG showed the highest number of higher Mw particles, indicating that these two molecules are more prone to aggregation.

[0430] conclusion In conclusion, combining this data with an overall ranking of the most stable candidates for the hIgG4 S228P format appears to be in the following order: S-SL048-106, S-SL048-118 and S-SL048-116.

[0431] Example 13 - Epitope mapping of AS20 by HDX-MS In this example, hydrogen deuterium exchange mass spectrometry (HDX-MS) was performed on the chimeric AS20 IgG4 antibody along with human BSSL to map its epitope.

[0432] Methods and Materials 40 μL of a 2 mg / mL solution of human BSSL (SEQ ID NO: 138) in PBS was mixed with 92.5 μL of 1.7 mg / mL chimeric AS20 hIgG4 antibody (containing HC SEQ ID NO: 139 and LC SEQ ID NO: 140) in a 1:1 molar ratio. The BSSL / antibody complex was concentrated to 40 μL using a 10K centrifugal filter unit (Amicon Ultra, Merck). In parallel, a sample containing only BSSL without added antibody was similarly prepared. The samples were analyzed on an automated HDX-MS system (CTC PAL / Biomotif HDX) where the samples were automatically labeled, quenched, digested, washed and separated at 2°C. More specifically, the samples were mixed with 3 μL of BSSL (or BSSL / antibody complex) and 22 μL of deuterated PBS and incubated at 4°C for four labeling time points: 5 min, 30 min, 90 min and 180 min. 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 about 2.3 and the temperature to about 4°C. 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 for 3 min at 350 μl / min. Digested peptides were then separated by a linear gradient of 8–55% ACN in 0.1% formic acid for 18 min using a 2 mm ID × 50 mm length HALO C18 / 1.8 μm analytical column operated at 95 μL / min. An Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific) operating at 70,000 resolution, m / z 400 was used for analysis. The software Mascot was used for peptide identification, and all HDX-MS data were processed using HDExaminer (Sierra Analytics, USA). Statistical analysis was performed using 95% confidence intervals.

[0433] result Deuteration kinetics of 66 peptides were followed by HDX-MS, covering 57.9% of the protein. Deuterium labeling (5, 30, 90 and 180 min) and differential deuterium uptake kinetics were calculated for BSSL alone and in the presence of AS20. Several peptides close to the N-terminus showed statistically lower deuterium uptake in the presence of AS20 antibody, clearly mapping epitopes to this region. By considering overlapping peptides, the spatial resolution could be reduced, resulting in the three peptide regions shown in Table 15.

[0434] Table 15 - List of AS20 epitope regions suggested by HDX-MS experiments [Table 15]

[0435] conclusion In summary, the AS20 epitope was successfully mapped to the N-terminal region of BSSL. A cluster of three discontinuous peptide regions was identified with significantly lower deuterium uptake in the presence of AS20. These regions were mapped together on the three-dimensional structure of BSSL, showing that these peptides together constitute the conformational epitope of AS20. Example 21 describes the crystal structure of the AS20 Fab fragment in complex with hBSSL. Analysis of the three-dimensional structure allows us to confirm the results from the HDX-MS data and specifically define the amino acids involved in the interaction.

[0436] Example 14 - Epitope mapping of pre-designated antibody candidates by HDX-MS In this example, hydrogen deuterium exchange mass spectrometry (HDX-MS) was performed to map the BSSL epitopes of five pre-specified antibody candidates: 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 (mAb11,8 HC SEQ ID NO:127, LC SEQ ID NO:128).

[0437] Methods and Materials A 2 mg / mL solution of BSSL in PBS was mixed with different antibodies (5 mg / mL in 25 mM histidine, 150 mM NaCl, 0.02% P80, pH 6.0) in a 1:1 molar ratio. Samples were concentrated and buffer exchanged into PBS using 10K centrifugal filter units (Amicon Ultra, Merck). In parallel, samples containing only BSSL without added antibodies were subjected to the same procedure.

[0438] Samples were analyzed on an automated HDX-MS system (CTC PAL / Biomotif HDX) where samples were automatically labeled, quenched, digested, washed and separated at 2° C. More specifically, samples were mixed with 3 μL of BSSL (or BSSL / antibody complex) and 22 μL of deuterated PBS and incubated at 4° C. for four labeling time points: 5 min, 30 min, 90 min and 180 min. 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 about 2.3 and the temperature to about 4° C. 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 for 3 min at 350 μl / min. Digested peptides were then separated by a linear gradient of 8–60% ACN in 0.1% formic acid for 15 min using a 2 mm ID × 50 mm length HALO C18 / 1.8 μm analytical column operated at 95 μL / min. An Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific) operating at 70,000 resolution, m / z 400 was used for analysis. The software Mascot was used for peptide identification, and all HDX-MS data were processed using HDExaminer (Sierra Analytics, USA). Statistical analysis was performed using 95% confidence intervals.

[0439] result Deuterium kinetics of 54 peptides were followed by HDX-MS, covering 64% of the protein. Deuterium labeling (5, 30, 90 and 180 min) and differential deuterium uptake kinetics were calculated for BSSL alone and in the presence of any of the five antibodies. Several peptides close to the N-terminus showed statistically lower deuterium uptake in the presence of the antibody for each of the pre-specified candidates. By considering overlapping peptides, the spatial resolution could be reduced, resulting in the two peptide regions shown in Table 16.

[0440] Table 16 - List of common epitope regions suggested by HDX-MS experiments [Table 16]

[0441] In addition to the epitope region common to all pre-specified antibodies, one extra peptide was identified as having statistically lower deuterium incorporation in three antibodies, namely peptide 10 (amino acids 84-101; NIWVPQGRKQVSRDLPVM (SEQ ID NO: 4)) for S-SL048-46, peptide 24 (amino acids 174-180; VKRNIAA (SEQ ID NO: 5)) for S-SL048-116, and peptide 39 (amino acids 283-295; HYVGFVPVIDGDF (SEQ ID NO: 6)) for S-SL048-11 (Table 17). However, these signals were relatively weak.

[0442] It should be noted that, although spread out in the primary sequence, two epitope regions common to all five antibodies (amino acids 1-12 and 42-55) cluster together in the 3D structure (Figure 10). Peptide 24 (amino acids 174-180) is relatively close to this core region, whereas peptides 10 (amino acids 84-101) and 39 (amino acids 283-295) are more spread out.

[0443] Table 17 - Summary of peptides with differential deuterium incorporation kinetics in the presence of BSSL alone and any of the five antibodies [Table 17]

[0444] conclusion The epitopes of five prespecified antibody candidates were successfully mapped to the N-terminal region of BSSL. Two discontinuous peptide regions were identified with significantly lower deuterium uptake in the presence of the five antibodies.

[0445] Overlapping peptides can allow for a reduction in spatial resolution. For example, in the AS20 experiment (Example 13), the peptide corresponding to amino acids 1-6 was found to be unchanged upon deuterium exchange, whereas a longer overlapping peptide was (AKLGAVYTEGGF, amino acids 1-12, SEQ ID NO: 3). In other words, the epitope residues could be reduced to YTEGGF (amino acids 7-12) (SEQ ID NO: 1). In contrast, in this example, such a reduction could not be performed because the peptide corresponding to amino acids 1-6 was not detected.

[0446] In addition to the two common epitope regions, one additional peptide each was identified as a possible epitope region in three of the pre-specified candidates (S-SL048-46, S-SL048-116 and S-SL048-11). Although statistically significant, the magnitude of signal change for these peptides is relatively low and may not be important for the interaction. Nevertheless, the fact that peptide VKRNIAA (amino acids 174-180, SEQ ID NO:5) clusters in the core region of the three-dimensional space may suggest that this region is also part of the epitope. Of note, this peptide also overlaps with one peptide detected in the original mapping of AS20. The signal change of two more surrounding peptides, peptide 10 (amino acids 84-101, SEQ ID NO:4) and peptide 39 (amino acids 283-295, SEQ ID NO:6), can be explained by stabilization upon antibody binding (an "allosteric" effect).

[0447] Taken together, the data strongly suggest a shared common core consisting of two peptide regions somewhere between sequences 1-12 (SEQ ID NO:3) and 42-55 (SEQ ID NO:2) for all pre-specified candidates. These regions were also identified in previous epitope mapping experiments of AS20 described in Example 13 and are found to be important for the interaction in the crystal structure of the AS20 complex described in Example 21.

[0448] Example 15 - Immunogenicity evaluation This example describes the immunogenicity evaluation carried out by Abzena 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).

[0449] Methods and Materials The Lipum-provided sequences were analyzed for immunogenic potential using Abzena's iTope™ MHC class II prediction in silico algorithm. The iTope™ software predicts favorable interactions between peptide amino acid side chains and specific binding pockets (pocket positions p1, p4, p6, p7, and p9) in the open-ended binding groove of 34 human MHC class II alleles. These alleles represent the most common HLA-DR alleles found worldwide, with no weighting due to which are most commonly found in any ethnic group. Twenty of the alleles contain an "open" p1 conformation, and 14 contain a "closed" conformation in which the glycine at position 83 is replaced by a valine. Placement of key binding residues is achieved by in silico generation of 8 amino acid overlapping 9-mer peptides spanning the test protein sequence.

[0450] The results should be evaluated in light of the fact that all prediction methods of MHC class II binding inherently over-predict the number of T cell epitopes since they do not allow for other important processes during antigen presentation such as protein / peptide processing, recognition by the T cell receptor or T cell tolerance to peptides.

[0451] The locations of the p1 anchor residues in AS20 (including the first residue of the MHC class II core 9mer ligand) in S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116, S-SL048-118 and hIgG S228P formats were determined.

[0452] If 50% or more of the MHC class II-binding peptides (i.e., 17 or more of 34 alleles) had high binding affinity (score >0.6), such peptides were defined as “accidental high affinity” MHC class II-binding peptides.

[0453] MHC class II-binding peptides with scores >0.55 (but not the majority >0.6) and binding to >50% of alleles were defined as "chance intermediate affinity."

[0454] These criteria were modified in the case where a large aromatic amino acid (i.e., F, W, Y) occurs at the p1 anchor position such that the open p1 pocket of 20 of the 34 alleles allows binding of a large aromatic residue. If this occurs, the chance peptide is defined as binding to 10 or more of the subset of 20 alleles.

[0455] The p1 anchor positions of germline medium and high affinity binding peptides were also analyzed and are not expected to be a problem in healthy individuals due to T cell tolerance.

[0456] Positive iTope™ hits were BLAST searched against the TCED™ database of known positive peptides to reveal regions representing closely homologous peptides from the T cell epitope database (i.e., peptides known to induce T cell activation in the ex vivo EpiScreen™ T cell epitope mapping assay). Kabat numbering was used to label antibody sequences.

[0457] result TCED™ analysis of peptides previously tested in the ex vivo EpiScreen™ T cell assay revealed strong homology to several iTope™ MHC binding peptides. In Table 18, + indicates mismatched residues where the replacement is an amino acid with similar physicochemical properties, and - indicates other mismatched residues. The locations of key MHC class II pocket positions in both iTope™ and TCED™ sequences are shown in the bottom row of Table 18.

[0458] Table 18 - Results of TCED™ analysis [Table 18]

[0459] Table 19 shows the total number of chance medium and high affinity MHC class II binding peptides and TCED™ hits in iTope™ for each candidate sequence (AS20 is shown for reference only).

[0460] Table 19 - Summary of immunogenicity analysis results [Table 19]

[0461] All five candidate humanized clones contained fewer non-germline coincident MHC class II peptides compared to AS20 hIgG4 S228P. All variant sequences contained at least two TCED™ hits and partial homology (minimum 6 out of 9 positions) to peptides known to induce T activation in the EpiScreen™ ex vivo assay. Of the five variants, S-SL048-106 hIgG4 S228P represented the lowest immunogenicity risk based on the number of non-germline coincident high and medium affinity MHC class II binding peptides. Candidates were ranked according to: fewer coincident hits 106<46<11=118<116 more coincident hits.

[0462] It should be noted that in silico immunogenicity analysis inherently over-predicts.

[0463] Example 16 - Manufacturability assessment This example describes an in silico manufacturability assessment performed by Abzena of 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).

[0464] Methods and Materials The amino acid sequences of S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116 and S-SL048-118 in the hIgG4 S228P format were analyzed using Abzena's in silico confidence prediction algorithm. The identified confidence is then analyzed by structural content. Briefly, for each V-domain sequence, the following was analyzed: · Presence of deamidation sites; · Presence of isomerization sites; ·Possible oxidation sites; · Presence of N-linked glycosylation sites; · Presence of free cysteine.

[0465] In this specification, the IMGT CDR definitions and numbering are used throughout unless otherwise indicated.

[0466] Presence of deamidation sites Deamidation of asparagine residues can lead to structural changes, altered pharmacokinetics and efficacy, and possible immunogenicity. Asparagine residues as potential deamidation sites were analyzed for both amino- and carboxy-adjacent amino acid content using a method based on

[14] .

[0467] Presence of isomerization sites Aspartate isomerization sites were predicted by analyzing the sequences of known isomerization motifs (DG, DS, DT or DD) focusing on the CDR regions.

[0468] Possible oxidation sites Structural models of both the heavy and light chain variable domains were generated and methionine and tryptophan residues were identified and evaluated to determine whether they were potential candidates for oxidation due to their surface exposed nature. Oxidation of individual residues can affect the biological activity of the antibody and can have biological consequences, such as reduced efficacy or altered pharmacokinetics.

[0469] Presence of N-linked glycosylation sites The VH and Vκ sequences were analyzed based on the consensus N-linked glycosylation motif: -NXS / T- (X can be any amino acid except proline).

[0470] Presence of free cysteine The sequences were analyzed to identify unpaired cysteine ​​residues. The VH and Vκ domains each typically contain two canonical cysteines that form intrachain disulfide bonds in the folded molecule. Additional cysteines are predicted to be detrimental to folding and potentially cause problems such as aggregation.

[0471] result The analysis did not identify any potential N-linked glycosylation sites within any of the VH or Vκ sequences of the five candidate antibodies. No unpaired cysteines were identified within any of the VH or Vκ sequences of the five candidate antibodies. No potential oxidation sites were identified within the VH or Vκ sequences of the five candidate antibodies, and no potential deamidation sites (T 1 / 2 <10 days) or moderate (T 1 / 2 <25 days) were not identified.

[0472] Two potential isomerization sites were identified within the VH for all five clones within the CDRs or CDR-proximal regions. · Asp54(DG) is located within VH CDR2, so isomerization may affect antigen binding. · Asp72 is located close to the CDR in a region sometimes called "CDR4", so isomerization may affect antigen binding.

[0473] Table 20 - Sequence confidence summary [Table 20] A representative S-SL048-106 Fv with potential post-translational fidelity determination is shown in FIG.

[0474] In summary, no free cysteines, oxidation sites or N-linked glycosylation sites were identified in any of the five lead candidates. No likely deamidation sites were identified in S-SL048-11, S-SL048-116 or S-SL048-118. S-SL048-46 and S-SL048-106 contain a possible deamidation site within VHCDR2. Two possible isomerization sites were identified within the heavy chain of each of the five lead mutants.

[0475] Example 17 - AS20 Preparation and Binding Properties of hIgG1 LALA-PG and Anti-NP hIgG1 LALA-PG This example describes the generation and binding properties of AS20 and anti-NP (clone B1-8) antibodies of the human IgG1-LALA-PG subclass, hereafter referred to as AS20 hIgG1 LALA-PG (HC containing SEQ ID NO:115 and LC containing SEQ ID NO:116) and anti-NP hIgG1 LALA-PG (HC containing SEQ ID NO:117 and LC containing SEQ ID NO:118). The anti-NP antibody was included as an isotype control.

[0476] Materials and Methods Expression and purification IgG4 is the most Fc-inactive naturally occurring human subclass. However, several publications have shown that IgG4 can interact with FcR and complement in mice

[15] and humans. Therefore, human IgG1 with mutations at three positions, namely L234A, L235A and P329G (abbreviated as hIgG1 LALA-PG), was selected for this study, since it has been reported to be the most Fc-silent mutant available, i.e., to have no immune effector functions [7, 16].

[0477] Production of material was outsourced to Absolute Antibody (Oxford, UK). The VH and VL sequence information of AS20 was sent to the company, where the gene was synthesized and cloned into a vector encoding the human IgG1-LALA-PG subclass. The antibody was transiently expressed in mammalian HEK293 cells and then 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.

[0478] Surface Plasmon Resonance (SPR) Single cycle kinetics were used to measure the affinity of the antibodies. AS20 hIgG1 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 antigen concentrations (1:3 dilutions starting from 50 nM for hBSSL and 1:2 dilutions starting from 800 nM for mBSSL) were injected over the surface. The sensor chip surface was regenerated with 10 mM glycine-HCl pH 2.1. The obtained single cycle kinetic data were fitted to a Langmuir 1:1 binding model and kinetic parameters were obtained using the software BIAevaluation. In the case of mBSSL, a steady state analysis was also performed by plotting the response level at equilibrium against each concentration, and the K D Values ​​were obtained by BIAevaluation software.

[0479] result AS20 hIgG1 LALA-PG and anti-NP hIgG1 LALA-PG were successfully generated (data not shown).

[0480] To determine the affinity of IgG, a single cycle kinetic SPR technique was used. An equilibrium dissociation constant (K) of 0.91 nM was observed for the binding of AS20 hIgG1 LALA-PG to hBSSL. D ) was calculated. For mBSSL, K D was determined to be 361 nM. As expected, no binding to BSSL was observed for anti-NP hIgG1 LALA-PG.

[0481] conclusion This example describes the quality checks that were performed to confirm that AS20 hIgG1 LALA-PG is functional, i.e., binding to BSSL is comparable to that of AS20 in hIgG4 S228P format.

[0482] SPR analysis of AS20 hIgG1 LALA-PG showed that binding to BSSL was very similar to that observed for AS20 in other IgG formats. K D was determined by SPR to be 0.91 nM, which is consistent with the K of 0.6 nM to 3 nM determined for other IgG formats of AS20. D The affinity of AS20 hIgG1 LALA-PG for mBSSL was found to be comparable to the K values ​​(described in Examples 1, 2, and 4). In a steady-state affinity analysis, the affinity of AS20 hIgG1 LALA-PG for mBSSL was found to be K D value, which is the same as the previously estimated K D (155 nM, Example 1) and the appearance of the sensorgrams was similar (not shown).

[0483] Thus, the data indicated that binding of AS20 hIgG1 LALA-PG was not affected by subclass change. The results also showed that the isotype control anti-NP hIgG1 LALA-PG did not bind to BSSL and therefore may be suitable for use as a negative control in future analyses.

[0484] Example 18 - Efficacy of AS20 hIgG1 LALA-PG in a mouse model of rheumatoid arthritis In this example, the effect of AS20 hIgG1 LALA-PG (heavy chain SEQ ID NO:115 and light chain SEQ ID NO:116) was investigated in an in vivo mouse model of rheumatoid arthritis (RA), namely collagen antibody-induced arthritis (CAIA). An 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.

[0485] Model description – CAIA in mice Collagen antibody-induced arthritis (CAIA) in mice is an arthritis model that is both B-cell and T-cell independent. The disease was induced with antibodies against collagen type II (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 joint. A boost injection of LPS is required to reach a significant severity and incidence of disease. The disease course is highly predictable and is initiated after the LPS boost. The disease reaches a maximum severity around day 15, after which it decreases in severity until eventual cure. The study was approved by the local animal ethics committee Malmo / Lund, Sweden (M118-15).

[0486] Materials and Methods Induction of disease DBA / 1 mice (male, 8–9 weeks) were injected iv with a cocktail of monoclonal anti-CII antibodies (CIA-MAB-50, MD Bioproducts) at 2 mg / mouse on day 0. On day 5, mice were injected ip with LPS (50 μg / mouse) to promote disease.

[0487] Experimental groups and administration of test articles AS20 hIgG1 LALA-PG and isotype control antibody 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). Test articles (antibodies) were administered ip starting one day before disease induction (day -1) and then every 4 days on days 3, 7, 11 and 15. Based on the average 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 isotype control (anti-NP hIgG1 LALA-PG) at 90 mg / kg. The relatively high doses were chosen to compensate for the lower 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 dose on day -1 was given as a bolus dose, i.e., all test articles were given as a double dose divided into two injections, the first injection in the morning and the second in the afternoon. The following doses (days 3, 7, 11 and 15) were given as single doses. Animals were weighed before each dose and the dose volume of 20 ml / kg was based on the individual weight of the animal.

[0488] Table 21 - Experimental Groups [Table 21]

[0489] Disease evaluation Disease was assessed daily from day 3 in a blinded manner using a gross scoring system of all 4 paws ranging from 0 to 15 (1 point for swollen or red toes, 1 point for swollen or red middle toes or knuckles, and 5 points for swollen ankles), resulting in a maximum total score for each mouse of 60. Due to ethical restrictions, animals with a score above 45 were removed from the experiment.

[0490] blood sampling On study withdrawal day 19, blood was collected from all mice by cardiac puncture under deep anesthesia (96 hours after the last injection on day 15) into microfuge tubes containing LiHeparin. Samples were immediately placed on ice and centrifuged (2000 x g for 5 minutes at 4°C) within 20 minutes of collection. Plasma samples were aliquoted and frozen on dry ice. 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 battery of safety biomarkers (groups 1-3, n=42).

[0491] PK collection of satellite animals Two satellite animals were included per dose group of antibody-treated animals to obtain information on PK profiles and ADAs. Blood samples were collected from the sublingual vein (2 animals per dose group) 1 hour before and 24 hours after dosing on day 7 (i.e., day 8). Blood samples were also collected (2 animals per dose group) 1 hour before and 24 hours after dosing on day 15 (i.e., day 16).

[0492] Tissue collection Upon study cessation, spleens were removed from all animals and weighed. Fourteen spleens from the isotype control treatment group (Group 2) and 14 animals from the 90 mg / kg AS20 hIgG1 LALA-PG group (Group 3) were homogenized and analyzed by FACS as described in Example 19.

[0493] Drug exposure in plasma Liquid chromatography tandem mass spectrometry (LC-MS / MS) was used to analyze the exposure of AS20 IgG1 LALA-PG in plasma samples collected from satellite animals (see above) and all animals upon study discontinuation (day 19).

[0494] Anti-drug antibodies (ADA) Immunogenicity assessment was performed at study termination (day 19) by analysis of ADA in plasma samples collected from satellite animals (see above) and from all animals.

[0495] ADA was measured using enzyme-linked immunosorbent assay (ELISA). Briefly, maxisorp plates were coated with AS20 hIgG1 LALA-20 or isotype control antibody and blocked with 5% BSA, 0.05% Tween-20 in 1x PBS. Plasma samples or mouse plasma spiked with positive control antibody were added and plates were incubated for 2 hours at room temperature. Plates were washed and secondary antibody peroxidase AffiniPure goat anti-mouse IgG+IgM(H+L) (Jackson ImmunoResearch) was added and incubated for 1 hour at room temperature, after which plates were washed extensively and finally TMD substrate (Sigma-Aldrich) was used for detection.

[0496] Measurement of safety biomarkers 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 using clinical chemistry methods for 14 safety biomarkers (albumin, alanine aminotransferase, alkaline phosphatase, amylase, bilirubin, blood urea nitrogen, calcium, creatinine, globulin, glucose, phosphate, potassium, sodium and total protein) at the Department of Chemical and Drug Safety, RISE, Sodertalje, Sweden, using cassette #500-0038 and an Abaxis Vetscan system.

[0497] result Severity of arthritis CAIA-induced and vehicle-treated animals developed moderate to severe disease with 100% incidence. Similar results were seen in isotype control-treated animals. Efficacy of AS20 hIgG1 LALA-PG administered ip every 4 days from day -1 to termination was evaluated at three doses (10, 30 and 90 mg / kg). AS20 hIgG1 LALA-PG at 90 mg / kg and 30 mg / kg showed a significant ameliorative effect on disease compared to isotype control on days 7-14, 16, 18 and 7-12, respectively. A slight reduction in disease severity, although not statistically significant, was seen with AS20 hIgG1 LALA-PG administered at 90 mg / kg compared to vehicle (Figures 18 and 19).

[0498] Two animals, one in the AS20 hIgG1 LALA-PG 10 mg / kg group and one in the isotype control group, were removed pre-termination (day 12) for ethical reasons (high score). These animals were included in the maximum score but excluded from the mean, AUC and % inhibition (Figure 19, Table 22).

[0499] Statistics of disease parameters are summarized in Table 22.

[0500] Table 22 - Statistics for CAIA severity parameters [Table 22] 1 Cumulative incidence. Mice were considered to have developed disease if they received ≥ 1 point on 2 consecutive days of scoring. 2 Mean CAIA scores at all scoring time points. 3 Area under the curve. 4 Percent change in total disease burden in each animal relative to the isotype control treatment group. Calculated by taking the difference between the mean AUC of the isotype control group and the AUC of each individual animal, dividing by the AUC of the isotype control group, and multiplying by 100 * (-1) compared to the isotype control.* p < 0.05; ** p<0.01.

[0501] Health assessment General health assessments were performed daily from day 3 to termination, along with disease assessments. Mice were weighed twice weekly as part of general health assessments. No adverse effects from treatment were observed in the animals (data not shown).

[0502] Drug exposure in plasma Plasma exposure of AS20 hIgG1 LALA-PG at the end of the study on day 19 was within the overall expected concentration range based on previous single-dose pharmacokinetic evaluation. The mean concentration for the 10 mg / kg dose group was determined to be 199 μg / mL (approximately 1.3 μM, 58-309 μg / mL), for the 30 mg / kg dose group the corresponding mean concentration was determined to be 614 μg / mL (approximately 4.1 μM, 229-970 μg / mL), and for the 90 mg / mL dose group the corresponding mean concentration was determined to be 1408 μg / mL (approximately 9.4 μM, 520-2557 μg / mL). Thus, a slightly nonlinear dose-exposure relationship was observed. No indication of changes in plasma exposure over time of treatment due to immunogenicity was detected. The mean concentration of hIgG1 LALA-PG in plasma samples from mice administered the isotype control was determined to be 1815 (approximately 12 μM, 1224-2511 μg / mL).

[0503] Anti-drug antibodies (ADA) No worrisome ADAs were detected in the study samples. Although the majority of samples tested positive, titers were not higher in the AS20 hIgG1 LALA-PG group than in the control group. Given the early onset of response (approximately day 7) and unchanged drug exposure over time, it is possible that the detected anti-drug signal in the samples was mainly caused by nonspecific / low affinity IgM.

[0504] Safety Biomarkers Glucose was increased in plasma after treatment with both AS20 hIgG1 LALA-PG and isotype control antibody. None of the liver injury biomarkers were increased by high dose AS20 hIgG1 LALA-PG. There was a trend towards increased plasma creatinine in the AS20 hIgG1 LALA-PG treatment group compared to the isotype control group, although this was not statistically significant (p<0.06). Other renal markers, i.e. blood urea, electrolytes or total protein, were not different between AS20 hIgG1 LALA-PG and isotype control treated mice.

[0505] conclusion CAIA-inducing isotype control treated mice developed moderate to severe disease with 100% incidence, and the same results were seen in vehicle treated mice.

[0506] AS20 hIgG1 LALA-PG administered at 90 mg / kg and 30 mg / kg demonstrated significant ameliorative effects on disease compared to isotype control on days 7-14, 16-18, and 7-12, respectively. Although not statistically significant, a slight reduction in disease severity was seen with AS20 hIgG1 LALA-PG administered at 90 mg / kg compared to vehicle.

[0507] Plasma exposure of AS20 IgG1 LALA-PG at the end of the study was generally within the expected concentration range. No indication of changes in plasma exposure over time on treatment due to immunogenicity was detected. No worrisome ADAs were detected in study samples.

[0508] None of the liver injury biomarkers were increased by high dose AS20 IgG1 LALA-PG. There was a trend towards increased plasma creatinine in the AS20 hIgG1 LALA-PG treatment group, although this was not statistically significant. Other renal markers, i.e. blood urea, electrolytes or total protein, did not differ between AS20 hIgG1 LAL.

[0509] Example 19 - FACS analysis of spleens from CAIA experiments In this example, the effect 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) was examined 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.

[0510] Materials and Methods This study is part of the in vivo experimental study (Example 18) described above. Briefly, CAIA was induced in DBA / 1 mice (male, 8-9 weeks old) by intravenous (iv) administration of an antibody to collagen type II, followed by intraperitoneal (ip) administration of LPS to promote disease development. Mice were treated with ip administration of AS20 hIgG1 LALA-PG or isotype control antibody (anti-NP hIgG1 LALA-PG) every 4 days starting 1 day before disease induction (day -1). At study termination (day 19), spleens of animals treated with the highest dose of AS20 hIgG1 LALA-PG (90 mg / kg) or isotype control (90 mg / kg) were removed, weighed and homogenized as described below. A FACS panel was designed to identify T cells, B cells, NKT cells, neutrophils, eosinophils, NK cells, dendritic cells, monocytes and macrophages.

[0511] Single cell suspension To obtain a single cell suspension, spleens were passed through a 70 μm cell strainer in RPMI-1640 culture medium (Thermo Fisher, HyClone). Cells were pelleted and resuspended in 1 ml of Milli-Q water, red blood cells were lysed (10 s), and 1 ml of 2× PBS was added, followed by 10 ml of 1× PBS. Cells were washed with 10 ml of HBSS (Thermo Fisher, HyClone) and finally resuspended in the appropriate volume of HBSS to obtain a 10×10 6 cells / ml was obtained.

[0512] FACS protocol 1×10 6 Cells were seeded into each well of a 96-well round-bottom plate, centrifuged at 850×g for 1 min at 4° C., washed with FACS buffer (1×PBS, 3% FBS, 2 mM EDTA), and pelleted again. Purified rat anti-mouse CD16 / CD32 (Fc block) diluted 1:50 in FACS buffer was added to each well and cells were incubated for 15 min, then washed again with FACS buffer. Antibody mixes were prepared by adding the following antibodies to the appropriate volume of FACS buffer: FITC hamster anti-mouse CD3ε (1:100) PE Rat Anti-Mouse CD86 (1:100) PE-CF594 Rat anti-mouse Ly-6C (1:50) PE-Cy7 Rat anti-mouse Ly-6G (1:200) Biotin hamster anti-mouse CD49b (1:200) APC-Cy7 hamster anti-mouse CD11c (1:50) Alexa Fluor 647 Rat Anti-Mouse IA / IE (MHCII) (1:200) Alexa Fluor 700 mouse anti-mouse CD45.2 (1:100) BV421 Rat anti-mouse Siglec-F (1:200) BV605 Rat anti-CD11b (1:200) BV650 Rat anti-mouse CD19 (1:100) BV786 hamster anti-mouse CD80 (1:200) eF506 Fixable Viability Dye (1:400)

[0513] 25 μl of antibody mix was added to the cells in each well and incubated on ice for 20 min protected from light. Cells were washed with FACS buffer and 25 μl of secondary antibody mix (PerCP-Cy5.5 streptavidin, diluted 1:200 in FACS buffer) was added and cells were incubated on ice for 20 min protected from light. Cells were then washed twice with FACS buffer, resuspended in 250 μl FACS buffer, transferred to FACS tubes and finally analyzed on a CytoFLEX flow cytometer platform (Beckman Coulter).

[0514] Gating Strategy Cell subsets were identified as follows: T cells: CD45.2 + , CD11b - , CD3 + B cells: CD45.2 + , CD11b - , CD19 + NKT cells: CD45.2 + , CD11b - , CD3 + , CD49b + Neutrophil:CD45.2 + , CD11b + , Ly6G + Eosinophils: CD45.2 + , CD11b + , Ly6G - , SiglecF + NK cells: CD45.2 + , CD11b + , Ly6G - , SiglecF - , CD49b + Dendritic cells: CD45.2 + , CD11b + , Ly6G - , SiglecF - , CD11c + , MHCII + Monocytes: CD45.2 + , CD11b + , Ly6G - , SiglecF - , CD11C - , Ly6C + Macrophages: CD45.2 + , CD11b + , Ly6G - , SiglecF - , CD11C - , Ly6C - , MHCII +

[0515] Furthermore, the expression of CD80 and CD86 on dendritic cells was analyzed.

[0516] result Body weight was not significantly different between the AS20 hIgG1 LALA-PG treatment group (Group 3, Example 18) and the isotype control group (Group 2, Example 18) 2 days prior to disease induction (AS20, 23.9±1.4 g; isotype control, 23.8±1.2 g) nor at study discontinuation 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 treatment group compared to the isotype control group (110±23 mg vs. 88±21 mg; p=0.02), and CD45 + The total number of leukocytes was also higher in the AS20 group compared to the isotype control group (44.5 × 10 6 vs. 39.0 x 10 6 ;p=0.008).

[0517] The total number of B cells in the spleen was higher in AS20 hIgG1 LALA-PG-treated mice compared to isotype control-treated mice (27.1 ± 3.5 × 10 6 vs. 21.0±5.8×10 6; p = 0.004). In contrast, the total number of NK cells was reduced in AS20 hIgG1 LALA-PG-treated mice compared to isotype control-treated mice (0.44 ± 0.09 × 10 6 vs. 0.54±0.09×10 6 ; p = 0.01). The total number of NKT cells was also reduced in AS20 hIgG1 LALA-PG treated mice compared to isotype controls, although this was not statistically significant (0.14 ± 0.03 × 10 6 vs. 0.16±0.03×10 6 ; p=0.08). No significant differences were observed in the total numbers of neutrophils, eosinophils, monocytes, macrophages, dendritic cells or T cells between AS20 IgG1 LALA-PG and isotype control treated mice (Figure 20).

[0518] CD45 in spleen + The percentage of B cells among the IgG1 cells was higher in AS20 IgG1 LALA-PG-treated mice compared to isotype control-treated mice (60.9±5.4% vs. 53.0±10.0%, p=0.02). In contrast, CD45 + The percentage of T cells, NKT cells and NK cells among the cells was found to be lower in AS20 hIgG1 LALA-PG treated mice compared to isotype control 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). There was a significant difference in CD45 expression between AS20 IgG1 LALA-PG treated mice and isotype control treated mice. + No significant differences were observed in the percentages of neutrophils, eosinophils, monocytes, macrophages, or dendritic cells among leukocytes (Figure 21). There were no significant differences between AS20 IgG1 LALA-PG-treated and isotype control-treated mice in cells expressing CD80 or CD86, and no significant differences in CD45 + There was no significant difference in the total number or proportion of cells (data not shown).

[0519] conclusion Treatment with AS20 hIgG1 LALA-PG (90 mg / kg dose) significantly reduced the total number of NK cells and the percentage of T cells, NKT cells and NK cells in the spleens of mice with CAIA.

[0520] Example 20 - Analysis of the effect of pre-designated antibody candidates on BSSL enzyme activity In this example, the effect of five pre-specified antibody candidates in hIgG4 S228P format on BSSL enzyme activity was investigated: 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). Comparison was made with 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) in hIgG4 S228P format and isotype control anti-NP antibody (heavy chain SEQ ID NO: 133 and light chain SEQ ID NO: 134).

[0521] Methods and Materials Preincubation of BSSL with pre-specified antibody candidates Native human BSSL (SEQ ID NO: 138; Table 2) was used in this example. The BSSL stock solution was diluted 10-fold with MilliQ (MQ)-H2O to a concentration of 0.42 mg / ml.

[0522] Antibodies were diluted in MQ-H2O to a starting concentration of 0.3 μg / μl, followed by 1:1 serial dilutions in MQ-H2O to give six concentrations 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 mixtures were incubated for 1 h at +4° C. After incubation, 20 μl of MQ-H2O was added to each BSSL / antibody reaction.

[0523] Triglyceride hydrolysis assay In a round-bottomed glass vessel with a diameter of 30 mm suitable for ultrasound therapy, 50 μl 3 H-labeled triolein (triolein [9,10-3H(N)], 91CI / mmol) NET431001MC Perkin Elmer (Waltham, MA) was mixed with 25 mg of unlabeled triolein (Sigma Cat. No. 92860), the solvent was evaporated under nitrogen gas (N2) at room temperature, 1.0 ml of 10% gum arabic (Sigma Cat. No. 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 vessel, the vessel was cooled in ice water and sonicated for 10 min in 50% pulse mode using a Soniprep 150 (MSE, UK) with a 9 mm diameter flat tip probe at medium setting placed a few mm below the surface of the liquid, and 2.5 ml of 18.7% BSA (Sigma Cat. No. A7906), 2.5 ml of 1.0 M Triglyceride (TG) emulsions were prepared by adding NaCl, and 3.25 ml of MQ-H2O to the emulsion. The emulsions were used the same day they were prepared. 10 μl of pre-incubated BSSL / antibody solution (see above) was then mixed with 150 μl of TG emulsion, 10 mM sodium cholate (Sigma Cat. No. C-1254), and MQ-H2O in a total volume of 200 μl in a 13×100 mm glass tube. Samples were prepared in duplicate. The tubes were incubated at 37° C. for 15 min, after which the reaction was 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 min. From the upper aqueous phase containing the hydrolyzed free fatty acids, 1400 μl was removed and mixed with 2.0 ml of Optiphase Hisafe3 scintillation cocktail (Perkin Elmer, Waltham, MA) in a 6 ml polyethylene vial (Perkin Elmer) and hydrolyzed. 3The amount of 3H-labeled free fatty acids was measured by liquid scintillation counting in a WinSpectral 1414 (Wallac, Turku, Finland).

[0524] Cholesterol ester hydrolysis assay Add 40 μl of sonicated water to a round-bottomed glass vessel with a diameter of 30 mm suitable for sonication. 14 Cholesterol ester (CE) emulsions were prepared by adding C-labeled cholesteryl oleate (oleate-1-14C, NEC6380050UC, Perkin Elmer), evaporating the solvent under nitrogen gas at room temperature, adding 2.0 ml of 0.2 M Tris-HCl pH 7.5 and 0.85 ml of MQ-HO to the vessel, chilling the vessel in ice water, and sonicating for 10 min in 50% pulse mode using a Soniprep 150 (MSE) with a 9 mm diameter flat tip probe at the medium setting placed a few mm below the surface of the liquid until an emulsion was obtained. The emulsions were used on the same day they were prepared. Next, 10 μl of the pre-incubated BSSL / antibody solution (see above) was mixed with 100 μl of CE emulsion and MQ-H2O to a total volume of 200 μl in a 13 × 100 mm glass tube. Samples were prepared in duplicate. The tubes were incubated at 37 °C for 30 min, after which the reaction was 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 min. 400 μl was removed from the upper aqueous phase containing the hydrolyzed free fatty acids and mixed with 2.0 ml of Optiphase Hisafe3 scintillation cocktail (Perkin Elmer) in a 6 ml polyethylene vial (Perkin Elmer) to obtain the hydrolyzed free fatty acids. 3 The amount of 3H-labeled free fatty acids was measured by liquid scintillation counting in a WinSpectral 1414 (Wallac).

[0525] result Enzyme activity was assessed by measuring the release of free fatty acids (radioactively labeled) after 15 minutes (triglyceride hydrolysis assay) or 30 minutes (cholesterol ester hydrolysis assay) of incubation, respectively. See Figure 17. For technical reasons, samples had to be analyzed in two consecutive sets, but the two antibodies (AS20 and the isotype control anti-NP antibody) were included in both sets. Data are expressed relative to the value obtained without adding antibodies to the reaction, which was set to 100% (Tables 23 and 24).

[0526] Table 23 - Effect of five BSSL-specific hIgG4 S228P and hIgG4 S228P subtype control antibodies on BSSL enzyme activity (triglyceride hydrolysis) [Table 23]

[0527] Table 24 - Effect of five BSSL-specific hIgG4 S228P and hIgG4 S228P subtype control antibodies on BSSL enzyme activity (hydrolysis of cholesterol esters) [Table 24]

[0528] conclusion None of the antibodies tested, namely, the five pre-designated candidates, AS20 in hIgG4 S228P format, AS20 CDR graft and anti-NP, showed any significant effect on the enzymatic activity of BSSL.

[0529] Example 21 - Epitope mapping of AS20 by X-ray crystallography X-ray crystallography was used to determine the three-dimensional structure of AS20 in complex with hBSSL. In this experiment, the antibody was cleaved to generate Fab fragments and a new C-terminally truncated hBSSL (t-hBSSL) construct was generated, corresponding to amino acids 1-530 of hBSSL, followed by AHHHHHH (SEQ ID NO: 146).

[0530] Materials and Methods Cloning, expression and purification of t-hBSSL Previously, human BSSL was crystallized in a truncated form lacking the flexible C-terminal tail. Therefore, to be able to generate crystals of AS20 Fab and hBSSL, a new truncated BSSL construct was made containing a C-terminal his-tag for purification. The construct gp67-BSSL-6xH, consisting of amino acids 1-530+AHHHHHH (BSSL numbering based on the sequence after removal of the signal peptide), was ordered from GeneArt and cloned into pFastBac tGFP Dual vector prepared for ligation-independent cloning (LIC). LIC was performed using the InFuSion cloning kit and transformed into Stellar competent cells.

[0531] Recombinant bacmid DNA was produced in DH10Bac E. coli cells. Transfection of bacmid into Sf9 cells was carried out for 120 h at 27°C. P1 virus was collected from the growth medium and then used to generate P2 virus stock. After 96 h, P2 virus stock was harvested by centrifugation. 400 ml of Sf9 cells (1.5 × 10 6 10 cells / ml) were infected with 2 ml of P2 virus stock and grown for 72 hours post-infection. The medium (P3 stock) was harvested by centrifugation and filtered. For large-scale expression, 35 ml of P3 stock virus was transferred to 2130 ml of Sf9 cells (1.6×10 6 Expression was carried out for 72 hours at 27°C. At harvest, the cell density was 2.34 x 10 6cells / ml, 75% expressed GFP, and viability was 89%. Cultures were centrifuged to remove cells. t-hBSSL was purified from the medium by batch IMAC using Ni Sepharose 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, pooled, and concentrated.

[0532] Complex formation between AS20Fab fragment and t-hBSSL Generation of AS20 antibody was performed at Absolute Antibody (Oxford, UK). Fab fragments were generated following the papain digestion protocol from the immobilized papain supplier (Thermo Scientific, product number 20341). A total of 15 mg of AS20 was concentrated to 17 mg / ml and buffer exchanged into 20 mM sodium phosphate, 10 mM EDTA pH 7, 20 mM cysteine. The antibody was incubated with immobilized papain (0.6 ml slurry) for 3 hours at 37°C, followed by overnight incubation at 4°C. As cleavage was not complete, the sample was incubated for a further 3 hours at 37°C and then incubated at RT 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 RT for approximately 30 minutes before being loaded onto a Superdex 200 column in 50 mM Hepes pH 7.0, 500 mM NaCl. The first major peak at approximately 60 ml elution volume contained the complex of t-hBSSL and AS20 Fab fragment. The relevant fractions were concentrated (10K MWCO) and diluted with 50 mM Hepes pH 7.0 buffer to reduce the salt concentration to 250 mM.

[0533] Crystallization and structural analysis of AS20 Fab in complex with t-hBSSL After purification of the Fab t-hBSSL complex, the sample was kept on ice at 4°C for approximately 36 hours while being transported between laboratories. The complex was then concentrated using 500 μL of Vivaspin with 30000 MWCO (Vivaspin 500, VS0122 from Sartorius) from 4 mg / mL to a final concentration of 17.3 mg / mL. The concentration was measured at A280 on a nanodrop with an extinction coefficient of 174,375 and Mw of 106.242 kDa.

[0534] Crystallization experiments were set up in Swiss CI XTAL SD-3 3-well plates with 35 μL reservoir solution and 150+50, 100+100, 50+150 nL droplets of protein + reservoir using a Mosquito liquid handling robot. Crystals were obtained at 20°C under conditions B9 and B10 using Molecular Dimensions' screen Morpheus. Crystals with rod-like morphology appeared during the first 12 h and grew larger during the next 24 h. These were cryo-cooled and quickly transferred to reservoir solution containing additional 10% glycerol before being placed in liquid nitrogen. Data were collected at the Biomax beamline with a MaxIV. Auto-processed data files were used to solve the structure by molecular replacement in Phaser using 1f6w.pdb (hBSSL) and 4n0y.pdb (Fab fragments) as search models. Water molecules were removed as they occur in the very C-terminal part of BSSL, and for 4n0y.pdb only the Fab chains were kept, which were modelled on polyalanine. Manual model building was performed in Coot and the model was refined with Refmac 5 (all of the CCP4 suit).

[0535] result Crystallization and structure determination of AS20 Fab t-hBSSL The AS20 Fab fragment could be crystallized together with t-hBSSL in Morpheus condition B9, which contains 30 mM NaBr, 30 mM NaFI, 30 mM NaI, 0.1 M Tris (base), 0.1 M bicine (buffer set to pH 8.5 with these two buffers), 20% PEG550 MME and 10% PEG 20K. The crystals belonged to space group C2, diffracting to 2.5 Å with unit cell sizes of 323, 67, 123, 90, 101.7 and 90. The structure was solved by molecular replacement, and two complete complexes were present in the asymmetric unit. The crystals packed around the two copies are different, resulting in small variations in the t-hBSSL structure. Notably, the last residues, including the 6-His tag, are found in molecule A but not B, due to fewer crystal contacts in this region and more room for flexibility of the C-terminus. The region between residues 272 and 283 is also highly disordered in B and cannot be modeled. Apart from these discrepancies, the two t-hBSSL models superimpose very well.

[0536] Analysis of the interaction interface between AS20 Fab and t-hBSSL The BSSL structure has been described as having a large core region consisting of a twisted 11-stranded β-sheet surrounded by alpha helices and connecting loops [9]. At the N-terminus there is a smaller three-stranded β-sheet. The structure has been likened to an oven glove on a left hand with the palm containing the active site triad close to the "thumb". From this analogy, the small N-terminal β-sheet is located at the back of the hand close to 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 the alpha C, the third α-helix in the structure

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

[0537] The epitope region is listed in Table 25 and includes residues 7-12 (strands 1 and 2, SEQ ID NO: 1), 42-55 (part of the loop region leading to strand 3 of the sheet, SEQ ID NO: 2) and 174-180 (C-terminal part of alpha C, SEQ ID NO: 5). The epitope is rather flat, with only a few characteristic residues protruding, namely Tyr7, Phe12 and Gln52 (major interactions listed in Table 245). The loop region from 47 to 54 forms a well-defined and uniform surface. Proline 47 is important for stacking interactions with Tyr31 of the Fab, but overall the surface is flat here. Many of the residues in the epitope sequence are important for the folding of BSSL, but do not interact specifically with AS20.

[0538] Table 25 - List of BSSL residues involved in the interaction of BSSL with Fab AS20 with the most important interactions ranked in the top row. BSSL residues important for BSSL folding in the epitope region are also listed. [Table 25]

[0539] conclusion This example describes the crystallization and structural analysis of AS20 Fab and t-hBSSL. The structures reveal that the epitope region is located in the same region as previously identified by HDX-MS as described in Example 13. This is a three-dimensional epitope consisting of a small beta sheet, a neat loop region leading into strand 3 of the sheet, and the C-terminal part 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), which are spread apart in the sequence but close together in the structure. The most well-defined residues are Tyr7, Phe12, and Gln52, which protrude from the surface.

[0540] The sequences of the five pre-specified antibodies were analyzed in the context of the AS20 Fab t-hBSSL structure. Sequence differences, which are largely conservative, and the results of HDX-MS mapping (Example 14) indicate that all five antibodies bind to the same epitope on BSSL.

[0541] Example 22-S-SL048-116 Complex formation, crystallization, structure determination and epitope analysis of Fab-BSSL complex Materials and Methods S-SL048-116 cleavage by FabRICATOR S-SL048-116 antibody (heavy chain SEQ ID NO: 125 and light chain SEQ ID NO: 126) in PBS was cleaved and F(ab')2 purified using the FragIT kit (Genovis) according to the manufacturer's instructions.

[0542] Reduction of F(ab')2 fragments and purification of Fab' fragments Large scale reduction of the purified F(ab')2 fragment was performed with cysteamine at a final concentration of 50 mM in PBS pH 7.2 containing 5 mM EDTA for 2 h at room temperature. Four-fifths of the resulting sample was purified on a HiLoad 26 / 60 Superdex 200 prep 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.

[0543] Alkylation of Fab' fragments and subsequent purification The remaining fifth of the reduced sample was treated with an equal volume of 375 mM iodoacetamide at room temperature for 30 min to block free cysteines by alkylation. The resulting alkylated sample was purified exactly the same as the non-alkylated sample. The total yield was 2 mg.

[0544] Expression and purification of BSSL P2 virus stocks used for expression were obtained from SciLifeLab (Stockholm, Sweden). The ExpiSf9 insect cell line was used in ExpiSf CD medium. Expression using the ExpiSf Protein Expression Kit (ThermoFisher Scientific) was performed according to the manufacturer's recommendations using the virus load recommended by SciLife Lab.

[0545] Culture supernatant from 2L expression was supplemented with EDTA-free protease inhibitor tablets (Merck Millipore), NiSO4 and imidazole (final concentration 15 mM). Any particulates were removed by centrifugation at 13000×g. The supernatant was loaded onto a 5 ml HisTrap Excel column (GE Healthcare) at 4° C. overnight at a flow rate of 2 ml / min. The column was washed with 6 column volumes (CV) IMAC buffer A (50 mM Tris-HCl, 500 mM NaCl; pH 7.5) containing 15 mM imidazole. The column was washed again with 10 CV of IMAC buffer A containing 25 mM imidazole. Bound proteins were eluted with a 20 CV linear gradient to 100% IMAC buffer B (50 mM Tris-HCl, 500 mM NaCl, 0.5 M imidazole, pH 7.5). The eluted protein was concentrated, centrifuged, and loaded onto a HiLoad 26 / 600 Superdex 200prep grade gel filtration column (GE Healthcare) pre-equilibrated with SEC buffer (50 mM HEPES, 500 mM NaCl, 2 mM EDTA; pH 7). Fractions from the peak of interest (1E6-1F8) were pooled and concentrated. The total yield was 6.2 mg, and the purity of the sample was approximately 85%.

[0546] BSSL:Fab' complex formation and purification Prior to complex preparation, BSSL was evaluated by SEC on a HiLoad 26 / 600 Superdex 200 prep grade column to establish that concentration / storage did not induce oligomerization. The complex was mixed at a molar ratio of 1:1.3 BSSL:Fab' and incubated on ice for 1 hour. The incubated complex was run on the same SEC column using the same buffer as for BSSL. Fractions of interest (B3-C4) were pooled and buffer exchanged into 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 ready for crystallization. The purity of the sample was >90% as estimated by SDS-PAGE analysis.

[0547] BSSL:Alk-Fab' complex formation and purification The complexes were mixed at a molar ratio of 1.1:1 BSSL:Alk-Fab' and incubated on ice for 1 hour. The incubated complexes were run on the same SEC column using the same buffer as for the BSSL:Fab' complex. Fractions of interest were pooled and buffer exchanged into 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 ready for crystallization.

[0548] Crystallization and freezing of Fab-BSSL complexes The best diffracting crystals were grown at 20°C from 14.5 mg / ml of Fab-BSSL complex in buffer (50 mM HEPES, 250 mM NaCl, 2 mM EDTA, pH 7.0) and mixed with a reservoir (16% (w / v) PEG4000, 0.1 M sodium citrate pH 5 and 6% (v / v) ethanol) and the following seed solution: 150 nl of complex + 37 nl of seeds (crushed crystals in 0.1 M sodium citrate pH 5.0, 20% (w / v) PEG4000 and 0.2 M ammonium sulfate) + 113 nl of reservoir.

[0549] Droplets were pipetted into an MRC plate with a 40 μl reservoir. Crystals appeared within a few days and were frozen in a cryosolution containing 20% ​​(v / v) glycerol, 16% (w / v) PEG4000, 0.1 M sodium citrate pH 5.0 and 3% (v / v) ethanol.

[0550] Data collection The dataset was collected at 100K on a BioMAX station, MAX IV, Lund, Sweden (λ = 0.97625 Å) equipped with an Eiger 16M hybrid pixel detector. The dataset was collected using an exposure time of 0.011 s and an oscillation of 0.1° per image, for a total of 360°. The data was processed to 2.5 Å in space group P21 using an autoPROC pipeline.

[0551] result Structure determination The Fab-BSSL structure was determined using molecular replacement using Phaser software with the 2.3 Å structure of the catalytic domain of human bile salt-activated lipase BSSL (from PDB: 1F6W) and the homologous 2.86 Å Fab structure (from PDB: 3NFP) as template. One complex was found in the asymmetric unit. The structure was refined with Refmac5, followed by Buster, and model building was performed with Coot. The final model included three protein chains with the heavy (H) and light (L) chains of BSSL (chain A) and S-SL048-116 Fab (Figure 22). The final model included amino acids 1-531 of chain A, excluding a flexible loop (amino acids 117-123) that was not seen in the electron density. The model included amino acids 1-227 of chain H and amino acids 1-211 of chain L. The first amino acid in chain H, Gln 1, is modeled to best fit the electron density as PCA pyroglutamic acid. In chain H, the two free cysteines (Cys133 and Cys225) are modeled as non-alkylated cysteines, although alkylation treatment of S-SL048-116 Fab was necessary for crystallization. There was no extra density to fit the methylamide groups that would have been attached to the sulfur atoms of the cysteines. Presumably, the cysteine ​​residues were at least partially alkylated, but the attached atoms are very flexible and not clearly seen in the electron density map. In addition, 87 water molecules are modeled.

[0552] Epitope analysis Epitope analysis was performed using the coordinates of the Fab-BSSL complex. The analysis was performed using the CONTACT software from the CCP4 suite of programs. S-SL048-116-Fab binds to BSSL via both the heavy and light chains. In the heavy chain, all three CDR loops are involved (CDR1, CDR2, and CDR3), whereas in the light chain, only CDR1 and CDR3 contact BSSL (Figure 23, Table 26). Also, Ile2, close to the N-terminus of the light chain, makes hydrophobic van der Waals interactions with BSSL. A network of hydrogen bonds stabilizes the interactions between the heavy chain and BSSL (7 hydrogen bonds in total) and between BSSL and the light chain (5 hydrogen bonds in total) calculated using PISA(QT) analysis. PISA(QT) analysis reveals a solvent accessible region of 426 Å. 2 is buried at the interface between the BSSL and the heavy chain, with an area of ​​468 Å 2 is buried at the interface between the BSSL and the light chain.

[0553] Table 26 - Summary of all interacting residues between S-SL048-116-Fab and BSSL from 0 to 4 Å [Table 26]

[0554] conclusion The results obtained in this example for S-SCL048-116 are consistent with those obtained from the HDX-MS study in Example 14.

[0555] Example 23 - Efficacy of S-SL048-116 in a mouse model of rheumatoid arthritis In this example, the effect of S-SL048-116 (heavy chain SEQ ID NO: 125 and light chain SEQ ID NO: 126) was investigated in an in vivo mouse model of rheumatoid arthritis (RA), namely collagen antibody-induced arthritis (CAIA). The CAIA model is described in Example 18. The study was approved by the local animal ethics committee Malmo / Lund, Sweden (02896-20).

[0556] Materials and Methods Induction of disease DBA / 1 mice (male, 8 weeks old) were injected iv with a cocktail of monoclonal anti-CII antibodies (CIA-MAB-50, MD Bioproducts) at 2 mg / mouse on day 0. To promote disease, mice were injected ip with LPS (50 μg / mouse) on day 5.

[0557] Experimental groups and administration of test articles 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). Test articles (antibody and vehicle) were administered ip every 4 days starting one day before disease induction (day -1) on days 3, 7, 11 and 15. Based on the average weight of the animals on day -2, S-SL048-116 was administered at three different doses, namely 10, 30 and 90 mg / kg. Human BSSL (K D The lower affinity (K = 0.5 nM) of S-SL048-116 for mouse BSSL compared with D = 40 nM). The experimental groups are summarized in Table 27. The first dose on day -1 was a bolus dose, i.e. the test article was given as a double dose, split into two injections, with the first injection in the morning and the second in the afternoon. The following doses (days 3, 7, 11 and 15) were given as single doses. Animals were weighed before each dose and the dose volume of 20 ml / kg was based on the individual weight of the animal.

[0558] Table 27 - Experimental Groups [Table 27]

[0559] Disease evaluation Disease was assessed daily from day 3 in a blinded manner using a gross scoring system of all 4 paws ranging from 0 to 15 (1 point for swollen or red toes, 1 point for swollen or red middle toes or knuckles, and 5 points for swollen ankles), resulting in a maximum total score for each mouse of 60. Due to ethical restrictions, animals with a score above 45 were removed from the experiment.

[0560] result Severity of arthritis CAIA-induced and vehicle-treated animals developed moderate to severe disease with 100% incidence. The efficacy of S-SL048-116 (SOL-116) administered ip every 4 days from day -1 to termination at three doses (10, 30 and 90 mg / kg) was evaluated. S-SL048-116 administered at 90 mg / kg showed an ameliorative effect on disease severity compared to vehicle (Figures 24 and 25).

[0561] For ethical reasons (high score), two animals were removed prior to termination, one in the vehicle group (day 15) and one in the 90 mg / kg group (day 12). These animals are included in the maximum score but are excluded from the mean, AUC and % inhibition (Figure 25, Table 28). One mouse in the vehicle group did not recover from the LPS boost, i.e., it was hypothermic, had a kyphotic posture and was very light in weight. This mouse is excluded from all results as it was removed prior to the onset of disease (day 7).

[0562] Statistics of disease parameters are summarized in Table 28.

[0563] Table 28 - Statistics for CAIA severity parameters [Table 28] 1 Cumulative incidence. Mice were considered to have developed disease if they received ≥ 1 point on 2 consecutive days of scoring. 2 Mean CAIA scores at all scoring time points. 3 Area under the curve. 4Percent change in total disease burden in each animal relative to the vehicle control treatment group. Calculated by taking 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).

[0564] Health assessment General health assessments were performed daily from day 3 through termination, along with disease assessments. Mice were weighed twice weekly as part of the general health assessments. No adverse effects from treatment were noted in the animals.

[0565] conclusion CAIA-induced vehicle-treated mice developed moderate to severe disease with 100% incidence. The efficacy of S-SL048-116 administered i.p. at three different doses, namely 10, 30 and 90 mg / kg, was evaluated on treatment start day -1. S-SL048-116 administered at 90 mg / kg had an ameliorative effect on disease severity compared to vehicle. No adverse effects were observed with treatment with S-SL048-116 or vehicle alone.

[0566] Example 24 - Analysis of cellularity in blood, spleen and mesenteric lymph nodes comparing BSSL knockout (KO) and wild type (WT) mice In this example, the abundance of different leukocyte subsets in blood, spleen, and mesenteric lymph nodes (MLN) from BSSL-deficient knockout (KO) mice and wild-type littermates was examined by fluorescence-activated cell sorting (FACS).

[0567] Materials and Methods Leukocytes were isolated for analysis from blood, spleen and MLN from 10 BSSL KO mice and 10 wild-type littermates (15-19 weeks old). Total leukocyte counts were determined for spleen and MLN using manual counting in a Burcher chamber. Cells isolated from blood, spleen and MLN were incubated with FC-Block (CD16 / C032 clone 2.4G2) followed by incubation with two different antibody cocktails, stain A and stain B.

[0568] Dyeing A The antibodies / clones / fluorochromes used for staining A were CD19 (ID3 / BB515), γδTCR (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).

[0569] The cell population identified by staining A was αβ T cells:CD45.2 + , TCRβ + CD4 αβ T cells: CD45.2 + , TCRβ + , CD4 + CD8 αβ T cells:CD45.2 + , TCRβ + , CD8 + γδT cells: CD45.2 + , TCRγδ + NKT cells: CD45.2 + , TCRβ + , NK1.1 + B cells: CD45.2 + , CD19 + NK cells: CD45.2 + , TCRβ - , TCRγδ-, NK1.1 + It was.

[0570] Staining B The antibodies / clones / fluorochromes used for staining B were MHC II (MS / 114.15.2 / Alexa488), FcεRlα (MAR-1 / PE), Siglec F (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).

[0571] The cell population identified by staining B was Myeloid cells: CD45.2 + , CD11b + Neutrophil:CD45.2 + , CD11b + , Ly6G + Eosinophils: CD45.2 + , CD11b + , Ly6G - , Siglec F + , S.S.C. 高 Monocytes: CD45.2 + , CD11b + , Ly6G - , Siglec F - , S.S.C. 低 , Ly6C + , MHCII - Macrophages: CD45.2 + , CD11b + , Ly6G - , Siglec F-, SSC 低 , Ly6C - , MHCII + Mast cells: CD45.2 + , CD11b + , FcεRlα + , CD117 + Basophil: CD45.2+ , CD11b + , FcεRlα + , CD117 - It was.

[0572] After incubation (20 min on ice), cells were washed, resuspended in FACS buffer and different cell subsets were identified by FACS.

[0573] result Total white blood cell counts in the spleen and MLN The number of leukocytes in the spleens of KO mice was found to be significantly reduced to approximately 50% of that in WT mice. In the MLN, no significant difference in the number of leukocytes was observed between KO and WT mice (Figure 26).

[0574] αβ T cells The total number of αβ T cells in the spleen was found to be reduced in KO mice compared to WT mice. This reduction was due to the expression of total CD45 + Similar to what was observed for leukocytes. No significant differences were observed in the numbers of αβ T cells in the MLNs. Total CD45 + The proportion of αβ T cells among CD4+ T cells was found to be higher in the spleen and blood of KO compared to WT mice, but no difference was observed in the MLN. + / CD8 + The ratio was higher in the KO mice compared to the WT mice.

[0575] γδ T cells The total number of γδ T cells in the spleen was found to be reduced in KO mice compared to WT mice. This reduction was due to the expression of total CD45 + This was similar to that observed for leukocytes. In MLNs, this reduction was even more pronounced in the spleen. + The proportion of γδ T cells in the spleen and blood was also found to be lower in the KO mice compared to the WT mice.

[0576] NKT cells The total number of NKT cells in the spleen was found to be reduced in KO mice compared to WT mice. This reduction was due to the expression of total CD45 + The results were similar to those observed for leukocytes. No significant differences in the numbers of NKT cells were observed in the MLNs. + The proportion of NKT cells in the spleen and MLN was lower in KO mice compared to WT mice.

[0577] B cells The total number of B cells in the spleen was found to be reduced in KO mice compared to WT mice. This reduction was due to the expression of total CD45 + The results were similar to those observed for leukocytes. No significant differences were observed in the numbers of B cells in the MLNs. CD45 in the spleen and blood + The proportion of B cells among the cells was found to be lower in KO mice compared to WT mice, but no such difference was observed in the MLN.

[0578] NK cells The total number of NK cells in the spleen was found to be reduced in KO mice compared to WT mice. This reduction was due to the expression of total CD45 + The difference was more pronounced in NK cells compared to that observed for leukocytes and other lymphocyte subsets. No significant difference was observed in NK cell numbers in MLNs. In the spleen and blood, CD45 + The proportion of NK cells among the cells was found to be lower in the KO mice compared to the WT mice, although no such difference was observed in the MLN (Figure 27).

[0579] Bone marrow cells The total number of bone marrow cells was decreased in both the spleen and MLN in KO mice compared with WT mice. + The proportion of bone marrow cells in the cells was found to be lower in the KO mice compared to the WT mice, but no such difference was observed in the blood.

[0580] neutrophil No significant difference was observed in the number of neutrophils in the spleen or MLN compared with KO and WT mice. The percentage of neutrophils in the blood was higher in KO mice, but no significant difference was observed in the spleen and MLN.

[0581] Eosinophils No significant difference was observed in the number of eosinophils in the spleen or MLN compared to KO and WT mice. The percentage of eosinophils in the blood was found to be higher in KO, but did not show significant differences in the spleen and MLN.

[0582] Monocytes The total number of monocytes was found to be lower in KO compared to WT mice in both the spleen and MLN, with no significant difference in the percentage of monocytes between KO and WT mice in any of the organs examined.

[0583] Macrophages The total number of macrophages was found to be lower in KO compared to WT mice in both the spleen and MLN, with no significant difference in the percentage of macrophages between KO and WT mice in any of the organs examined.

[0584] Mast cells No significant differences were observed in the number of mast cells in the spleen and MLN between KO and WT mice. The percentage of mast cells in the spleen was higher in KO mice compared to WT mice. No differences were observed in the blood or MLN.

[0585] basophil The total number of basophils in the spleen was found to be lower in the KO compared to the WT mice. No difference in the number of basophils was observed in the MLN. The percentage of basophils in the blood was found to be higher in the KO compared to the WT mice. No such difference was observed in the spleen or MLN.

[0586] conclusion CD45+ In addition to the overall effect on leukocytes, the data suggest further effects specifically on NK cells, namely CD45 + Both the total number and percentage of NK cells in the blood and spleen of untreated BSSL-deficient mice were lower compared to wild-type littermates. These data suggest that treatment with AS20 hIgG1 LALA-PG (90 mg / kg dose) significantly reduced the CD45 + This demonstrates that the total number of NK cells and the proportions of T cells, NKT cells and NK cells among the cells were significantly reduced (Example 19).

[0587] Table 29 - List of sequences and SEQ ID NOs used herein [Table 29] TIFF0007676356000030.tif239161 TIFF0007676356000031.tif239161 TIFF0007676356000032.tif239161 TIFF0007676356000033.tif239162 TIFF0007676356000034.tif239161 TIFF0007676356000035.tif239161 TIFF0007676356000036.tif238162 TIFF0007676356000037.tif239161 TIFF0007676356000038.tif239160 TIFF0007676356000039.tif239161 TIFF0007676356000040.tif238161 TIFF0007676356000041.tif239162 TIFF0007676356000042.tif239162 TIFF0007676356000043.tif239160 TIFF0007676356000044.tif238162 TIFF0007676356000045.tif239161 TIFF0007676356000046.tif238161 TIFF0007676356000047.tif239161 TIFF0007676356000048.tif239161 TIFF0007676356000049.tif239160 TIFF0007676356000050.tif239160 TIFF0007676356000051.tif85160

[0588] References

Table 30

Claims

1. specifically binds to bile salt-stimulated lipase (BSSL); Three complementarity determining regions (CDRs) (HCDRs) of the heavy chain variable region (HCVR); and The three CDRs (LCDRs) of the light chain variable region (LCVR) 1. An isolated antibody or antigen-binding fragment thereof comprising: The first HCDR consists of an amino acid sequence according to SEQ ID NO:7; The second HCDR consists of an amino acid sequence according to SEQ ID NO:8; The third HCDR consists of an amino acid sequence according to SEQ ID NO:9; The first LCDR consists of an 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 an amino acid sequence according to SEQ ID NO: 11; or The first HCDR consists of an amino acid sequence according to SEQ ID NO:7; The second HCDR consists of an amino acid sequence according to SEQ ID NO: 18; The third HCDR consists of an amino acid sequence according to SEQ ID NO:9; The first LCDR consists of an 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 an amino acid sequence according to SEQ ID NO: 21; or The first HCDR consists of an amino acid sequence according to SEQ ID NO:7; The second HCDR consists of an amino acid sequence according to SEQ ID NO:8; The third HCDR consists of an amino acid sequence according to SEQ ID NO:9; The first LCDR consists of an 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 an amino acid sequence according to SEQ ID NO: 11; or The first HCDR consists of an amino acid sequence according to SEQ ID NO:7; The second HCDR consists of an amino acid sequence according to SEQ ID NO: 19; The third HCDR consists of an amino acid sequence according to SEQ ID NO:9; The first LCDR consists of an 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 an amino acid sequence according to SEQ ID NO: 22; An isolated antibody or antigen-binding fragment thereof.

2. said HCVR consists of an 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; 2. The isolated antibody or antigen-binding fragment thereof of claim 1.

3. said HCVR consists of an 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; 2. The isolated antibody or antigen-binding fragment thereof of claim 1.

4. said HCVR consists of an 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; 2. The isolated antibody or antigen-binding fragment thereof of claim 1.

5. said HCVR consists of an 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; 2. The isolated antibody or antigen-binding fragment thereof of claim 1.

6. said HCVR consists of an 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; 2. The isolated antibody or antigen-binding fragment thereof of claim 1.

7. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, and a chimeric antibody or an antigen-binding fragment thereof.

8. The antigen-binding fragment may be a single chain variable fragment (scFv), a Fab fragment, an F(ab') ２ Fragment, F(ab') ３ 8. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a Fab' fragment, a Fv fragment, and a dAb fragment.

9. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the isolated antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

10. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the isolated antibody or antigen-binding fragment thereof is an isotype class selected from the group consisting of IgG, IgA, IgM, IgD and IgE.

11. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 10, wherein the isolated antibody or antigen-binding fragment thereof comprises at least one Fc-silencing mutation that inhibits interaction with an Fc receptor.

12. The isolated antibody or antigen-binding fragment thereof of claim 11, wherein the isolated antibody or antigen-binding fragment thereof is of the IgG isotype class and at least one Fc silencing mutation is selected from the group consisting of L234A, L235A and P329G.

13. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the isolated antibody or antigen-binding fragment thereof comprises at least one stabilizing mutation that prevents or reduces in vivo Fab arm exchange.

14. 14. The isolated antibody or antigen-binding fragment thereof of claim 13, wherein the isolated antibody or antigen-binding fragment thereof is of the IgG4 isotype subclass and at least one stabilizing mutation is S228P.

15. A pharmaceutical composition comprising the isolated antibody and / or antigen-binding fragment thereof of any one of claims 1 to 14 and a pharma- ceutically acceptable carrier or excipient.

16. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, or a pharmaceutical composition according to claim 15, for use as a medicament.

17. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, or a pharmaceutical composition according to claim 15, for use in the treatment and / or prevention of an inflammatory disease.

18. 18. The isolated antibody or antigen-binding fragment thereof, or pharmaceutical composition for use according to claim 17, wherein the inflammatory disease is a chronic inflammatory disease.

19. 18. The isolated antibody or antigen-binding fragment thereof, or pharmaceutical composition for use according to claim 17, wherein the inflammatory disease is a systemic inflammatory disease.

20. 18. The isolated antibody or antigen-binding fragment thereof, or pharmaceutical composition for use according to claim 17, wherein the inflammatory disease is an autoimmune disease.

21. 18. The isolated antibody or antigen-binding fragment thereof, or pharmaceutical composition for use according to claim 17, wherein the inflammatory disease is an autoinflammatory disease.

22. 18. The isolated antibody or antigen-binding fragment thereof, or pharmaceutical composition for use according to claim 17, wherein the inflammatory disease is a natural killer (NK) cell-mediated inflammatory disease.

23. 18. The isolated antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 17, wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), psoriatic arthritis, inflammatory bowel disease (IBD) such as Crohn's disease or ulcerative colitis (UC), and hepatic spondylosis.

24. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

25. 25. An expression vector comprising the polynucleotide of claim 24.

26. A cell comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14, a polynucleotide according to claim 24, and / or an expression vector according to claim 25.

27. 1. A method for producing an antibody or antigen-binding fragment thereof, comprising: Culturing the cell of claim 26 comprising the expression vector of claim 25 under conditions in which the antibody or antigen-binding fragment thereof is expressed by the cell; and isolating the antibody or antigen-binding fragment thereof from the cell or the medium in which the cell is cultured; The method includes:

28. 1. An in vitro method for detecting the presence and / or quantifying the amount of bile salt stimulated lipase (BSSL) in a sample, comprising: contacting said sample in vitro with the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 14; and Detecting the presence or absence of BSSL in the sample in vitro and / or quantifying the amount of BSSL in the sample based on the amount of isolated antibody or antigen-binding fragment thereof bound to BSSL. The method includes:

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