RU2826992C2 - Novel antibodies to bssl - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are recovered antibodies and antigen-binding fragments thereof, which bind to human bile salt stimulated lipase (hBSSL). Antibodies and their antigen-binding fragments bind to an epitope located in the N-terminal part of hBSSL and identified as containing amino acid residues 7–12 and amino acid residues 42–55. Also disclosed are polynucleotides coding said antibodies or fragments thereof, expression vectors and host cells containing said polynucleotides, a method of producing antibodies. Invention also relates to the medical use of the antibodies and/or antigen-binding fragments thereof, particularly in the treatment or diagnosis of inflammatory conditions, as well as to related pharmaceutical compositions.

EFFECT: invention provides specific binding of BSSL without significant effect on enzymatic activity and with low risk of immunogenicity.

41 cl, 33 dwg, 29 tbl, 24 ex

Description

AREA OF TECHNOLOGY

The present invention relates to novel isolated antibodies and antigen-binding fragments thereof that bind to a previously uncharacterized epitope of bile salt-stimulated lipase (BSSL) located in the N-terminal portion of the BSSL protein. The present document also relates to the medical use of the antibodies and antigen-binding fragments thereof, in particular in the treatment of inflammatory conditions, as well as related pharmaceutical compositions. The present document also describes the use of antibodies or antigen-binding fragments thereof as molecular tools in the detection of BSSL and/or for the diagnosis of BSSL-associated diseases.

LEVEL OF TECHNOLOGY

Inflammatory conditions, including autoimmune and autoinflammatory diseases, continue to pose a significant threat to human health. Despite advances in the treatment of inflammatory conditions, optimized treatments are still being sought.

Inflammatory conditions include a wide range of disorders and diseases that are characterized by inflammation, including autoimmune diseases and autoinflammatory diseases. Inflammation can, for example, occur as a response to infections, injuries, allergens and/or toxins, or as a response to the body itself, such as autoimmune processes. An autoimmune disease occurs when the body's immune system attacks and mistakenly destroys healthy body tissue. It is reported that more than 80 autoimmune diseases are known.

Some inflammatory conditions are chronic. Chronic inflammation occurs when the inflammatory response is prolonged, leaving the body in a constant state of 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).

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

JIA, also known as juvenile rheumatoid arthritis (JRA), is the most common form of arthritis in children and adolescents. JIA develops before age 16, and the cause of JIA is largely unknown. The primary focus of treatment for JIA is to help the child return to normal levels of physical and social activity. Most children receive an NSAID and intra-articular corticosteroid injections. Methotrexate, a DMARD, is a powerful drug that helps suppress joint inflammation in most JIA patients with polyarthritis, although this drug is reported to be less helpful in systemic arthritis, and many children receive TNFα-inhibiting drugs such as etanercept.

IBD is a term used to describe disorders that involve chronic inflammation of the digestive tract. IBD includes UC and CD.

The goal of IBD treatment is to reduce the inflammation that causes the signs and symptoms. In the best-case scenario, this may result not only in symptom relief but also in long-term remission and a reduced risk of complications. Treatment for IBD typically involves medications, such as anti-inflammatory drugs (NSAIDs), immune suppressors, and/or biologic agents, as well as surgery.

Bile salt-stimulated lipase (BSSL), also known as bile salt-dependent lipase (BSDL), carboxylester lipase (CEL), or bile salt-activated lipase (BAL), is a lipolytic enzyme encoded by the CEL gene and expressed in the exocrine pancreas and secreted into the intestinal lumen in all species studied to date that promotes lipid digestion.

In several species, including humans, primates, dogs, cats, and mice, BSSL is also expressed in lactating mammary glands and secreted in milk. Furthermore, BSSL has been found to be present at low but significant levels in the serum of healthy humans and is involved in lipoprotein metabolism and modulation of atherosclerosis. BSSL has also been found to play a role in inflammatory processes.

BSSL can be isolated from suitable tissue such as breast milk. Alternatively, recombinant BSSL can be produced by standard methods by isolating DNA encoding BSSL.

BSSL-encoding DNA can be successfully 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).

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

Document [2] describes antigen-binding compounds that bind BSSL or fetoacinar pancreatic protein (FAPP). The compounds are described to recognize the C-terminal peptide (epitope J28) of BSSL. FAPP is an oncofetal form of BSSL characterized by a carbohydrate-dependent epitope J28. It is stated that the antigen-binding compounds induce apoptosis and/or slow down the proliferation of tumor cells expressing the BSSL or FAPP polypeptide. Document [2] describes compounds that are capable of directly affecting tumor cells, in particular pancreatic tumor cells expressing BSSL or FAPP, and inducing their death via apoptosis and/or stopping their proliferation.

Papers [3, 4] describe the discovery that BSSL plays a role in inflammatory processes and that inhibition or elimination of BSSL protects against the development of chronic arthritis in animal models. Papers [3, 4] describe that BSSL protein is present in inflammatory cells and inflamed tissue and that BSSL-deficient mice are protected from developing an inflammatory disease, an example of which is collagen-induced arthritis (CIA).

Although treatments for inflammatory conditions such as autoinflammatory disease and autoimmune disease have improved significantly in recent years with the introduction of new drugs and drug classes such as antibodies, most regimens and drugs still have in common that they aim to suppress the immune system, such as all TNFα inhibitors and corticosteroids. This in turn increases the risk of secondary infections and complications.

Therefore, there is still a significant clinical need for new selective, preferably biological, drugs for the treatment, prophylaxis and prevention of inflammatory diseases that target different and/or novel targets involved in inflammatory signals and processes and that do not primarily act through suppression of the immune system and are therefore expected to have fewer and/or less severe adverse effects.

ESSENCE OF THE INVENTION

The general object of the present invention is to provide antibodies or antigen-binding fragments thereof that specifically bind to bile salt-stimulated lipase (BSSL), such as human BSSL (hBSSL).

This and other objects are achieved by the embodiments described herein.

The present invention is defined in independent claims. Additional embodiments of the invention are defined in dependent claims.

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

Another aspect of the present invention relates to an isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) consisting of an amino acid sequence selected from ZH1-[GYTFTSYN]-ZH2-[X ₅₃ GVIX ₅₇ PGDGX ₆₄ TSYX ₆₈ QKFX ₇₂ ]-ZH3-[ARDYYGSSPLGY]-ZH4 and a light chain variable region (LCVR) consisting of an amino acid sequence selected from ZL1-[X ₂₄ ASX ₂₇ SISYX ₃₉ N]-ZL2-[AX ₅₇ SX ₆₆ LX ₆₈ ]-ZL3-[HQRSSX ₁₁₅ PT]-ZL4. In this aspect, each region of ZH1, ZH2, ZH3 and ZH4 independently represents zero, one or more independently selected amino acid residues, 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, and X ₇₂ is selected from K and Q. In addition, each region of ZL1, ZL2, ZL3 and ZL4 independently represents zero, one or more independently selected amino acid residues, 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.

A further aspect of the present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of BSSL, preferably hBSSL. The epitope comprises a first surface comprising 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.

Another aspect of the present invention relates to a pharmaceutical composition comprising an isolated antibody and/or antigen-binding fragment thereof as defined above and a pharmaceutically acceptable carrier or excipient.

Further aspects of the present invention relate to an isolated antibody or antigen-binding fragment thereof as defined above or to a pharmaceutical composition as defined above for use as a medicament and for use in the treatment and/or prevention of an inflammatory disease.

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

Another related aspect of the present invention defines a method for treating and/or alleviating and/or preventing and/or preventing an inflammatory disease. Said method comprises administering a therapeutically effective amount of an isolated antibody or antigen-binding fragment thereof according to the above or a pharmaceutical composition according to the above to a subject in need thereof.

Further aspects of the present invention relate to a polynucleotide encoding an antibody or antigen-binding fragment thereof as defined above, an expression vector comprising such a polynucleotide, and a cell comprising an antibody or antigen-binding fragment thereof as defined above, as well as a polynucleotide as defined above and/or an expression vector as defined above.

Another aspect of the present 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 comprises contacting the sample with an isolated antibody or antigen-binding fragment thereof as described above and determining the presence or absence of BSSL in the sample and/or determining the amount of BSSL in the sample based on the amount of the isolated antibody or antigen-binding fragment thereof bound to BSSL.

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

Another aspect of the present 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 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.

The antibodies and antigen-binding fragments thereof of the present invention bind to a previously uncharacterized epitope in hBSSL other than the active site of the enzyme. Therefore, the antibodies and antigen-binding fragments thereof can bind to hBSSL without competing with the enzymatic activity of hBSSL. The antibodies and antigen-binding fragments thereof of the present invention are useful for treating and/or preventing inflammatory conditions, while alleviating the above and other shortcomings of existing treatments.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

The embodiments together with other purposes and their advantages can be better understood by reading the following description together with the accompanying graphic materials, in which:

Figure 1 shows the interaction between hBSSL and immobilized AS20 mIgG1. The sensorgram is fitted to a 1:1 binding model. Due to the good fit, the sensorgram and the fitted line cannot be distinguished.

Figure 2 shows the steady-state analysis of the interaction between mBSSL and immobilized AS20 mIgG1. The K _D value is taken from the half-maximal response, where max was automatically corrected for the large volume effect with an offset of -173 RU (top). The volume effect is not removed from the figure.

Figure 3a shows a graph illustrating the binding of AS20 scFv ELISA to non-biotinylated and biotinylated human BSSL. The absorbance values displayed (y-axis) are the average of duplicates. Figure 3b shows a graph illustrating the binding of AS20 scFv ELISA to mouse and human BSSL, as well as an irrelevant protein. The absorbance values displayed (y-axis) are the average of duplicates.

Figure 4 shows a histogram of the results of a bead-based multiplex assay (LUMINEX®) analyzing the ability of AS20 scFv (left columns) to bind to human BSSL and 30 different irrelevant proteins. A positive control scFv, irrelevant scFv_1 (right columns), putatively binding to b-irrelevant protein_1, was also included in the assay.

Figure 5 shows the results of an HTRF-based competition assay analyzing the binding of AS20 scFv to mouse and human BSSL.

Figure 6 is a sequence comparison between the AS20 graft, AS20 CDRs, and the AS20 humanization library backbone. * indicates an in-frame stop introduced in LCDR3 to ensure that only clones mutagenized in this region are displayed on the phage. X indicates mutagenized positions in the AS20 humanization library. The boundaries for the CDRs are defined according to Kabat and the residue numbering is defined according to IMGT nomenclature [5]. eHCDR2, eLCDR1, and eLCDR2 denote the extended regions of HCDR2, LCDR1, and LCDR2, including the amino acid position outside the corresponding CDR region according to IMGT.

Figure 7 shows graphs showing trends in size exclusion chromatography data at A) +40 °C and B) +4 °C, as described in Example 12.

Figure 8 shows graphs showing the mean Tm1 (circle) and Tm2 (square) values plotted for each candidate. The bars indicate the standard deviation; a) + 4°C b) +40°C.

Figure 9 shows the results of DLS analysis showing the intensity dependence on size for the candidates. The samples were analyzed after storage for 30 days at -80 °C (solid arrow), + 4 °C (dashed arrow) and + 40 °C (solid arrow), respectively.

Figure 10 shows a graphical representation of the BSSL structure with the epitope regions of the putative antibody candidates indicated (amino acid residues 7-12 for S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116, S-SL048-118; aa 42-55 for S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116, S-SL048-118; aa 84-101 for S-SL048-46; aa 174-180 for S-SL048-116; aa 283-295 for S-SL048-11).

Figure 11 shows svFv S-SL048-106 with potential post-translational biases highlighted.

Figure 12 is a sketch of t-hBSSL showing the tack appearance and the epitope outlined in dotted line and colored in light gray around the back of the tack. Strands are shown as arrows and helices as whorls.

Figure 13, left panel, shows t-hBSSL in a surface view in dark gray, with sequences that interact with AS20-Fab in light gray. The variable regions of the heavy and light chains are shown as a ribbon (light chain in gray and heavy chain in black). The right panel shows the same view, but t-hBSSL is shown as "sticks" with the active site triad highlighted in black. In the left panel, the active site is hidden beneath the surface.

Figure 14 is a table showing the amino acid sequence differences between the 38 scFv candidates.

Figures 15A and 15B provide a summary of the design of a combinatorial scFv library for the heavy chain variable region as described in Example 5.

Figures 16A and 16B provide a summary of the design of a combinatorial scFv library for the light chain variable region as described in Example 5.

Figure 17 is a graph showing the BSSL activity assays 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. Note that the chimeric AS20 is designated in the Figure as AS20.

Figure 18 illustrates the severity of arthritis. Development of CAIA in mice after injection of CIA-MAB-50 and administration of (A) isotype control anti-NP hIgG1 LALA-PG (90 mg/kg), AS20 hIgG1 LALA-PG (90, 30 and 10 mg/kg) every 4 ^days from day -1 to day 15. (B) Isotype control and AS20 hIgG1 LALA-PG 90 mg/kg. (C) Isotype control and AS20 IgG1 LALA-PG 30 mg/kg. (D) Isotype control and AS20 hIgG1 LALA-PG 10 mg/kg. Two animals, one in the AS20 hIgG1 LALA-PG 10 mg/kg group and one in the isotype control group, were excluded from the study early (Day 12) due to ethical concerns (high score). Data from these animals were included in the results up to the day of their exclusion from the study. Data are presented as mean ± SEM. * p <0.05; ** p < 0.01.

Figure 19 shows the plots of disease parameters. CAIA disease parameters including animals treated ip 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 day -1 to day 15. (A) Mean CAIA score (sum of scores during the experiment divided by the number of scoring days). (B) Maximum CAIA score. (C) Overall disease burden (AUC). (D) Percent inhibition. Two animals, one in the AS20 hIgG1 LALA-PG 10 mg/kg group and one in the isotype control group were excluded from the study early (Day 12) due to ethical concerns (high score). These animals were included only in the maximum score. Data are presented as mean ± SEM. * p <0.05; ** p <0.01.

Figure 20 shows the cell subset plots against the total cell count. Data are presented as mean ± SD.

Figure 21 shows cell subset percentage plots. Data are presented as mean ± SD.

Figure 22 shows the structure of the Fab-BSSL complex, drawn as a sketch. The dimeric complex of S-SL048-116 Fab with a light chain and a heavy chain and BSSL. The same complex is rotated to the right by 180°.

Figure 23 shows the interactions between BSSL and S-SL048-116 Fab. The variable Ig domains of the light chain and heavy chain Fab interact with BSSL. Two essential amino acids for the epitope, Arg 176 and Gln 52, are drawn as a ball and stick.

Figure 24 illustrates the severity of arthritis. Development of CAIA in mice after injection of CIA-MAB-50 and administration of (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. Three animals were discontinued early from the study due to ethical concerns, two in the vehicle group (day 7 and day 15) and one in the 90 mg/kg group (day 12). Data from these animals were included in the results up to the day of their exclusion from the study, and data from the animal excluded on day 7 were completely excluded from the analysis. Data are presented as mean ± SEM.

Figure 25 shows CAIA disease parameters including animals treated i.p. with vehicle and S-SL048-116 (SOL-116) at different doses (90, 30, and 10 mg/kg). (A) Mean CAIA score (sum of scores during the experiment divided by the number of days of scoring). (B) Maximum CAIA score. (C) Overall disease burden (AUC). (D) Percent inhibition. Three animals were excluded from the study early due to ethical concerns, two in the vehicle group (day 7 and day 15) and one in the 90 mg/kg group (day 12). These animals were included in the maximum score only. Data are presented as mean ± SEM.

Figure 26 shows the total leukocyte counts in the spleen (A) and mesenteric lymph nodes (B). Data are presented as mean ±SEM. **p < 0.01.

Figure 27 shows the ratio of NK cells to CD45+ cells in (A) spleen, (B) blood, and (C) mesenteric lymph nodes. Data are presented as mean ± SEM. ****p<0.001.

DETAILED DESCRIPTION

Definitions

The term "isolated" when used in connection with antibodies, such as in the expression "isolated antibody" and the like, means that the antibody has been separated from its original environment. As used herein, the term "isolated antibody" is intended to mean an antibody that is substantially free of other antibodies having different antigen specificities, such as an isolated antibody that specifically binds BSSL, particularly human BSSL (hBSSL), is substantially free of antibodies that specifically bind antigens other than BSSL. However, an isolated antibody that specifically binds hBSSL may have cross-reactivity with other antigens, such as BSSL molecules from other species, such as mouse or rat BSSL (mBSSL). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. For example, an isolated antibody or antigen-binding fragment thereof may be purified to a purity of greater than 95% or 99%, 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 or reversed-phase high-performance liquid chromatography (HPLC). One of skill in the art will recognize that isolated antibodies or antigen-binding fragments thereof are referred to herein, even if the word "isolated" is not explicitly mentioned each time the term "antibodies or antigen-binding fragments thereof" and the like is used.

In the context of this document, the term "isolated humanized antibody" and the like refers to an isolated antibody that has been previously humanized.

As used herein, the term "antigen-binding fragment" is intended to refer to 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 portion 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 consecutive amino acid sequence of the antibody from which it is derived, or may consist of different portions of the amino acid sequence of the antibody joined together with or without 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, complementarity determining regions (CDRs), and nanobodies.

"Variable single chain fragment" or "single chain variable fragment" ("scFv") is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins, linked by a short linker peptide, typically about 10 to 25 amino acids. scFv of the same or different types (having affinity for the same or different epitopes) can be combined in various ways, as known to those skilled in the art. Non-limiting examples of such combinations are tandem di-scFv, diabodies, tandem tri-scFv or tri(a)bodies.

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

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

As used herein, the terms "which binds to", "having an affinity for", "affinity" and the like refer to the property of an antibody or antigen-binding fragment thereof to bind to a target molecule. Standard assays for assessing the binding ability of an antibody or antigen-binding fragment to a target molecule include, for example, enzyme-linked immunosorbent assays (EIA) such as enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), surface plasmon resonance (SPR) analysis, LUMINEX® multiplex assay and flow cytometry analysis. As illustrated in the experimental section, the binding kinetics, such as binding affinity, of antibodies can also be assessed using standard assays known in the art, such as the BIACORE® system assay.

By the terms "specifically binds to", "specifically binding to", etc. it is meant that the molecule in question, such as an antibody or an antigen-binding fragment thereof, binds specifically to the target antigen without any significant binding to other molecules. The specificity of an antibody or an antigen-binding fragment thereof can be determined based on affinity and/or avidity. Affinity, represented by the equilibrium constant for dissociation of an antigen from an antibody or an antigen-binding fragment thereof (K _D ), is a measure of the strength of binding between an antigenic determinant, i.e. an epitope, and the antigen-binding site of an antibody or an antigen-binding fragment thereof. The lower the K _D value, the stronger the binding strength between the antigenic determinant and the antibody or an antigen-binding fragment thereof. Alternatively, affinity can also be expressed as an affinity constant (K _A ), which is 1/K _D . As will be appreciated by one of skill in the art, affinity can be determined in a known manner depending on the particular antigen of interest.

Typically, antibodies or antigen-binding fragments thereof bind to their antigen with an equilibrium dissociation constant (K _D ) of 10 ^-5 to 10 ^-12 mol/liter (M) or less, and preferably 10 ^-7 to 10 ^-12 M or less, and more preferably 10 ^-8 to 10 ^-12 M, i.e., with an affinity constant (K _A ) of 10 ⁵ to 10 ¹² M ^-1 or greater, and preferably 10 ⁷ to 10 ¹² M ^-1 or greater, and more preferably 10 ⁸ to 10 ¹² M ^-1 . Any K _D value greater than 10 ^-4 M (or any K _A value below 10 ⁴ M ^-1 ) is generally considered to indicate non-specific binding. Preferably, the antibody or antigen-binding fragment thereof according to the embodiments will bind to BSSL with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 5 nM.

The terms "detection", "detection", etc. include any means of detection, including direct and indirect detection.

In this document, all amino acids in the variable regions, including the CDRs described herein, are therefore numbered according to the unique IMGT numbering as defined in the publication by Marie-Paule Lefranc [5].

The term "Kabat numbering" etc. refers to the numbering scheme of amino acid residues in antibodies based on the variable regions.

As used herein, the terms "monoclonal antibody", "monoclonal antibodies", etc. refer to an antibody/antibodies that have monovalent affinity, meaning that each antibody molecule in a monoclonal antibody sample binds to the same epitope on an antigen. Monoclonal antibodies are produced by identical immune cells that are clones of a unique parent cell, such as a hybridoma cell line.

In the context of this document, the term "polyclonal antibodies" refers to a set of antibodies that react against a specific antigen, but the set may contain different antibody molecules, for example, identifying different epitopes on the antigen. Polyclonal antibodies are typically produced by inoculating a suitable mammal and subsequently purified from the serum of that mammal.

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

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

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

In the context of this document, the term "at least one" should be interpreted as one or more.

As will be appreciated by those skilled in the art, reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter as such. For example, a description that uses "about X" includes a description of "X."

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length and include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleotides may 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.

As used herein, the term "host cell" includes a single cell or cell culture that may be or has been a recipient of any vector of this document. Host cells include the progeny of a single host cell, and these progeny may not necessarily be completely identical, in morphology or in total DNA complement, to the original parent cell due to natural, accidental or deliberate mutation and/or alteration. A host cell includes cells transfected or infected with a vector containing a nucleic acid of this document. Host cells may be prokaryotic or eukaryotic cells.

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

In the context of this document, the terms "% identity" or "% identity" 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]. The comparison is performed in a window corresponding to the shortest of the aligned sequences. The shortest of the aligned sequences may in some cases be the target sequence. In other cases, the query sequence may be the shortest of the aligned sequences. At each position, amino acid residues or nucleotides are compared, and the percentage of positions in the query sequence that have identical matches in the target sequence is reported as % identity.

In the context of this document, 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.

It should be understood that the aspect and embodiments described herein include "consisting of" and/or "consisting essentially of" the aspects and embodiments. As used herein, singular words also include plural words unless otherwise indicated.

"hIgG1 LALA-PG" as described in [7].

"hIgG4 S228P, hIgG4 S241P" as described in [8].

"G-SP140-8", clone binding to SP140, negative control.

"expiHEK293 cells", human cells derived from the 293 cell line and the core component of the Expi293™ expression system.

The aim of the present invention is to provide antibodies and/or antigen-binding fragments thereof that can be used to treat and/or prevent inflammatory conditions, while alleviating the previously mentioned and other disadvantages of existing treatment methods. In addition, the aim of the present invention is to provide agents for use in diagnosing inflammatory conditions and for studying the bile salt-stimulated lipase (BSSL) protein.

In more detail, the present invention relates to new 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 document also relates to the medical use of the antibodies and antigen-binding fragments thereof, in particular in the treatment of inflammatory conditions, as well as related pharmaceutical compositions. The present document also describes the use of antibodies or antigen-binding fragments thereof as molecular tools in the detection of BSSL and/or for the diagnosis of BSSL-associated diseases.

In an embodiment, whenever BSSL is referred to, it also includes human BSSL (hBSSL), unless it is clear from the context that hBSSL is not intended to be included.

This document describes a new group of anti-BSSL antibodies, including antigen-binding fragments thereof, that bind to a previously unidentified epitope on hBSSL. The antibodies may be humanized or their CDR sequences may be grafted to a non-human framework. The antibodies or antigen-binding fragments thereof can also bind to mouse or rat BSSL (mBSSL), although the affinity for hBSSL and mBSSL may differ due to amino acid differences in one of the epitopes to which the antibodies and/or their antigen-binding fragments bind.

As is well known, antibodies are immunoglobulin molecules capable of specific binding 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 comprising at least two heavy (H) chains (HC) and two light (L) chains (LC) linked by disulfide bonds. The heavy chain variable regions (HCVR) and the light chain variable regions (LCVR) contain a binding domain that interacts with the antigen. The constant regions of antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system, such as effector cells, and the first component of the classical complement system, i.e. complement component 1q (C1q).

The antibodies or antigen-binding fragments thereof described herein can be used to inhibit or reduce at least some biological activities of a BSSL protein bound thereto. The binding can, for example, significantly or completely inhibit some biological activities of the BSSL protein. These effects of the antibodies or antigen-binding fragments thereof of the present invention were highly unexpected given that said antibodies or antigen-binding fragments thereof do not bind to the active site of BSSL. Thus, 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, for example, do not significantly inhibit or reduce the ability of BSSL to hydrolyze cholesterol esters (EC 3.1. 1.13).

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

In addition, the antibodies or antigen-binding fragments thereof described herein can be used for diagnostic purposes to diagnose pathological conditions associated with BSSL, such as inflammatory conditions associated with BSSL.

As demonstrated in the experimental section, several approaches were used to attempt to develop a humanized BSSL antibody or antigen-binding fragment thereof, but despite an initial quantity of approximately 1,000,000 scFv candidates, an unexpectedly low number were found to exhibit sufficient binding affinity to the hBSSL protein. From the 1,000,000 candidates, 68 initial candidates were identified, which were then narrowed down to 38 candidates and ultimately to 5 candidates selected for further evaluation and characterization. Accordingly, anti-BSSL antibodies or antigen-binding fragments thereof cannot be easily humanized using standard protocols and still have sufficient binding affinity to hBSSL. Thus, humanization of anti-BSSL antibodies or antigen-binding fragments was a major challenge that was overcome as described herein to obtain humanized antibodies or antigen-binding fragments thereof that still exhibit sufficient binding affinity to hBSSL. The antibodies or antigen-binding fragments thereof of the present invention share common structural and functional features as described elsewhere herein.

Generation of antibodies and antigen-binding fragments

The antibodies and antigen-binding fragments thereof of the present invention were prepared in a multi-step manner with the aim of finding antibodies and antigen-binding fragments thereof that had sufficiently good binding affinity to hBSSL. It was also an aim to provide humanized antibodies and antigen-binding fragments thereof. It was also found that some of the identified antibodies and antigen-binding fragments thereof bind with sufficient binding affinity to mBSSL. Although the present invention is not limited to humanized antibodies that bind to BSSL, humanized antibodies that bind BSSL and antigen-binding fragments thereof are described herein.

The antibodies and their antigen-binding fragments were derived from the sequence of the non-human monoclonal antibody mBSSL. DNA encoding the heavy and light chain immunoglobulins was obtained from a non-human hybridoma expressing this antibody and engineered to contain non-murine, e.g., human, immunoglobulin sequences using standard molecular biology techniques.

However, it was unexpectedly found that the CDR graft constructed as described 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. Thus, in order to obtain antibodies and antigen-binding fragments thereof with sufficiently high binding affinity for hBSSL, it was necessary to further modify the framework region (FW) of the antibody by introducing mutations in both the CDRs and the adjacent FW regions, as described in Example 5. This was achieved by constructing a humanization library used to select and isolate scFv fragments binding murine and human BSSL using phage display. Such phage display methods for isolating human antibodies are known in the art, see, for example, US 5,223,409; US 5,403,484; US 5,571,698; US 5,427,908; US 5,580,717; US 5,969,108; US 6,172,197; US 5,885,793; US 6,521,404; US 6,544,731; US 6,555,313; US 6,582,915 and US 6,593,081.

The resulting isolated antibody and its antigen-binding fragment had a minimized content of animal-derived CDRs, with only essential residues of non-human germline being tolerated. The remaining CDRs were converted to the human v gene sequence, with the exception of several novel variations, by introducing species-neutral essential residues de-novo, 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 a human or non-human scaffold to produce a humanized or non-humanized antibody and/or antigen-binding fragment thereof, depending on the intended use of the antibody.

In humanized antibodies, the constant regions and a portion of the variable regions, with the exception of the CDR sequences, have a framework that is 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 have been grafted onto human framework sequences. Such humanized antibodies may also be referred to as CDR grafts in the context of the present invention. An advantage of using humanized antibodies is that they reduce the risk of immunogenic reactions that may occur when a framework from another species is used when such an antibody is administered to a human. This opens up possibilities for their use in medicine in humans. The antibodies or antigen-binding fragments thereof described herein are useful in diagnostic applications and for detecting BSSL protein, such as hBSSL, in various types of samples, as described in more detail elsewhere herein.

The document thus also describes a method for producing an isolated antibody or antigen-binding fragment thereof according to the present invention. Said method comprises culturing a host cell expressing the antibody or antigen-binding fragment thereof under conditions allowing expression of the antibody or antigen-binding fragment thereof from an expression vector contained in the host cell and containing a polynucleotide encoding the antibody or antigen-binding fragment thereof. Said method also comprises isolating the antibody or antigen-binding fragment thereof from the host cell or from a culture medium in which the host cell is cultivated.

Epitope binding by antibodies or their antigen-binding fragments

The isolated antibodies or their antigen-binding fragments were found to bind to a previously unrecognized epitope of the human BSSL protein, which is located in the N-terminal portion of the BSSL protein. The epitope may form a conformational epitope of BSSL.

It was unexpectedly found that the antibodies and antigen-binding fragments thereof obtained according to the present invention bind to a previously unrecognized epitope of the hBSSL protein. Even more unexpectedly, it was found that the epitope is not located near the active site of BSSL for lipid metabolism, but rather in the N-terminal portion of BSSL. This provides several advantageous effects. One of them is that the antibodies or antigen-binding fragments thereof are less likely to cause negative side effects, since they do not significantly affect the enzymatic lipase activity of BSSL. Another advantage is that the antibodies or antigen-binding fragments thereof are suitable for studying the BSSL protein and its lipase activity, since it is not significantly affected by the antibodies or antigen-binding fragments thereof of the present invention.

The structure of BSSL has been described as having a large core region consisting of a twisted 11-stranded beta-sheet surrounded by alpha helices and connecting loops ([9], Figure 12). At the N-terminus there is a smaller 3-stranded beta-sheet. The structure can be compared to a left-handed glove in which the palm contains the active site triad near the "thumb". In this similarity, the small N-terminal beta-sheet is located on the back of the hand near the "little finger", see Figure 12. The part of the BSSL structure that interacts with the Fab molecule is located on the small N-terminal beta-sheet and in the C-terminal portion of the alpha-C, the third alpha helix in the structure, see Figure 13. In other words, the antibody binding region is not near the active site of BBSL, but on the opposite side from BSSL.

The identified epitope region includes residues 7-12 (chain 1 and 2) and 42-55 (loop region leading into chain 3 of the sheet). The epitope is fairly flat, with only a few characteristic residues projecting, namely Tyr7, Phe12, and Gln52 (the major interactions are listed in Table 25). The loop region 47-54 is well defined and forms a uniform surface. Proline 47 is important for the stacking interaction with Tyr31 of the antibody, but overall the surface is flat. In a preferred embodiment, the epitope region also includes residues 174-180 (C-terminus of alpha-C).

An aspect of the present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope of BSSL, preferably hBSSL. Said epitope comprises a first surface comprising or defined by the 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. Said epitope also comprises a second surface comprising or defined by the 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.

The first peptide comprising the amino acid sequence of SEQ ID NO: 1 defines a first epitope surface in BSSL, and the second peptide comprising the amino acid sequence of SEQ ID NO: 2 defines a second epitope surface in BSSL. Therefore, 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.

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 to SEQ ID NO: 3. SEQ ID NO: 3 is a longer amino acid sequence comprising as a part the amino acid sequence according to SEQ ID NO: 1.

The isolated antibody or antigen-binding fragment thereof may further specifically bind to another peptide and a surface, i.e. a third peptide and a third surface of BSSL, such as hBSSL. 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 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 another 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% and more preferably at least 92% identity with SEQ ID NO: 6.

In one embodiment, an isolated antibody or antigen-binding fragment thereof may specifically bind to a first peptide comprising, for example, consisting of SEQ ID NO: 1, a second peptide comprising, for example, consisting of SEQ ID NO: 2, and a third peptide comprising, for example, consisting of SEQ ID NO: 4, or to an amino acid sequence having the corresponding identities defined herein. Alternatively, instead of binding to the shorter amino acid sequence of SEQ ID NO: 1, such an antibody or antigen-binding fragment thereof may bind to the longer amino acid sequence of SEQ ID NO: 3 or to an amino acid sequence having the identity defined herein.

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

In a further embodiment, the isolated antibody or antigen-binding fragment thereof specifically binds to a first peptide comprising, for example, consisting of SEQ ID NO: 1, a second peptide comprising, for example, consisting of SEQ ID NO: 2, and a third peptide comprising, for example, consisting of SEQ ID NO: 6, or to an amino acid sequence having the corresponding identities defined herein. Alternatively, instead of binding to the shorter amino acid sequence of SEQ ID NO: 1, such an antibody or antigen-binding fragment thereof may bind to the longer amino acid sequence of SEQ ID NO: 3 or to an amino acid sequence having the identity defined herein.

Another aspect of the present invention is directed to a BSSL epitope, such as an hBSSL epitope, comprising a first peptide comprising, for example, consisting of the 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. The BSSL epitope also comprises a second peptide comprising, for example, consisting of the 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.

In one embodiment, the first peptide comprises, for example, consists of, the amino acid sequence according to SEQ ID NO: 3 or an amino acid sequence having the identity defined herein.

The epitope may further comprise a third peptide comprising, for example, an amino acid sequence according to SEQ ID NO: 4 or an amino acid sequence having a defined identity, an amino acid sequence according to SEQ ID NO: 5 or an amino acid sequence having an identity defined herein, or an amino acid sequence according to SEQ ID NO: 6 or an amino acid sequence having an identity defined herein.

Such epitopes may be useful for developing antibodies or antigen-binding fragments thereof that bind to a BSSL protein, such as hBSSL, for example, for use in treating and/or preventing conditions associated with BSSL, and/or for use as molecular tools for studying the BSSL protein. Thus, the present invention is also directed to the use of such epitopes for developing an antibody or antigen-binding fragment thereof. Since these epitopes are not located in the catalytic center of the BSSL protein lipase, they are an attractive target for the production of anti-BSSL antibodies or antigen-binding fragments.

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

Antigen-binding parts of antibodies and their antigen-binding fragments

A full-length antibody contains two heavy chains and two light chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (HVCR) and the first, second, and third constant regions ( _CH1 , _CH2 , and _CH3 ). In this document, the terms _VH, VH, and HCVR are used interchangeably. Each light chain contains a light chain variable region (LVCR) and a light chain constant region ( _CL ). In this document, the terms _VL, VL, and LCVR are used interchangeably.

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 (FRs or FWs). Each HCVR and LCVR consists of three CDRs and four FRs/FWs arranged from N-terminus to C-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4.

An extended CDR (eCDR) as used herein refers to an amino acid sequence that contains at least one additional amino acid residue in addition to the amino acids of the CDR as defined in accordance with the IMGT nomenclature.

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

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 sequence of its HCDR and/or LCDR, or the amino acid structure of the regions containing the HCDR and/or LCDR. It will be understood by the skilled artisan that minor changes, such as substitutions of one, two, three, four or even more amino acid residues in the amino acid sequence, can occur without affecting the functional properties, such as its binding capacity or binding affinity for BSSL, such as hBSSL, of the isolated antibody or antigen-binding fragment thereof. It will be understood that the first HCDR, the second HCDR, the third HCDR, the first LCDR, the second LCDR and the third LCDR can be independently selected from the listed amino acid sequences.

Thus, one aspect of the invention relates to an isolated antibody or antigen-binding fragment thereof comprising three HCVR CDRs (HCDRs) and three LCHV CDRs (LCDRs). In this aspect, the first HCDR comprises, preferably consists of, the 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 to SEQ ID NO: 7, the second HCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 8 or an amino acid sequence having at least 75% identity to SEQ ID NO: 8, and the third HCDR comprises, preferably consists of, the 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 to SEQ ID NO: 9. Furthermore, in this aspect, the first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 10 or an amino acid sequence having at least 80% identity to SEQ ID NO: 10, the second LCDR comprises, preferably consists of, the amino acid sequence of ATS or an amino acid sequence having at least 66% identity to the amino acid sequence of ATS, for example AAS, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11 or an amino acid sequence having at least 87% identity to SEQ ID NO: 11.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In one embodiment, when the extended second LCDR comprises, preferably consists of, the 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%, such as at least 83.3% identical to SEQ ID NO: 15.

In one embodiment, when the extended second LCDR comprises, preferably consists of, the 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%, such as at least 83.3% identical to SEQ ID NO: 17.

In one embodiment, when the extended second LCDR comprises, preferably consists of, the 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%, such as at least 83.3% identical to SEQ ID NO: 28.

In one embodiment, when the extended second LCDR comprises, preferably consists of, the 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%, such as at least 83.3% identical to SEQ ID NO: 29.

The isolated antibodies or antigen-binding fragments thereof as described herein may also or alternatively be structurally described by the amino acid sequence of their HCVR and/or LCVR. It will be appreciated by the skilled artisan that the HCVR and LCVR may be independently selected from the listed amino acid sequences. As explained above, it will be appreciated by the skilled artisan that minor changes, such as substitutions, including deletion or addition of amino acids, of one, two, three, four or even 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 ability to bind to hBSSL. The change may be in the amino acid sequence of the CDRs, in the amino acid sequence outside the CDR regions, referred to herein as framework regions, or in both the amino acid sequence of the CDRs and the amino acid sequence outside the CDR regions of the HCVR or LCVR.

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

In a particular embodiment, 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 of SEQ ID NO: 30, SEQ ID NO: 34, and SEQ ID NO: 36. In another 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 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 of SEQ ID NO: 36.

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 of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 38.

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

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 of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36. The antibody or antigen-binding fragment thereof further comprises 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 ...8%, such as at least 99% identical to any of SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36. a sequence that is at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to any of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 38.

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

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

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

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

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

One aspect of the present invention relates to an isolated antibody or antigen-binding fragment thereof comprising an HCVR consisting of an amino acid sequence selected from i) ZH1-[GYTFTSYN]-ZH2-[X ₅₃ GVIX ₅₇ PGDGX ₆₄ TSYX ₆₈ QKFX ₇₂ ]-ZH3-[ARDYYGSSPLGY]-ZH4, or an amino acid sequence that is at least 92% identical to the sequence defined in i), such as 93% or more, such as 94% or more, such as 95% or more, such as 96% or more, such as 97% or more, such as 98% or more, such as 99% or more sequence identity defined in i), and an LCVR consisting of an amino acid sequence selected from 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 which is at least 87% identical to the sequence defined in ii), such as 88% or more, such as 89% or more, such as 90% or more, such as 91% or more, such as 92% or more, such as 93% or more, such as 94% or more, such as 95% or more, such as 96% or more, such as 97% or more, such as 98% or more, such as 99% or more identical to the sequence defined in ii).

In this aspect, each region of ZH1, ZH2, ZH3, and ZH4 independently represents zero, one, or more independently selected amino acid residues, and each region of ZL1, ZL2, ZL3, and ZL4 independently represents zero, one, or more independently selected amino acid residues. 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, 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. The numbering of the amino acid residues complies with the IMTG numbering standard, i.e., for position "X _n " in sequence i) or ii) as defined above, n is an integer and denotes the position of amino acid residue X according to IMGT numbering.

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

The present document provides antibodies or antigen-binding fragments thereof, wherein X _n in sequence i) is independently selected from the group of possible residues listed below in List A. It will be understood by the skilled artisan that X _n can be selected from any of the listed groups of possible residues and that this selection is independent of the choice of amino acids in X _m , where n≠m. Thus, any of the listed possible residues at position X _n can be independently combined with any of the listed possible residues at any other variable position according to List A.

List A: List of possible amino acid residues in the sequence i)

X ₅₃ may represent I;

X ₅₃ may represent M;

X ₅₇ may represent N;

X ₅₇ can represent Y;

X ₅₉ may represent G;

X ₅₉ may represent S;

X ₆₄ can represent A;

X ₆₄ can represent S;

X ₆₈ can be selected from A and T;

X ₆₈ can be selected from A and N;

X ₆₈ can be selected from T and N;

X ₆₈ can represent A;

X ₆₈ can represent N;

X ₆₈ may represent T;

X ₇₂ may represent K; and

X ₇₂ may represent Q.

Similarly, provided herein are antibodies or antigen-binding fragments thereof, wherein X _k in sequence ii) is herein independently selected from a group of possible residues according to list B below. It will be clear to the skilled artisan that X _k may be selected from any of the listed groups of possible residues and that this selection is independent of the choice of amino acids in X _l , where k≠l. Thus, any of the listed possible residues at position X _k from list A may be independently combined with any of the listed possible residues at any other variable position according to list B.

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

X ₂₄ can represent S;

X ₂₄ can represent R;

X ₂₇ may represent S;

X ₂₇ may represent P;

X ₃₉ may represent M;

X ₃₉ may represent L;

X ₄₀ may represent H;

X ₄₀ can represent N;

X ₅₇ may represent A;

X ₅₇ may represent T;

X ₆₆ can be selected from K and S;

X ₆₆ can be selected from R and S;

X ₆₆ can be selected from R and K;

X ₆₆ can represent K;

X ₆₆ can represent R;

X ₆₆ can represent S;

X ₆₈ can be selected from A and P;

X ₆₈ can be selected from A and Q;

X ₆₈ can be selected from P and Q;

X ₆₈ can represent A;

X ₆₈ may represent P;

X ₆₈ may represent Q.

X ₁₀₅ may represent H;

X ₁₀₅ may represent Q.

X ₁₁₅ can be selected from S and T;

X ₁₁₅ can be selected from S and Y;

X ₁₁₅ can be selected from T and Y;

X ₁₁₅ may represent S;

X ₁₁₅ may represent Y; and

X ₁₁₅ may represent T.

For the purpose of clarification, it should be pointed out that the choice of amino acid residues for amino acid positions in sequence i) from list A is independent of the choice of amino acid residues for amino acid positions in sequence ii) from list B. For the avoidance of doubt, list A and list B provide several specific and individual examples in accordance with the present invention, and the examples provided may be freely combined.

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

Similarly, each of ZL1, ZL2, ZL3 and ZL4 may be zero, one or more independently selected amino acid residues. The identity and number of amino acid residues in each of said ZL1, ZL2, ZL3 and ZL4 may be independently selected. In addition, ZL1 may be linked via an amino acid linker or another linker to ZH1 or ZH4. Also, ZL4 may be linked via an amino acid linker or another linker to ZH1 or ZH4. Thus, the sequence defined in i) and the sequence defined in ii) may be part of a single amino acid sequence, in other words, may be part of a single polypeptide.

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%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to the sequence of SEQ ID NO: 39.

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%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to the sequence of SEQ ID NO: 40.

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%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to the sequence of SEQ ID NO: 41.

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%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to the sequence of SEQ ID NO: 42.

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

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%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to the sequence of SEQ ID NO: 44.

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%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to the sequence of SEQ ID NO: 45.

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%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical to the sequence of SEQ ID NO: 46.

It will be appreciated by the skilled artisan that the % identity of each region of ZH1, ZH2, ZH3, ZH4, ZL1, ZL2, ZL3 and ZL4 to the amino acid sequence of its corresponding SEQ ID NO: above is independent of the % identity of any other region of ZH1, ZH2, ZH3, ZH4, ZL1, ZL2, ZL3 and ZL4 to its corresponding SEQ ID NO:. Thus, for example, ZH1 may exhibit 95% identity to SEQ ID NO: 39, and ZH2 may exhibit 99% identity to SEQ ID NO: 40.

During the humanization process that produces the novel anti-BSSL antibodies or antigen-binding fragments thereof provided herein, several mutations were introduced into both the CDRs and adjacent FW regions. For the purposes of the present invention, each HCVR and LCVR antibody consists of three CDRs, of which one, two or three CDRs may be extended CDRS (eCDRs), and four FRs/FWs arranged from the N-terminus to the C-terminus in the following order as defined in Table 1.

Table 1 – IMGT Numbering

IMGT Numbering VH-FW1 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 CDR-H1 27, 28, 29, 30, 35, 36, 37, 38 VH-FW2 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 eCDR-H2 53, 54, 55, 56, 57, 58, 59, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 CDR-H2 56, 57, 58, 59, 62, 63, 64, 65 VH-FW3 66, 67, 68, 69, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 10, 101, 102, 103, 104 CDR-H3 105, 106, 107, 108, 109, 110, 112, 113, 114, 115, 116, 117 VH-FW4 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 VL-FW1 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 eCDR-L1 24, 25, 26, 27, 28, 29, 37, 38, 39, 40 CDR-L1 27, 28, 29, 37, 38 VL-FW2 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 eCDR-L2 56, 57, 65, 66, 67, 68 CDR-L2 56, 57, 65 VL-FW3 66, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 CDR-L3 105, 106, 107, 108, 109, 115, 116, 117 VL-FW4 118, 119, 120, 121, 122, 123, 124, 125, 126, 127

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

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

It will be understood by the skilled person that various modifications and/or additions can be made to the antibody or antigen-binding fragment thereof as described herein to adapt said antibody or antigen-binding fragment thereof to a particular use without departing from the scope of the present invention. For example, the antibody or antigen-binding fragment thereof can have an amino acid sequence that has been extended and/or comprises additional amino acids at the C-terminus and/or N-terminus, for example at the C-terminus and/or N-terminus of its heavy or light chain. Thus, the antibody or antigen-binding fragment thereof can comprise any suitable number of additional amino acid residues, for example at least one additional amino acid residue. Each additional amino acid residue can be added individually or together, for the purpose of, for example, optimizing and/or simplifying the production, purification, in vivo or in vitro stabilization, binding or detection of the polypeptide. Such additional amino acid residues can include one or more amino acid residues added for the purpose of chemical coupling. An example is the addition of a cysteine residue. Additional amino acid residues may also provide a "tag" for purification or detection of the antibody or antigen-binding fragment thereof, such as a His ₆ tag, a (HisGlu) ₃ tag, a "myc" tag (c-myc), or a FLAG tag.

An isolated antibody or antigen-binding fragment thereof of the present invention may be selected from, among others, 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.

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.

It may be desirable to reduce or eliminate the effector function of antibodies or antigen-binding fragments thereof, for example, to prevent target cell death or unwanted cytokine secretion. This may be particularly appropriate when antibodies or antigen-binding fragments thereof are designed to interact with cell surface receptors and prevent receptor-ligand interactions, i.e., antagonists. Other examples where effector function may need to be reduced include preventing antibody-drug conjugates from interacting with Fc receptors (FcγRs) resulting in off-target cytotoxicity.

Therefore, 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 or more, preferably all three Fc silencing mutations L234A, L235A, and P329G.

In addition, antibodies or their antigen-binding fragments of the IgG4 isotype are considered potential candidates for immunotherapy when reduced effector functions are desired. IgG4 antibodies are known to be dynamic molecules capable of undergoing a process known as Fab-arm exchange (FAE), and without being limited by theory, this is believed to result in the production of functionally monovalent bispecific antibodies (bsAbs) of unknown specificity and therefore, potentially, reduced therapeutic efficacy. This may result in undesirable pharmacodynamic unpredictability for human immunotherapy.

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

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 can be selected from the group consisting of the IgG1 and IgG4 isotype subclasses.

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.

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

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

Binding affinity

In one embodiment, an isolated antibody or antigen-binding fragment thereof according to this document may have an affinity for hBSSL of no more than a K _{D of} 1×10 ^-7 M, preferably no more than a K _D of 1×10 ^-8 M. For example, an isolated antibody or antigen-binding fragment thereof may have an affinity for hBSSL of no more than a K _{D of} 5 nM, such as no more than a K _{D of} 3 nM.

As demonstrated in the experimental section, the isolated antibody or antigen-binding fragment thereof may have an affinity for hBSSL of no more than a _KD of 1.7 nM, such as no more than a _{KD of} 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, or no more than 0.6 nM. In one example, an isolated antibody or antigen-binding fragment thereof that binds to hBSSL according to the present disclosure has an affinity for hBSSL in the range of _{KD of} 0.6-1.7 nM, such as in the range of _{KD 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.

Pharmaceutical compositions

The term "pharmaceutical composition" refers to a preparation that is in a form that provides effectiveness of the biological activity of the active ingredient contained therein and that does not contain additional components that are unacceptably toxic to the subject to which the composition is to be administered. The pharmaceutical compositions of the present invention comprise an antibody and/or an antigen-binding fragment thereof, such as an scFv, as defined herein, and a pharmaceutically acceptable carrier or excipient.

An antibody or antigen-binding fragment thereof, such as an scFv, as defined herein, can be prepared by methods known in the art in the form of, for example, tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols for inhalation and for parenteral use (including intravenous, subcutaneous or intramuscular administration), sterile aqueous or oily solutions or suspensions or sterile emulsions.

Thus, provided herein is a composition comprising an isolated antibody or antigen-binding fragment thereof as described herein and at least one pharmaceutically acceptable excipient or carrier. For example, the excipient may be a diluent. In one example, the pharmaceutical composition may further comprise at least one additional active agent, such as at least two additional active agents, such as at least three additional active agents. Non-limiting examples of additional active agents that may be useful in such a combination include immune response modifying agents.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is nontoxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

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

The pharmaceutical composition according to the present invention may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention 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 maintaining the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures and by including various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenolsorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be ensured by including agents that delay absorption, such as aluminum monostearate and gelatin.

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

The present invention also provides a kit of parts comprising an antibody or an antigen-binding fragment thereof, or a pharmaceutical composition according to the present invention, means for administering the antibody or an antigen-binding fragment thereof, or a pharmaceutical composition, and, optionally, a package insert with instructions for use. The means for administering the antibody or an antigen-binding fragment thereof or a pharmaceutical composition can be, for example, a syringe. The term "package insert" is used to denote instructions typically included in commercial packages of therapeutic products that contain information on indications, use, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

Medical use of isolated antibodies or their antigen-binding fragments.

The antibody or antigen-binding fragment thereof according to the present invention can be used to effectively reduce the pro-inflammatory effect of BSSL in a subject such as a human.

The advantage of the antibodies and antigen-binding fragments thereof according to the present invention is that they do not bind to the active site of the BSSL protein, which is responsible for the lipase activity of BSSL. This is demonstrated and described in detail in the experimental part. Thus, the risk of negative side effects is reduced, since the lipase activity does not change significantly.

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

The present invention 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 invention 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 invention is also directed to a method for the treatment and/or amelioration and/or prevention and/or prophylaxis of an inflammatory disease. Said method comprises administering a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof, such as an scFv, or the pharmaceutical composition to a subject in need thereof.

Various in vivo models can be used to predict the effect of antibodies and antigen-binding fragments in the treatment and/or prevention of inflammatory disease. Typically, these models use mice and administer various substances to induce an immune response. The effect of antibodies or their antigen-binding fragments on such an immune response can then be studied after administration of the antibodies/antigen-binding fragments.

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 usually administered with a booster dose of CII in incomplete Freund's adjuvant (IFA) on day 21. This model induces autoimmune arthritis. Arthritis usually occurs 21-28 days after the first CII injection. This model is B- and T-cell dependent (adaptive immunity).

Another model is the "collagen antibody-induced arthritis" (CAIA). In this model, a mixture of CII antibodies is administered, usually with a booster dose of lipopolysaccharide (LPS) on day 5. This model is independent of B and T cells (innate immunity). The protocol for in vivo testing of the efficacy of the antibodies and antigen-binding fragments thereof of the present invention in the treatment and/or prevention of inflammatory disease using the CAIA model is described in the experimental section of this document.

Another model is the "glucose-6-phosphate isomerase-induced arthritis" model. In this model, a peptide corresponding to the glucose-6-phosphate isomerase sequence is administered to induce an immune response. This model is T-lymphocyte dependent.

Another model is the pristane-induced arthritis (PIA) model, in which pristane is administered. This model is T-lymphocyte dependent.

A "dextran sodium sulfate-induced colitis" model may also be used, in which dextran sodium sulfate (DSS) is administered through drinking water.

All of the above methods are well known to those skilled in the art. These methods can be used to test in vivo the efficacy of the antibodies or antigen-binding fragments thereof of the present invention.

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

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

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 called fatty liver disease, and hyperinflammation.

In a specific embodiment, the inflammatory disease is an inflammatory condition caused 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. This virus causes coronavirus disease 2019 (COVID-19). Patients with severe COVID-19 often suffer from systemic hyperinflammation, including elevated levels of various inflammatory cytokines, such as interleukin 2 (IL-2), IL-7, IL-6, granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-γ inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1-α (MIP-1α), and TNF-α.

RA primarily affects the joints, but can also be a systemic inflammatory disease that may be characterized by extra-articular manifestations in several organs. Therefore, RA can be considered a systemic inflammatory disease.

In addition, the isolated antibody or antigen-binding fragment thereof, such as an scFv fragment, or pharmaceutical composition described herein may be an alternative to existing biological therapies for patients who do not respond or who temporarily respond to existing tumor necrosis factor alpha (TNFα) inhibitors, thereby reducing the need for corticosteroids and/or immunosuppressants and/or pharmaceuticals. Thus, the use of the isolated antibody or antigen-binding fragment thereof, such as an scFv fragment, described herein prevents and/or reduces adverse effects and/or side effects of alternative treatment regimens in patients, which is a key issue in medical care in general and is especially important for young and pediatric patients, as well as immunocompromised and/or elderly patients.

Treatment and/or prevention using an isolated antibody and/or antigen-binding fragment thereof or a pharmaceutical composition as described herein is typically passive immunotherapy, meaning that the antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising such antibodies and/or antigen-binding fragments thereof, is administered to a subject in need thereof. However, other types of immunotherapeutic methods can also be used, such as gene therapy, in which, instead of administering an antibody or antigen-binding fragment thereof, a gene construct capable of expressing such an antibody or antigen-binding fragment thereof is directly administered to a subject.

The subject of the present invention may be a human or a 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, domestic 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.

As used herein, the term "treatment" (and grammatical variations thereof such as "treat" or "treatment") refers to a clinical intervention in an attempt to alter the natural course of a disease in an individual receiving treatment, and may be performed either prophylactically or during a clinical pathological process. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms (improving quality of life), reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease, and causing remission or improved prognosis. The antibody or antigen-binding fragment thereof of the present invention may be used to delay the development of a disease or to slow the progression of a disease.

By "reduction" or "inhibition" is meant the ability to cause an overall reduction of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more. The term "reduction" or "inhibition" may refer to the symptoms of the disease being treated. The term "reduction" or "inhibition" also includes delaying the onset of a disease, particularly an inflammatory disease.

Methods of administration

An isolated antibody or antigen-binding fragment thereof, such as an scFv, or a pharmaceutical composition of the present invention may be administered in a standard manner for a pathological condition that it is desired to treat and/or prevent, such as 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 an scFv, or a pharmaceutical composition for use as described herein may be formulated for parenteral administration, such as intravenous or subcutaneous administration, in particular subcutaneous administration.

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

Although the administration schedule may be tailored to the particular disease and subject being treated, typically an isolated antibody or antigen-binding fragment thereof, such as an scFv, or a pharmaceutical composition as defined herein is administered 1-3 times per week, such as 1-2 times per week, such as once per week, although other administration schedules are also possible.

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, combination therapy may comprise an antibody or antigen-binding fragment thereof, such as an scFv, of the present invention, in combination with at least one other anti-inflammatory or immunosuppressant agent. It should be understood that combination therapy includes both sequential and simultaneous administration. As used herein, the term "simultaneous" is used to mean the administration of two or more therapeutic agents, wherein at least part of the administration overlaps in time. Accordingly, simultaneous administration includes an administration schedule wherein the administration of one or more agents continues after the cessation of administration of one or more other agents.

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

A therapeutically effective amount of an antibody or antigen-binding fragment thereof, such as an scFv, may vary depending on factors such as the pathological condition, the age, sex and weight of the individual, and the ability of the antibody or antigen-binding fragment thereof, such as an scFv, to elicit a desired response in the subject. A therapeutically effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the administered substance. When an antibody or antigen-binding fragment thereof, such as an scFv, is used to prevent a pathological condition (prophylactic purposes), the prophylactically effective amount is usually, but not necessarily, less than the therapeutically effective amount because the prophylactic dose is administered to subjects before or at an earlier stage of the disease.

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

Expression system

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

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

Therefore, in one embodiment, said polynucleotide is selected from the group consisting of SEQ ID NO: 174-202 and any combination and/or variant thereof. A variant of any of SEQ ID NO: 174-202 used in the present invention comprises a polynucleotide encoding the same antibody or antigen-binding fragment thereof as the polynucleotide defined in any of SEQ ID NO: 174-202, but may have at least one synonymous substitution, i.e., a replacement of at least one base with another, such that the resulting amino acid sequence is not modified. Therefore, such a synonymous substitution replaces at least one base in a codon in said polynucleotide with another codon, both of which encode an amino acid residue. For example, a polynucleotide according to any of SEQ ID NO: 174-202 or a combination thereof may be codon-optimized for expression in a particular host cell.

A polynucleotide encoding an antibody or an antigen-binding fragment thereof as described herein can be introduced into an expression vector. The expression vector enables the reproduction of the polynucleotide introduced therein. Said vector can be a self-replicating nucleic acid structure, as well as a vector incorporated into the genome of the host cell into which it has been introduced. Thus, the present invention is also directed to such an expression vector comprising a polynucleotide encoding an antibody or an antigen-binding fragment thereof.

The expression vector preferably comprises a polynucleotide encoding an antibody or an antigen-binding fragment thereof, operably linked to at least one regulatory element. In some embodiments, the regulatory element is or comprises a promoter. A promoter is a DNA sequence that is bound by proteins that initiate transcription of an RNA molecule from a DNA (gene) located downstream. Another example of a regulatory element is an enhancer. An enhancer is a short region of DNA that can be bound by activators to increase the likelihood that transcription of a particular gene will occur.

Examples of expression vectors include a DNA molecule, an RNA molecule, a plasmid, an episomal plasmid, and a viral vector. Non-limiting but illustrative examples of viral vectors include a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, Semliki Forest virus, poliovirus, and a hybrid vector.

The expression vector can be introduced into a host cell for expression and/or reproduction of the vector containing the polynucleotide. In particular, the expression vector is intended for use in the treatment and/or prevention of an inflammatory disease, being expressed in a subject, so as to thereby produce antibodies or antigen-binding fragments thereof in said subject.

Thus, the present invention also provides a host cell comprising an expression vector. The host cell used may 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 obtained from it, regardless of the number of passages.

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

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

Antibodies or antigen-binding fragments thereof can be produced by introducing their sequence into an expression vector and allowing the expression vector to express the antibody or antigen-binding fragment thereof in a host cell, whereupon the produced antibodies or antigen-binding fragments are isolated/purified prior to use, for example, for therapeutic or diagnostic purposes, as described elsewhere herein. In addition, the vector itself can be administered to a subject for direct expression of the antibody or antigen-binding fragment thereof in the subject to be treated. In addition, the expression vector preferably comprises a promoter that controls expression of a polynucleotide encoding the antibody or antigen-binding fragment thereof.

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

Diagnostic use of isolated antibodies or their antigen-binding fragments.

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 methods such as, but not limited to, ELISA, Western blotting, RIA, surface plasmon resonance (SPR), and flow cytometric analysis.

An advantage of using the antibodies and antigen-binding fragments thereof according to the present invention is that they do not bind to the active site of BSSL and thus do not inhibit the lipase activity of the protein. Thus, it is possible to use the antibodies or antigen-binding fragments thereof to study the BSSL protein without significantly affecting the lipase activity, as demonstrated in the experimental section. Thus, the antibodies or antigen-binding fragments thereof are useful as molecular tools in studying the BSSL protein and/or its enzymatic activity in vitro/ex vivo and/or in vivo.

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

The above-described method may be in the form of an ex vivo or in vitro method. In such a case, said method comprises contacting ex vivo or in vitro a sample with an isolated antibody or antigen-binding fragment thereof according to the present invention.

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

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

In one embodiment, the method also includes obtaining a sample from a subject suspected of having a BSSL-associated disease.

In a particular embodiment, said method comprises comparing a quantitatively determined level of BSSL in a sample with a threshold value. In such a particular embodiment, step c) comprises concluding whether the subject is diagnosed with a disorder associated with BSSL or not, based on the comparison between the quantitatively determined level of BSSL in the sample and the threshold value. For example, if the amount of BSSL in BSSL exceeds the threshold value, a conclusion can be made as to whether the subject is diagnosed with a disorder associated with BSSL or not.

The cut-off value depends on the specific BSSL-related disorder and can be determined by quantifying the BSSL level in samples taken from subjects already diagnosed with the specific BSSL-related disorder and/or by quantifying the BSSL in samples taken from healthy subjects not suffering from the specific BSSL-related disorder. The cut-off value can then be determined based on these BSSL quantities from subjects suffering from the specific BSSL-related disorder and preferably based on BSSL quantities from healthy subjects.

The disorder associated with BSSL is typically an inflammatory condition, as described elsewhere herein. The inflammatory condition may be, for example, a chronic or systemic inflammatory condition, 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.

A sample potentially containing BSSL may be any type of sample, such as a sample obtained from a subject. Therefore, in one embodiment, said sample is a biological sample. An example of such a biological sample is a body fluid sample, such as a blood sample, a blood 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 pathological conditions associated with BSSL include both in vitro methods and in vivo methods, such as in situ hybridization.

As mentioned elsewhere in this document, antibodies or antigen-binding fragments thereof may be humanized or their CDR sequences (or portions thereof) may be grafted onto a non-human backbone. The latter may be useful in using antibodies or antigen-binding fragments thereof as a molecular tool to study the BSSL protein in species other than humans, for example to reduce negative immunogenic reactions to antibodies and/or their antigen-binding fragments.

Illustrative embodiments

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

In one embodiment, the first epitope comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 3 or an amino acid sequence having at least 80%, such as 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 3.

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

In one embodiment, the isolated antibody or antigen-binding fragment thereof further specifically binds to the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 80%, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

In one embodiment, the isolated antibody or antigen-binding fragment thereof further specifically binds to the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 80%, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

In one embodiment, the isolated antibody or antigen-binding fragment thereof further specifically binds to the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 80%, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

An 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) of a heavy chain variable region (HCVR) (HCDR). 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 of a light chain variable region (LCVR) (LCDR). The first LCDR comprises or consists of the amino acid sequence according to SEQ ID NO: 10 or an amino acid sequence that is at least 80% identical to SEQ ID NO: 10, the second LCDR comprises or consists of the amino acid sequence of ATS or an amino acid sequence that is at least 66% identical to the amino acid sequence of ATS, such as AAS, and the third LCDR comprises or consists of the amino acid sequence according to SEQ ID NO: 11 or an amino acid sequence that is at least 87% identical to SEQ ID NO: 11.

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, 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 NO: 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 NO: 11, 21 and 22.

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

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

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

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

In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs HCVRs. The first HCDR comprises, preferably consists of, the amino acid sequence of 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, the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence that is at least 77% identical to SEQ ID NO: 23, and the third HCDR comprises, preferably consists of, the amino acid sequence of 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 LCVRs. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 16 or an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, the second LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 15 or an amino acid sequence that is at least 66% identical to SEQ ID NO: 15, and the third LCDR comprises, preferably consists of, the 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.

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

In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs HCVRs. The first HCDR comprises, preferably consists of, the amino acid sequence of 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, the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence that is at least 83% identical to SEQ ID NO: 25, and the third HCDR comprises, preferably consists of, the amino acid sequence of 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 LCVRs. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 26 or an amino acid sequence that is at least 90% identical to SEQ ID NO: 26, the second LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 28 or an amino acid sequence that is at least 66% identical to SEQ ID NO: 28, and the third LCDR comprises, preferably consists of, the 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.

In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs HCVRs. The first HCDR comprises, preferably consists of, the amino acid sequence of 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, the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 77% identical to SEQ ID NO: 12, and the third HCDR comprises, preferably consists of, the amino acid sequence of 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 LCVRs. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 14 or an amino acid sequence that is at least 70% identical to SEQ ID NO: 14, the second LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 15 or an amino acid sequence that is at least 66% identical to SEQ ID NO: 15, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11 or an amino acid sequence that is at least 87% identical to SEQ ID NO: 11.

In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises three HCDRs HCVRs. The first HCDR comprises, preferably consists of, the amino acid sequence of 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, the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 77% identical to SEQ ID NO: 12, and the third HCDR comprises, preferably consists of, the amino acid sequence of 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 LCVRs. The first LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 16 or an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, the second LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 17 or an amino acid sequence that is at least 50% identical to SEQ ID NO: 17, and the third LCDR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 11 or an amino acid sequence that is at least 87% identical to SEQ ID NO: 11.

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

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

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

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

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

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

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 an amino acid sequence that is at least 98% identical thereto; such as selected from the group consisting of SEQ ID NOs: 30, 34 and 36, or an amino acid sequence that is at least 96% identical thereto; such as selected from the group consisting of SEQ ID NOs: 34 and 36, or an amino acid sequence that is at least 96% identical thereto.

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

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 an amino acid sequence that is at least 96% identical thereto; for example, selected from the group consisting of SEQ ID NOs: 31, 35, 37 and 38, or an amino acid sequence that is at least 96% identical thereto; for example, selected from the group consisting of SEQ ID NOs: 35, 37 and 38, or an amino acid sequence that is at least 96% identical thereto; for example, selected from the group consisting of SEQ ID NOs: 37 and 38, or an amino acid sequence that is at least 96% identical thereto.

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

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

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

In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises an HCVR and an LCVR. The HCVR and LCVR are 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 pair of amino acid sequences of SEQ ID NOs: 30 and 31; the pair of amino acid sequences of SEQ ID NOs: 32 and 33; the pair of amino acid sequences of SEQ ID NOs: 34 and 35; the pair of amino acid sequences of SEQ ID NOs: 36 and 37; and the pair of amino acid sequences of SEQ ID NOs: 36 and 38; for example, a pair of amino acid sequences selected from the group consisting of the pair of amino acid sequences of SEQ ID NOs: 36 and 37; and the pair of amino acid sequences of SEQ ID NOs: 36 and 38.

In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequence ZH1-[CDR-H1]-ZH2-[eCDR-H2]-ZH3-[CDR-H3]-ZH4, wherein each of ZH1, ZH2, ZH3, and ZH4 is zero, one, or more independently selected amino acid residues. In one embodiment, the heavy chain variable region consists of an amino acid sequence selected from i) ZH1-[GYTFTSYN]-ZH2-[X ₅₃ GVIX ₅₇ PGDGX ₆₄ TSYX ₆₈ QKFX ₇₂ ]-ZH3-[ARDYYGSSPLGY]-ZH4, wherein, independently of one another, 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; and X ₇₂ is selected from K and Q, and ii) an amino acid sequence that has at least 92% identity to the sequence,

defined in i). The light chain variable region comprises an amino acid sequence ZL1-[eCDR-L1]-ZL2-[eCDR-L2]-ZL3-[CDR-L3]-ZL4, wherein each of ZL1, ZL2, ZL3 and ZL4 is zero, one or more independently selected amino acid residues. In one embodiment, the light chain variable region consists of an amino acid sequence selected from iii) ZL1-[X ₂₄ ASX ₂₇ SISYX ₃₉ N]-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 that has at least 87% identity to the sequence defined in iii).

In one embodiment, ZH1 comprises, preferably consists of, the 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.

In one embodiment, ZH2 comprises, preferably consists of, the 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.

In one embodiment, ZH3 comprises, preferably consists of, the 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.

In one embodiment, ZH4 comprises, preferably consists of, the 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.

In one embodiment, ZL1 comprises, preferably consists of, the 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.

In one embodiment, ZL2 comprises, preferably consists of, the 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.

In one embodiment, ZL3 comprises, preferably consists of, the 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.

In one embodiment, ZL4 comprises, preferably consists of, the 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.

In one embodiment, the antibody is a full length antibody.

In one embodiment, the antibody is selected from the group consisting of human antibodies, humanized antibodies, and chimeric antibodies.

In one embodiment, the antigen-binding fragment is an antigen-binding fragment such as a variable single chain fragment, a Fab fragment, an F(ab') ₂ fragment, an F(ab') ₃ fragment, a Fab' fragment, an Fd fragment, an 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.

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.

In one embodiment, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of the class of isotypes IgG, IgA, IgM, IgD, and IgE; for example, IgG. In a particular embodiment, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of the subclass of isotypes IgG1 and IgG4.

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.

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

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

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

In one embodiment, the antibody is a humanized antibody.

In one embodiment, the isolated antibody or antigen-binding fragment thereof has an affinity for hBSSL of no more than a _KD of 1.7 nM.

In one embodiment, the isolated antibody or antigen-binding fragment thereof is capable of displacing the binding of hBSSL to monocytes, preferably CD14 ⁺ monocytes.

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

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

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

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

One embodiment relates to a method of treating and/or alleviating and/or preventing and/or prophylaxis of an inflammatory disease. In this method, a therapeutically effective amount of an isolated antibody and/or antigen-binding fragment thereof or a pharmaceutical composition according to the present invention is administered to a subject in need thereof.

In one embodiment, the inflammatory disease is a chronic inflammatory disease.

In one embodiment, the inflammatory disease is a systemic inflammatory disease.

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

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

In one embodiment, the inflammatory disease is hepatic steatosis.

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

In one embodiment, the isolated antibody and/or antigen-binding fragment thereof or pharmaceutical composition is administered parenterally, such as subcutaneously. Therefore, in a particular embodiment, the isolated antibody and/or antigen-binding fragment thereof or pharmaceutical composition is formulated for parenteral administration, such as subcutaneous administration.

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

In one embodiment, treatment and/or prevention is accomplished using passive immunotherapy.

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

An embodiment relates to a method for obtaining an isolated antibody or antigen-binding fragment thereof according to the invention. Said method comprises culturing a host cell according to the invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.

An embodiment relates to a method for detecting the presence or absence of BSSL and/or quantifying the level of BSSL in a sample. Said method comprises the following steps: a) obtaining a sample potentially containing BSSL, b) contacting the sample with an isolated antibody or antigen-binding fragment thereof according to the invention, and c) detecting the presence or absence of BSSL and/or quantifying the level of BSSL in said sample.

An embodiment relates to a method for diagnosing a disorder associated with BSSL. The method comprises the following steps: a) obtaining a sample from a subject suspected of having a disorder associated with BSSL, b) contacting the sample with an isolated antibody or antigen-binding fragment thereof according to the invention, c) detecting the presence or absence of BSSL and/or quantifying the level of BSSL in the sample, and d) concluding based on the results of step c) whether the subject is diagnosed with a disorder associated with BSSL or not.

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

An embodiment relates to a method for determining the enzymatic activity of BSSL. Said method comprises the following steps: a) obtaining a sample potentially containing 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 BSSL in the sample.

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%, such as 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1. The second epitope consists of a second surface comprising or consisting of an amino acid sequence according to SEQ ID NO: 2 or an amino acid sequence having at least 80%, such as 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 2.

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

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

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

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

Although the present invention has been described with reference to various typical aspects and embodiments, it will be understood by those skilled in the art that various changes can be made and elements can be replaced by equivalents without departing from the scope of the present invention. In addition, many modifications can be made to adapt a particular situation or molecule to the ideas of the present invention without departing from its basic scope. Therefore, it is intended that the present invention is not limited to any particular contemplated embodiment, but that this invention will include all embodiments falling within the scope of the appended claims.

EXAMPLES

Table 2 - BSSL material used in examples

Reagent Short name Characteristics Source/Supplier SEQ ID NO: Human BSSL hBSSL Native Astra Hässle AB 138 Biotinylated human BSSL b-hBSSL Biotinylated on glycosaccharides, 30-40 biotin/BSSL Astra Hässle AB, self-biotinylated 138 Biotinylated human BSSL hBSSL-b amine Human, biotinylated at free amine, ≈3-biotin/BSSL Astra Hässle AB, self-biotinylated 138 BSSL mice mBSSL Recombinant R&D Systems (Part No.: 5658-CE) 137

Example 1. Binding of IgG AS20 to human and mouse BSSL (SPR)

In this example, binding of the mouse AS20 IgG1 antibody (AS20 mIgG) to human and mouse BSSL was studied using surface plasmon resonance (SPR). Levels of AS20 mIgG (heavy chain variable region (HCVR) SEQ ID NO: 80 and light chain variable region (LCVR) SEQ ID NO: 114) were elevated in mice compared to full-length BSSL protein (SEQ ID NO: 138) purified from human milk. Reactivity to mouse BSSL was unknown.

Material and methods

hBSSL and mBSSL (Table 2) were used together with mouse IgG1 AS20 (AS20 mIgG1) (in-house produced, HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114) in the experiments described in this Example.

SPR measurements were performed using a BIACORE® T200 instrument (GE Healthcare). To minimize avidity effects, the antibody was immobilized on the sensor surface and BSSL was added as the analyte. AS20 mIgG1 was immobilized by amine coupling to the BIACORE® CM5 sensor chip (carboxylated dextran surface). The chips were activated by adding a 1:1 mixture of 0.2 M N-ethyl-N’-[(dimethylamino)peopyl]carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS) with a contact time of 7 minutes. The antibody was injected for 0.2-2.8 min, diluted to 14-50 μg/mL in 10 mM acetate-HCl, pH 5.0 (set one) or pH 6.0 (set two), to achieve final immobilization levels of 560 to 830 RU. Remaining activated carboxyl groups on the sensor surface were deactivated by injecting 1 M ethanolamine for 7 min.

The running buffer used in the first set of experiments was PBS pH 7.4 (10 mM phosphate, 2.5 mM KCl, 137 mM NaCl) supplemented with 0.05% (v/v) Tween 20. In the second set of experiments, the running buffer was 25 mM TrisHCl, pH 7.5, 150 mM _NaCl . 146 mM _H3PO4 was used as a standard regeneration solution. Kinetic studies and nonlinear regression analysis were performed according to the single cycle kinetics (SCK) method for the BIACORE® T200 instrument and evaluation software. The interaction between mBSSL and AS20 mIgG1 was analyzed using the steady state affinity model.

In the first set of experiments, three SCK experiments were performed with the highest concentrations of hBSSL in a concentration series of 300, 100, and 50 nM, respectively. The concentration series were made with dilutions of 1:3, 1:3.16 (semi-log), and 1:2.

In the second set of experiments, hBSSL was diluted to a 20 nM starting concentration in running buffer, followed by serial 1:1 dilutions in the same buffer to yield 5-point concentrations ranging from 20 nM to 1.25 nM. mBSSL was diluted to a 2000 nM starting concentration in running buffer, followed by serial 1:1 dilutions in the same buffer to yield 5-point concentrations ranging from 2000 nM to 125 nM.

Results

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

It was also noted that mouse BSSL interacted with immobilized AS20 mIgG1, but with an affinity that was nearly 100-fold weaker. A steady-state assay was performed to determine the affinity (Fig. 2 and Table 3).

Table 3 - Kinetic parameters of the interaction between AS20 mIgG1 and hBSSL, and AS20 mIgG1 and mBSSL

Interaction of AS20 mIgG with: k _on (M ^-1 s ^-1 ) k _off (s ^-1 ) K _D (nM) hBSSL* ¹ (9.7 ± 0.9) × 10 ⁴ (2.7 ± 0.1) × 10 ^-4 2.8 ± 0.2 hBSSL* ² 2.3 x 10 ⁵ 2.5 × 10 ^-4 1,1 mBSSL** ² BUT. BUT. 155

*Kinetics of one cycle;

**Steady-state analysis;

N.O. Not defined;

¹ First set of experiments;

² Second set of experiments.

Example 2 - Characterization of AS20 scFv production and binding to human and mouse BSSL (ELISA, SPR and LUMINEX®)

In this example, a single-chain variable fragment (scFv) version of AS20 mIgG1, designated AS20 scFv (containing HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114), was generated with preserved binding to human BSSL as assessed by enzyme-linked immunosorbent assay (ELISA), SPR, and LUMINEX®.

Material and methods

Small scale production and purification

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

ELISA

Non-biotinylated human BSSL (hBSSL) and biotinylated human BSSL (b-hBSSL) were coated either directly or via streptavidin onto a 384-well ELISA plate at two different concentrations, 1 μg/ml and 0.5 μg/ml in PBS at 4°C overnight. Purified scFv AS20 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) with concentrations ranging from 1 μg/ml to 4 ng/ml. Binding was detected using α-FLAG M2 antibody conjugated to horseradish peroxidase (HRP) (Sigma-Aldrich) followed by incubation with the chromogenic Ultra 3,3',5,5'-tetramethylbenzidine (TMB) ELISA substrate. Signal development was stopped by the addition of 1 M sulfuric acid and the optical density was measured at 450 nm.

In the second ELISA, 1 μg/mL human and mouse BSSL and negative control protein were coated directly onto a 384-well ELISA plate and incubated at 4°C overnight. Purified scFv AS20 and negative control scFv were added at two different concentrations: 1 μg/mL and 0.2 μg/mL. Binding signal detection was performed as described above. All samples in both ELISA sets were assayed in duplicate. hBSSL, b-hBSSL and mBSSL (Table 2) were used together with scFv AS20 (in-house, HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114) in the experiments described in this example.

SPR measurements

Affinity assessment of scFv clones was performed by SPR using BIACORE® T200 (GE Healthcare). α-FLAG M2 antibody was immobilized on the CM5 S chip via primary amine coupling using NHS-EDC chemistry, allowing capture of AS20 scFv via its 3xFLAG tag. A 3-fold dilution series consisting of five different concentrations, from 200 nM to 2 nM, of hBSSL and b-hBSSL were sequentially injected into the flow cells, allowing binding to the captured AS20 scFv. Surface regeneration was performed under acidic conditions using 10 mM glycine-HCl at pH 2.2. The obtained single cycle kinetic data were fitted to a 1:1 Langmuir binding model, and the kinetic parameters, k _a (1/M s), k _d (1/s), and K _D (M) were obtained using BIAevaluation software.

LUMINEX® assays

Biotinylated hBSSL was incubated with LUMINEX® Neutravidin-coupled beads and mixed with 30 different ID beads, each conjugated to an irrelevant protein. The mixed pool of beads was incubated with AS20 scFv present in bacterial supernatant diluted 1:10 in assay buffer (PBS supplemented with 3% BSA, 0.05% Tween 20 and 10 μg/ml Neutravidin). One positive control scFv, i.e., an scFv expected to bind to beads coated with one of the irrelevant proteins, was also included. Binding of scFv clones to a specific protein-conjugated bead was enabled by the use of R-PE-conjugated anti-FLAG M2 antibody followed by analysis on the FlexMAP 3D instrument.

Results

Small scale production and purification

Gel electrophoresis analysis of bacterially expressed and purified AS20 scFv demonstrated high purity with a single major protein band in good agreement with the expected molecular mass of the scFv (data not shown).

ELISA

AS20 scFv exhibited concentration-dependent binding to both non-biotinylated and biotinylated human BSSL (Fig. 3a). The signal intensity at a given scFv concentration was much higher for biotinylated BSSL than for non-biotinylated BSSL, which may be due to differences in coating conditions.

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

SPR measurements

A single-cycle kinetics approach was used to determine the affinity of AS20 scFv for non-biotinylated and biotinylated human BSSL. AS20 scFv exhibited very similar affinity for non-biotinylated human BSSL ( _KD = 0.6 nM) and biotinylated human BSSL ( _KD = 0.8 nM) (Table 4).

Table 4 - Kinetic parameters of the interaction between AS20 scFv and hBSSL, native and biotinylated

Interaction of AS20 scFv with: k _on (M ^-1 s ^-1 ) k _off (s ^-1 ) K _D (nM) hBSSL 1.9 x 10 ⁵ 1.2 x 10 ^-4 0.6 b-hBSSL 5.5 x 10 ⁴ 4.4 × 10 ^-5 0.8

Luminex Analysis

To determine whether scFv AS20 is subject to non-specific interactions with irrelevant proteins, a Luminex assay was performed in which scFv AS20 was assayed against 30 different irrelevant proteins as well as its cognate target. AS20 scFv showed binding only to human BSSL with no or very low binding to all other proteins included in the assay (Fig. 4).

Conclusion

The scFv AS20 was found to bind to both non-biotinylated human BSSL and biotinylated human BSSL with similar affinity (similar K _D values) in the subnanomolar range. The obtained K _D value for non-biotinylated BSSL is in good agreement with that reported in Example 1 for the full-length IgG antibody. The AS20 scFv also exhibited low off-target binding to 30 irrelevant proteins when analyzed using the Luminex-based approach. As shown by the ELISA results, the scFv AS20 exhibits binding to mouse BSSL, as demonstrated for the full-length IgG in Example 1.

Example 3 - Characterization of AS20 scFv binding to mouse BSSL via 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 application of a competitive HTRF-based assay as an alternative method for characterizing the interaction between scFv AS20 and mouse BSSL.

Material and methods

To study the interaction between AS20 scFv and native murine BSSL, a competition assay was developed. Binding was detected using a terbium-conjugated α-FLAG antibody donor molecule (Cisbio #611FG2TL), which reacts with the C-terminal FLAG tag of the scFv, and a streptavidin-conjugated acceptor molecule XL665 (Cisbio #610SAXL), which reacts with biotin moieties 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 scFv AS20 (in-house produced, HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114) in the experiments described in this Example.

2.5 nM scFv AS20 was pre-incubated for 2 h with the mouse or human BSSL ortholog, followed by the addition of 5 nM 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 signal/noise (615 nm) were measured using EnVision (PerkinElmer). The Delta R experimental output was calculated for each point using quadruplicates and two blanks.

Results

The data demonstrated that mouse BSSL competed with human BSSL-biotin for binding to scFv AS20 in a concentration-dependent manner (Fig. 5). The same observation was true for native human BSSL. A 50% reduction in Delta R was achieved with 150-200 nM mouse BSSL and approximately 2 nM human BSSL, suggesting that scFv AS20 has approximately 100-fold lower affinity for mouse BSSL compared to the human ortholog.

Conclusion

The data obtained demonstrated that AS20 scFv had approximately 100-fold lower affinity for mouse BSSL compared to the human ortholog. This was very consistent with the affinity obtained for the full-length antibody formats of the same clone using SPR; AS20 mIgG1 (Example 1) and chimeric AS20 (Example 4).

Example 4 - Production of Chimeric AS20

In this example, a chimeric AS20 was produced. More specifically, a chimera of the human IgG4 subclass was constructed.

Materials and methods

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

Results

Purity was determined by HPLC and was >98%. The sequence of chimeric AS20 was determined as SEQ ID NO: 139 for the heavy chain and SEQ ID NO: 140 for the light chain.

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

Example 5 - Design and construction of AS20 CDR graft antibody and AS20 humanization library

AS20 is a murine antibody, AS20 mIgG, see Example 1. Non-human antibodies have been shown to elicit immune responses in humans that can lead to neutralization of the administered antibody and, in turn, limit the effect of the antibody in treating disease. In order to overcome this potential problem, humanization of the antibody was performed. This Example describes two strategies for humanization of AS20, namely complementarity determining region (CDR) grafting and a library-based approach. The resulting CDR graft antibody is referred to herein as an AS20 CDR graft or CDR graft, and the resulting library is referred to herein as an AS20 humanization library. The library was subsequently used to select and isolate AS20-binding scFv fragments using phage display (see Example 6).

Materials and methods

Figures 15A and 15B provide a summary of the design of a combinatorial scFv library for the variable region of the heavy chain, and Figures 16A and 16B provide a summary of the design of a combinatorial scFv library for the variable region of the light chain.

The scFv format was chosen as the scaffold for both the graft CDR and the humanization library. The data presented in Example 2 demonstrated that AS20 scFv fully retained the binding capacity of its full-length parental IgG counterpart, suggesting that the scFv gene would be a good scaffold format for both the graft CDR and for constructing combinatorial libraries.

The human immunoglobulin (IHGV) heavy chain variable region germline gene with the highest sequence homology to the AS20 heavy chain variable region according to IMGT/DomainGapAlign (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi), IGHV1-46 with 73.5% homology (residues 1-104) was chosen as the IHGV scaffold. Homology was taken into account when selecting the light chain sequence, but pairs of heavy and light chains with favorable biophysical properties were also considered [10]. Together, this led to the choice of the human germline gene IGKV1-39. Also based on the IMGT/DomainGapAlign domain search, IGHJ4 and IKVJ2 were selected as junction fragments to obtain the complete heavy and light chain variable domains, respectively.

The AS20 CDR graft was generated by grafting six mouse CDR loops into human germline genes. For the heavy chain, the following regions were grafted into the IGHV1-46 scaffold: heavy chain complementarity determining region 1 (HCDR1) (SEQ ID NO: 7); extended HCDR2 (eHCDR2) (SEQ ID NO: 141); and HCDR3 (SEQ ID NO: 9). This yielded a CDR graft onto HCVR according to SEQ ID NO: 144. For the light chains, the following regions were grafted into the IGKV1-39 scaffold: extended light chain complementarity determining region 1 (eLCDR1) (SEQ ID NO: 142), eLCDR2 (SEQ ID NO: 143), and LCDR3 (SEQ ID NO: 21), yielding LCVR according to SEQ ID NO: 145. By fusing HCVR to LCVR via a glycine-serine linker ((Gly ₄ Ser) ₃ ), the gene encoding the corresponding scFv construct was generated. Two additional amino acids (Arg and Thr, both parts of the CL domain) were added at the end of LCVR to include a BsiWI restriction site. GenScript (Piscataway, NJ, USA) was employed for the synthesis and subcloning of the scFv gene. After synthesis, the scFv gene was cloned into the internal phagemid using the restriction enzymes SfiI and BsiWII.

The same HCVR and LCVR scaffold used in the CDR graft was also used to construct the AS20 humanization library scaffold (Figure 6). The mutagenesis strategy for the AS20 humanization library is summarized in Table 5. HCDR3 is considered to be the most important region for antigen binding. It was hypothesized that this loop is also likely to be important for the AS20-BSSL interaction and was therefore kept constant. Instead, 22 positions that differ between AS20 and the AS20 humanization scaffold in the other five CDRs were chosen for variation (Figure 6). In this case, double diversity was attempted first, i.e., allowing residues found in AS20 and human germline genes that create the humanization scaffold at a particular position into the process. However, not all amino acid pairs can be formed by oligo NNS without introducing additional amino acids, so additional chemical diversity was added at six positions (three in VH: 62, 64, 68 and three in VL: 27, 66, 68). An alternative strategy was undertaken for LCDR3. In this case, the diversity of rearranged functional antibodies encoded by the IGVK1-39/IKV1D-39 gene as found in the IMGT database (http://www.imgt.org/ligmdb/) was taken into account. More specifically, antibody sequences having an LCDR3 length of 8 amino acids, an LCDR3 length of AS20, were used as a guide for what diversity to introduce. The resulting consensus sequence was QQSYSTPT (amino acid residues 105-117, SEQ ID NO: 173). Based on the mouse AS20 and human consensus sequences, two amino acids were introduced at positions 105, 107, and 108. At position 115, again due to the constraint of the NNS oligo, four amino acids were introduced. As a result of the VJ gene addition process, the greatest diversity in LCDR3 was found at position 116. In an attempt to mimic this variability, but also constrained by the NNS codon usage, six amino acids (P, H, L, Y, S, and F) were involved in the process. This strategy allows for the capture of >50% of the diversity found among antibodies at this position. Overall, the above procedure generates a combinatorial theoretical diversity of approximately 1.2× ¹⁰⁹ different variants.

Table 5 - Positions targeted for mutagenesis in the AS20 humanization library. The positions in HCVR and LCVR are indicated at the top and bottom of the table, respectively, the amino acids in bold represent the amino acids found in AS20, while the underlined amino acids represent the corresponding diversity found in the human germline gene. The numbering is determined by IMGT nomenclature, and the IUPAC nucleotide code is used to define the codon. One primer each for the five target regions (HCDR1, HCDR2, LCDR1, LCDR2, and LCDR3) was used to introduce diversity.

Diversity was introduced into the library backbone gene using an optimized Kunkel mutagenesis methodology essentially as described in [11], using the AS20 humanization library backbone gene (Fig. 6) together with five mutagenic oligonucleotides (Table 6). To assess whether the intended diversity had been incorporated, E. coli TOP10 cells were chemically transformed with a small aliquot of the DNA obtained by the Kunkel mutagenesis methodology and 96 clones were selected and submitted for sequencing (GATC, Germany). The remaining DNA was then electroporated into SS320 cells (Lucigen, Middleton, WI, USA), yielding a highly diverse library containing approximately 1.7× ¹⁰¹⁰ clones, as measured by the number of bacterial colonies obtained after transformation. Transformed SS320 cells were harvested and stored with 15% glycerol at -80 °C. The bacterial glycerol stock solution was used to inoculate a total of 600 ml of 2×YT with antibiotics selective for both the phagemid and F' episome. Bacteria were grown to exponential phase and then infected with M13KO7 helper phages (New England Biolabs, Ipswich, MA, USA) at infections in multiples of five. The culture was expanded overnight and phages displaying scFvs were harvested by standard PEG/NaCl precipitation. The final library stock was dissolved in PBS supplemented with 0.5% BSA, 0.05% Tween-20.

Table 6 - Oligonucleotide primers used to construct the AS20 humanization library. Sequences are formatted using the IUPAC nucleotide code.

Primer name NT sequence SEQ ID NO: SL048_hum_AS20_H1 GATACACCTTCACCAGCTACWATATGCACTGGGTGCG 164 SL048_hum_AS20_H2 GACAAGGGCTTGAGTGGATRGGARTAATCWACCCTRGTRRTGGTKMCACAAGCTACRMTCAGAAGTTCMAGGGCCGCGTCACC 165 SL048_hum_AS20_L1 CGTCACCATCACCTGCAGKGCAAGTYMGAGCATTAGCTATWTGMATTGGTATCAGCAGAAAC 166 SL048_hum_AS20_L2 CCTAAGCTCCTGATCTATGMTRCATCCARSTTGSMAAGTGGGGTCCCATCAC 167 SL048_hum_AS20_L3 GATTTTGCAACTTATTACTGTCASCAGAGKTMTAGTWMTYHCACTTTTGGCCAGGGG 168

Results

The genes encoding the AS20 CDR graft and the AS20 humanization library backbone were synthesized and cloned into the pHAT4 phagemid vector. The AS20 humanization library was constructed using an optimized Kunkel procedure, yielding 1.7× ¹⁰¹⁰ transformants. Sequencing of 96 randomly selected clones confirmed the introduction of the target diversity (data not shown).

Conclusion

An AS20 CDR graft and an AS20 humanization library were successfully constructed. In both cases, IGHV1-46 and IGKV1-39 were used as human scaffold genes. The binding of the AS20 CDR graft to BSSL was assessed in both scFv (Example 6) and IgG formats (Examples 9 and 11). The AS20 humanization library was used to isolate humanized scFv fragments that bind BSSL by phage display and various binding screening assays (Example 6). Some of the selected clones showed binding to the cognate target (human BSSL) with affinity and specificity equivalent to the parental IgG, and even showed better affinity when binding to the mouse ortholog (Example 9). Sequence analysis of the selected clones (Example 11) revealed enrichment of specific residues at certain positions. Of interest is the fact that at several positions (e.g. VH:62, 64 and VL:115) there is preferential selection of amino acids beyond the double diversity, indicating that introducing additional diversity was a successful strategy.

Example 6 - Phage display selection on human and mouse BSSL and subsequent screening and sequencing

In this example, phage display selection was performed to ensure the isolation of scFv fragments specific for human and mouse BSSL.

Material and methods

Antigens

During the phage display selection, mouse BSSL and non-biotinylated and biotinylated human BSSL were used as target antigens. Specifically, two variants with different biotinylation degrees and different binding chemistry were prepared. They were amine BSSL-b and glyco BSSL-b. hBSSL, b-hBSSL, hBSSL-b amine, and mBSSL (Table 2) were used as the target for the phage display selection in this example.

Phage display selection

For all antigens, phage display was performed with four enrichment rounds using two in-house constructed synthetic human scFv phage libraries: the SciLifeLib2 library and the AS20 humanization library (see Example 5). SciLifeLib2 is a naïve synthetic human scFv library similar in design and construction to those previously described [12]. Selection pressure was increased by gradually decreasing the amount of antigen and increasing the number and intensity of washes between different rounds. For the two biotinylated human BSSL samples, selection was performed by immobilization on streptavidin-coated paramagnetic beads (Dynabeads M-280, ThermoFisher Scientific, #11206D) and most steps of the selection process were automated and performed by a Kingfisher Flex robot. Native antigens were selected by spotting on a 96-well plate (NUNC Maxisorp #442404). In some of the lanes, to preferentially select for cross-species reactive scFv, the antigen was alternated between human and mouse BSSL in different cycles. Phage elution was performed by trypsin or by competitive elution using mouse BSSL to select for human BSSL and vice versa. Combining these different parameters resulted in a design comprising a total of five different selection lanes for ScilifeLib2 and nine for the AS20 humanization library. Isolated phages were propagated in Top10F’ E. coli, either on agar plates at 37°C overnight (cycles 1 and 2) or in solution at 30°C overnight (cycles 3 and 4). Phage stocks were generated by infection with excess M13K07 helper phage (New England Biolabs, #N0315S) and scFv expression induced by the addition of IPTG. Overnight cultures were precipitated with PEG/NaCl, resuspended in selection buffer, and used for the next round of selection. Table 7 shows the phage display selection lanes.

Table 7 - Phage display lanes

Track Cycle 1 Cycle 2 Cycle 3 Cycle 4 Library Pre-adsorption 1 200 pmol hBSSL ¹ 50 pmol hBSSL ¹ 12.5 nMl hBSSL ¹ 2.5 nM hBSSL ¹ SciLifeLib2 (scFv) R1&R2 SAV 2 200 pmol hBSSL ² 50 pmol hBSSL ² 12.5 nM hBSSL ² 2.5 nM hBSSL ² 3 50 pmol hBSSL ^2.4 12.5 nM hBSSL ^2.4 2.5 nM hBSSL ^2.4 4 ^mBSSL3 12.5 nM hBSSL ^{2 mBSSL3} 5 ^{mBSSL3 mBSSL3 mBSSL3 mBSSL3} 6 200 pmol hBSSL ¹ 50 pmol hBSSL ¹ 12.5 nMl hBSSL ¹ 2.5 nM hBSSL ¹ AS20 (scFv) 7 200 pmol hBSSL ² 50 pmol hBSSL ² 12.5 nM hBSSL ² 2.5 nM hBSSL ² 8 50 pmol hBSSL ^2.4 12.5 nM hBSSL ^2.4 2.5 nM hBSSL ^2.4 9 ^mBSSL3 12.5 nM hBSSL ^{2 mBSSL3} 10 ^{mBSSL3 mBSSL3 mBSSL3 mBSSL3} 11 hBSSL ³ hBSSL ³ hBSSL ³ hBSSL ³ 12 hBSSL ^3.4 hBSSL ^3.4 hBSSL ^3.4 13 mBSSL ^3.4 mBSSL ^3.4 mBSSL ^3.4 14 ^{mBSSL3 mBSSL3} hBSSL ³

1 – selection performed on biotinylated BSSL (amino-linked, 10X) coupled to SAV magnetic beads (M280);

2 – selection performed on biotinylated BSSL (on carbohydrates, BH8520) linked to SAV magnetic beads (M280);

3 – selection performed on a native antigen applied to the surface of immunotubes;

4 – elution of phage by competition with other BSSL species (mouse or human) (in all other cases trypsin will be used for elution).

Re-cloning

To ensure production of soluble scFv, phagemid DNA was isolated from the third and fourth rounds of each selection lane. In the pools, genes encoding scFv fragments were subcloned into the screening vector, providing a signal for scFv secretion along with a triple-FLAG tag and a hexahistidine (His) tag at the C-terminus. The construct was subsequently transformed into E. coli TOP10.

Primary ELISA analysis and sequencing

For each selection lane, a total of 89 to 222 colonies from rounds 3 and 4 were picked and cultured in 96-well plates and grown overnight. Expressed scFvs (supernatants) were screened by ELISA for binding to the native form of the respective selection target. Experiments in this screening run included the AS20 scFv, which had been previously constructed, produced and characterized for binding to both human and mouse BSSL (see Example 2). An AS20 CDR graft was also included as an scFv (see Example 5). Clones scoring positive in ELISA were subjected to DNA sequencing (GATC Biotech, Cologne, Germany).

Secondary ELISA and HTRF analysis

All clones with unique sequence identified for each selection lane were further analyzed in a second screen by ELISA. In this experiment, the number of antigens was expanded to include native human and mouse BSSL as well as biotinylated human BSSL. Streptavidin was used as an irrelevant antigen. Binding of all scFvs was also assessed by HTRF (homogeneous time-resolved fluorescence).

Results

A total of 14 phage selection lanes were performed in parallel on four BSSL forms using the SciLifeLib 2 library and the AS20 humanization library. From each of the 14 lanes, between 89 and 222 clones were selected and analyzed. ELISA and HTRF binding screening and sequencing revealed a total of 68 unique scFv clones capable of binding to the BSSL ortholog for which they were selected. Surprisingly, no binding of the AS20 CDR of the scFv graft was detected.

Conclusion

Primary screening of a total of 2365 clones by ELISA resulted in a total of 467 scFv fragments with potential binding affinity for human and mouse BSSL that were submitted for sequencing. Secondary screening by ELISA followed by HTRF and resequencing yielded a total of 68 scFv clones with unique sequence. Their binding data suggested that their relative binding to human and mouse BSSL can be divided into three groups with recognition characteristics of one or both of these orthologs.

64 scFv isolates were obtained from the AS20 humanization library, while only four of them were obtained from SciLifeLib2, indicating that BSSL is a challenging target for selection by phage display using a naive antibody library.

Example 7 - ELISA and SPR affinity ranking of 68 anti-BSSL scFv

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 the earlier binding data (ELISA and HTRF), these results were taken into account when deciding on the selection of candidates for further development.

Material and methods

hBSSL and mBSSL (Table 2) were used as BSSL reagents in this Example.

ELISA

Human and mouse BSSL were coated at 4°C overnight, 1 μg/ml in PBS, in a 384-well ELISA plate. Two negative control proteins, streptavidin and BSA, were also included. FLAG-tagged scFv clones present in the bacterial supernatant 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. Binding was detected using HRP-conjugated α-FLAG M2 antibody (Sigma-Aldrich #A8592) followed by incubation with TMB ELISA substrate (ThermoFisher Scientific #34029). The development of the colorimetric signal was stopped by adding 1 M sulfuric acid, and the plate was analyzed at 450 nm.

SPR

Kinetic screening was performed on a BIACORE® T200 biosensor instrument (GE Healthcare). The α-FLAG M2 antibody (Sigma-Aldrich #F1804), which functions as a capture ligand, was immobilized on all 4 surfaces of the CM5-S sensor chip according to the manufacturer's recommendations.

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

Results

ELISA

Binding of 68 scFv clones to directly coated human and mouse BSSL could be confirmed for the majority of clones. As demonstrated in Example 6, BSSL-binding clones could be divided into three groups with recognition characteristics of either human BSSL, mouse BSSL, or human and mouse BSSL. The majority of clones showed preferential binding to human BSSL (data not shown).

SPR

Data analysis was performed by visual inspection of the sensorgrams (not shown). From their examination it became apparent that most clones had significantly higher affinity, lower K _D (M) values for human BSSL than for murine BSSL. Inspection of the sensorgrams also demonstrated that many of the humanized clones exhibited affinities in the same range as for scFv AS20. 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 for mouse BSSL for these particular clones was also similar to that observed for AS20.

Several clones obtained from phage display selection lanes sorted for mouse BSSL showed preferential binding to mouse BSSL over human BSSL, an example of which is clone S-SL048-66 (containing SEQ ID NO: 61 HCVR and SEQ ID NO: 95 LCVR)

Conclusion

In this example, binding of the previously identified 68 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 was found for AS20, the vast majority of clones exhibited higher calculated binding affinities, defined as the equilibrium dissociation constant, K _D (M), for human BSSL than for mouse BSSL. The calculated affinities for human BSSL ranged from low nanomolar to nanomolar values, with the reference AS20 scFv having a K _D value of 2 nM. A small set of scFv clones exhibited higher affinity for mouse BSSL than for human BSSL, with the highest affinity corresponding to a K _D value of 43 nM.

From all collected data, 38 clones (containing 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 scFv AS20 (HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114) were selected for transformation into human IgG4 S228P. All candidate clones were derived from the AS20 humanization library and none from SciLifeLib.

Example 8 - Conversion to hIgG4 S228P format and small-scale transient expression of 38 humanized BSSL-specific antibodies

In this experiment, the 38 most promising humanized scFv clones (containing 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 the human IgG4 S228P antibody format. In addition, AS20 (parental clone) (containing HCVR SEQ ID NO: 80 and LCVR SEQ ID NO: 114) and AS20 CDR graft (containing HCVR SEQ ID NO: 144 and LCVR SEQ ID NO: 145) were similarly converted to IgG4 S228P.

Briefly, human IgG4 is considered to be the most Fc-silencing natural IgG subclass in humans, i.e., it does not mediate major effector functions through the Fc portion of the antibody. Like IgG1, IgG4 has a serum half-life of 21 days. However, IgG4 naturally tends to dissociate in vivo into half-IgG4 molecules and can then recombine with other circulating IgG4 molecules. This half-molecule exchange can be avoided by introducing a stabilizing mutation in the hinge region, namely S228P (Eu numbering; identical to Kabat numbering S241P) [13].

The genes encoding VH and VL of 38 scFv clones, AS20 and AS20 CDR grafts were successfully transferred into a vector encoding the human IgG4 subclass S228P. ExpiHEK293 cells were transiently transfected, 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).

Material and methods

Sequencing

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

Table 8 - Antibodies used in this Example

SEQ ID NO:
HCVR SEQ ID NO:
LCVR SEQ ID NO:
hIgG4 S228 P heavy chain (HC) SEQ ID NO:
hIgG4 S228 P light chain (LC) S-SL048-10 47 81 S-SL048-11 30 31 119 120 S-SL048-12 48 82 S-SL048-13 49 83 S-SL048-14 50 84 S-SL048-17 51 85 S-SL048-18 52 86 S-SL048-38 53 87 S-SL048-40 54 88 S-SL048-41 55 89 S-SL048-43 56 90 S-SL048-45 57 91 S-SL048-46 32 33 121 122 S-SL048-47 58 92 S-SL048-48 59 93 S-SL048-65 60 94 S-SL048-66 61 95 S-SL048-74 62 96 S-SL048-75 63 97 S-SL048-79 64 98 S-SL048-81 65 99 S-SL048-86 66 100 S-SL048-89 67 101 S-SL048-103 68 102 S-SL048-104 69 103 S-SL048-105 70 104 S-SL048-106 34 35 123 124 S-SL048-107 71 105 S-SL048-108 72 106 S-SL048-109 73 107 S-SL048-110 74 108 S-SL048-112 75 109 S-SL048-115 76 110 S-SL048-116 36 37 125 126 S-SL048-118 36 38 127 128 S-SL048-125 77 111 S-SL048-131 78 112 S-SL048-134 79 113 AS20 80 114 129 130 AS20 CDR graft 144 145 131 132 anti-NP 133 134

Figure 14 shows the sequence differences of 38 humanized clones transformed with hIgG4 S228P. In the AS20 humanization library, a total of 20 positions were targeted for diversification in the heavy chain CDR1 and CDR2 and the light chain CDR1, CDR2, and CDR3. For comparison, the figure shows AS20 and the graft CDR design.

In-Fusion Cloning

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

Transfection into HEK293, expression and purification

Transfection of plasmid DNA into expiHEK293 cells was performed using the ExpiFectamineTM 293 Transfection Kit (ThermoFisher Scientific #A14525) in 4 ml cultures in a 24-well plate. After 5 days of culture at 37°C, 6% _CO2 , 80% relative humidity and 400 rpm, the medium supernatant was mixed with protein A-conjugated magnetic beads and purified on a KingFisher Flex. Immediately after elution in 0.1 M glycine, pH 2.7, neutralization was performed with 1 M Tris-HCl, pH 8.8, and buffer exchange to PBS was performed using a 96-well spinner plate for desalting. SDS-PAGE was performed to determine the purity and integrity of purified IgG and concentrations determined using an Implen NP80 UV-Vis spectrophotometer (Fisher Scientific).

Results

In-Fusion Cloning

38 unique scFvs, AS20 and AS20 CDR graft were successfully converted into full-length human IgG4 antibodies, as confirmed by sequencing.

Transfection into HEK293, expression and purification

Antibodies were 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).

Conclusion

All BSSL-binding antibodies were successfully re-cloned into hIgG4 format, expressed in HEK293 cells and purified using protein A-conjugated magnetic beads on a Kingfisher Flex. All demonstrated acceptable levels of purity when assessed by SDS-PAGE.

Example 9 – Binding of 38 hIgG4 S228P clones to human and mouse BSSL

This example describes a surface plasmon resonance (SPR) target binding assay of 38 hIgG4 S228P clones (Example 8, Table 8) that was performed to verify that binding to human and mouse BSSL was retained after conversion of scFv to IgG format.

Material and methods

Kinetic parameters of hIgG4 S228P clones were determined by SPR using the BIACORE® T200 System (GE Healthcare). Single cycle kinetics were used to measure the affinity of purified hIgG4 molecules for human and mouse BSSL. Anti-Fab antibody (GE Healthcare, #28958325) was immobilized on the CM5 S sensor chip via primary amine coupling using NHS-EDC chemistry. hIgG4 was captured by the anti-Fab antibody and subsequently five different concentrations of hBSSL (1:5 dilutions starting at 50 nM) or mBSSL (1:5 dilutions starting at 500 nM) were coated onto 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 BIAevaluation software. For mouse BSSL, steady state analysis was performed by plotting the response rate at equilibrium against each concentration, and K _D values were obtained using BIAevalution software.

Results

A single cycle kinetics approach was used to determine the affinity of the converted antibodies for non-biotinylated human and mouse BSSL. The obtained equilibrium dissociation constants (K _D ) are listed in Table 9. For hBSSL, the data generally fit a 1:1 binding model well. For mouse BSSL, the data did not always fit a 1:1 binding model very well. Instead, the data were analyzed using a steady state analysis. The determined K _D values for binding to human BSSL as well as mouse BSSL were in the same range as the K _D values determined for the same clones in scFv format (see Example 7). For the AS20 CDR graft clone, a low degree of binding to human BSSL was observed (data not shown). However, the model fit was not considered accurate and therefore no K _D value was obtained for this particular clone.

Table 9 - Measured equilibrium dissociation constants (K _D ). In some cases the data quality was considered too low to be reliable and are designated in this document as "n.d." (not determined).

K _D (nM), hBSSL K _D (nM), mBSSL S-SL048-10 2,2 57 S-SL048-11 0.7 24 S-SL048-12 6.4 57 S-SL048-13 0.9 57 S-SL048-14 0.9 89 S-SL048-17 4.3 68 S-SL048-18 2,3 68 S-SL048-38 1.0 67 S-SL048-40 1.0 87 S-SL048-41 1,2 79 S-SL048-43 1,2 137 S-SL048-45 9.6 82 S-SL048-46 3.7 24 S-SL048-47 8.5 But. S-SL048-48 0.8 41 S-SL048-65 But. 30 S-SL048-66 8.0 83 S-SL048-74 4.8 21 S-SL048-75 But 164 S-SL048-79 5.0 48 S-SL048-81 4.4 60 S-SL048-86 6.8 167 S-SL048-89 6.0 But. S-SL048-103 1.7 199 S-SL048-104 3.3 111 S-SL048-105 6.0 237 S-SL048-106 1,2 111 S-SL048-107 1,2 98 S-SL048-108 1.3 149 S-SL048-109 1.4 353 S-SL048-110 5.9 168 S-SL048-112 5.0 But. S-SL048-115 6.0 186 S-SL048-116 0.6 52 S-SL048-118 0.9 95 S-SL048-125 2.1 123 S-SL048-131 2.7 63 S-SL048-134 1.8 But. AS20 0.6 161 AS20 CDR graft But. But.

Conclusion

Overall, the results demonstrate that the binding affinity of the antibodies to human and mouse BSSL was not affected by recloning into the hIgG4 format. However, the AS20 CDR graft behaved differently. As described in Example 6, the AS20 CDR graft did not show binding to BSSL when expressed in the scFv format. However, a binding signal to human BSSL was observed in the IgG format.

Example 10 - Functional test of 28 hIgG4 S228P clones using flow cytometry-based displacement assay.

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

Material and methods

Preparation of leukothrombocyte film

Human blood was collected from a single healthy donor into 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 bucket centrifuge rotor.

Flow cytometry-based substitution analysis

28 BSSL-specific antibodies to hIgG4 and five reference antibodies at different concentrations (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-bottomed polystyrene tubes, 1× PBS (pH 7.4) was added to a final volume of 20 μl and the antibody/b-hBSSL mixtures were incubated for 30 min at +4°C to facilitate binding of the antibodies to BSSL. Then, buffy coat (50 μl) was added to each antibody/b-hBSSL mixture and incubation was continued for another 30 min at +4°C. Afterwards, 2 ml of FACS lysis solution (BD Biosciences) were added and the cells were incubated for 10 min at room temperature to lyse the red blood cells and fix the white blood cells. The cells were then centrifuged for 5 min at 200× g, the resulting pellets were washed with 2 ml of FACS buffer (1× PBS supplemented with 1% FCS and 0.1% NaN ₃ ) and centrifuged again. Finally, the supernatants were discarded and the cells were resuspended in the last drop of liquid, approximately 50 μl.

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

Results

CD14 ⁺ cells were first isolated to delineate the monocyte population. Binding of b-hBSSL to isolated CD14 ⁺ monocytes was then detected using BB515-labeled streptavidin and quantified as the mean fluorescence intensity (MFI) in the BB515 channel. The ability of BSSL-specific hIgG4 and reference antibodies to displace b-hBSSL binding to CD14 ⁺ monocytes was quantified as a decrease in BB515 MFI in monocytes following incubation with increasing concentrations of BSSL-specific hIgG4 or reference antibodies.

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

Antibody Ability to displace b-hBSSL binding to monocytes (percentage reduction relative to binding without competing antibodies)   0.5 mcg 1.0 mcg 1.5 mcg 2.0 mcg 3.0 mcg S-SL048-10 34 42 S-SL048-11 1 9 60 68 S-SL048-14 11 32 48 56 S-SL048-17 16 36 S-SL048-18 58 61 S-SL048-38 6 44 52 51 S-SL048-40 20 28 44 53 S-SL048-41 12 29 51 71 72 S-SL048-43 37 55 S-SL048-46 43 61 S-SL048-48 28 23 50 61 68 S-SL048-65 5 23 21 35 39 S-SL048-66 12 12 39 43 51 S-SL048-74 29 52 S-SL048-75 3 0 S-SL048-79 20 52 S-SL048-81 31 62 S-SL048-103 12 -1 26 69 S-SL048-104 2 44 S-SL048-106 52 61 S-SL048-107 37 49 S-SL048-108 41 48 S-SL048-109 38 51 S-SL048-112 28 54 S-SL048-116 58 64 S-SL048-118 23 58 S-SL048-125 9 33 S-SL048-131 31 58 AS20 CDR graft 8 -13 -8 AS20 IgG4 19 21 58 Anti-NP hIgG4 -5 0 -2 4 3 AS20 mIgG1 31 66 72 74 80 α-synuclein mIgG1 -8 -2 -4 -1 -7

Conclusion

The molecular weight of hBSSL (76 kDa) is approximately half that of IgG (150 kDa). Therefore, in this example, 1 μg BSSL and 2 μg IgG corresponded approximately to a 1:1 molar ratio. Using the highest antibody concentration (3 μg per reaction), we demonstrated that 9 of 28 BSSL-specific hIgG4 S228P antibodies inhibited (displaced) at least 60% of BSSL (1 μg per reaction) from binding to monocytes. The most effective antibody for displacing binding was AS20 mIgG1, whereas AS20 CDR graft hIgG4, anti-NP hIgG4, and anti-α-synuclein mIgG1 did not affect binding at all.

Example 11 - Preparation of 5 previously mentioned hIgG4 S228P antibodies and controls

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

Two controls were also included: AS20 and the AS20 CDR graft 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 hIgG4 S228P subclass format chosen is the same as that previously used for the evaluation of 38 BSSL-specific clones in Example 8.

Material and methods

Antibody production

Absolute Antibody (Oxford, UK) was commissioned to prepare the material. VH and VL sequence information for seven clones was sent to the company, where the genes were synthesized and cloned into vectors encoding the human IgG4-S228P subclass. The antibodies were transiently expressed in mammalian HEK293 cells and then purified by protein A affinity chromatography. Purity and integrity were assessed by SDS-PAGE, and endotoxin levels were determined by the LAL endotoxin chromogenic assay.

The amino acid sequences of the eight antibodies correspond to SEQ ID NO as shown in Table 11.

Table 11 - Antibodies used in this Example

Name of the antibody HC SEQ ID NO: LC SEQ ID NO: S-SL048-11, hIgG4 S228P 119 120 S-SL048-46, hIgG4 S228P 121 122 S-SL048-106, hIgG4 S228P 123 124 S-SL048-116, hIgG4 S228P 125 126 S-SL048-118, hIgG4 S228P 127 128 AS20, hIgG4 S228P 129 130 AS20 CDR graft hIgG4 S228P 131 132 Anti-NP hIgG4 S228P 133 134

Surface Plasmon Resonance (SPR)

Kinetic parameters of hIgG4 S228P clones were determined by SPR using the BIACORE® T200 System (GE Healthcare). Anti-Fab antibody (GE #28958325) was immobilized on the CM5 S sensor chip via primary amine coupling using NHS-EDC chemistry. hIgG4 antibodies were captured with anti-Fab antibody and then five different concentrations of hBSSL (1:5 dilutions, 0.08-50 nM) or mBSSL (1:2 dilutions, 50-800 nM) were spotted on the surface. The sensor chip surface was regenerated with 10 mM glycine-HCl pH 2.1. For binding to human BSSL, single cycle kinetic data were fitted to a 1:1 binding model and kinetic parameters were obtained using BIAevaluation software. For mouse BSSL, steady state analysis was performed by plotting the response rate at equilibrium against each concentration, and K _D values were obtained using BIAevalution software.

Results

Antibody production

Ten milligrams at a concentration of 4.6-5 mg/mL of eight antibodies in 25 mM histidine, 150 mM NaCl, 0.02% P80 (pH 6.0) were obtained from Absolute Antibody. Purity was determined by SDS-PAGE to >98% and endotoxin levels were determined by the LAL endotoxin chromogenic assay to <0.05 EU/mg. Monomer content of each clone was determined by analytical size exclusion chromatography to >98%.

SPR

SPR was used to determine the affinity of antibodies to human and mouse BSSL. Table 12 lists the various K _D values of the previously reported clones for binding to hBSSL and mBSSL. For comparison, the K _D values determined for the same clones produced in-house are also included (these experiments are described in Example 9).

Table 12 - Summary of SPR results for antibodies in hIgG4 S228P format. *These values were obtained from the experiments presented in Example 9. Numbers marked with # are considered unreliable and should be viewed with caution. In some cases, the quality of the data was considered too low to be reliable and are indicated in this document as "n.d." (not determined).

Own production batch* Absolute Antibody Protein Batch Clone hBSSL K _D
(nM) mBSSL K _D (nM) hBSSL K _D
(nM) mBSSL K _D
(nM) S-SL048-11 0.7 24 ^# 0.6 65 S-SL048-46 3.7 24 3.8 55 S-SL048-106 1,2 111 ^# 1,2 >500 S-SL048-116 0.6 52 0.5 40 S-SL048-118 0.9 95 0.5 70 AS20 0.6 161 0.6 223 CDR transplant But. But. 61 But.

Conclusion

The five previously mentioned candidates and three controls manufactured by Absolute Antibody were assayed for binding to BSSL by ELISA (data not shown) and SPR. The results demonstrated that binding to BSSL was generally very similar to that observed for the corresponding clones manufactured in-house in the same IgG format (see Example 9). The results demonstrated that the isotype control antibody anti-NP hIgG4 S228P did not bind to BSSL and could therefore be suitable for use as a negative control in future assays. The functionality of these antibody lots was also assessed in a displacement assay (Example 10). Very similar results were obtained for the different clones as described in Example 10 (data not shown).

In this example, we were able to measure the affinity of a CDR graft for the first time. In the AS20 CDR graft experiment, hIgG4 S228P (containing HC SEQ ID NO: 131 and LC SEQ ID NO: 132) showed clear binding to hBSSL at higher concentrations, although binding was significantly lower compared to AS20 hsIgG S228P (containing HC SEQ ID NO: 129 and LC SEQ ID NO: 130). The affinity for hBSSL measured in this assay was approximately 100-fold lower, while for mBSSL the affinity was too low to be detected. Thus, the affinity for the target was significantly lower when the CDRs were grafted onto a new scaffold. The observed stronger binding of BSSL via the AS20 CDR graft of hIgG4 S228P compared to the scFv AS20 CDR graft (containing HCVR SEQ ID NO: 144 and LCVR SEQ ID NO: 145, see Example 6) may be due to the different antibody formats.

Example 12 - Stability studies of five candidate antibodies

This example describes a stability study of S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116, and S-SL048-118 in hIgG4 S228P format to characterize the biophysical stability of the antibodies. Both AS20 hIgG4 S228P and hIgG1 LALA-PG were included for comparison (see Example 17). Also included were: AS20 CDR graft hIgG4 S228P and anti-NP hIgG1 LALA-PG isotype control. Analysis was performed by SDS-PAGE, analytical SEC, nano-DSF, and DLS.

Materials and methods

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

Table 13 - Antibodies used in this Example

Name of the antibody HC SEQ ID NO: LC SEQ ID NO: S-SL048-11 hIgG4 S228P 119 120 S-SL048-46 hIgG4 S228P 121 122 S-SL048-106 hIgG4 S228P 123 124 S-SL048-116 hIgG4 S228P 125 126 S-SL048-118 hIgG4 S228P 127 128 AS20 hIgG4 S228P 129 130 AS20 CDR graft hIgG4 S228P 131 132 AS20 hIgG1 LALA-PG 115 116 Anti-NP hIgG1 LALA-PG 117 118

Table 14 - Sample Analysis Schedule

Analysis Temp (°C) Day 0 1 2 9 16 22 30 SEC HPLC -80 X X +4 X X X X X X X +80 X X X X X X SDS PAGE -80 X X +4 X X X +80 X X DSF -80 X +4 X X X X X X X +80 X X X X X X DLS -80 X +4 X +80 X

Analytical size exclusion chromatography (SEC) was performed using a BioSEC column (300A, 7.8×300 mm; πPN 5190-2511, Agillent) coupled to an Agilent 1100 system using 0.15 M sodium phosphate (Na _x H _y PO ₄ ), pH 6.8 running buffer at a flow rate of 1 mL/min. Proteins were determined by measuring the optical density at A ₂₈₀ and A ₂₂₀ . 20 μg of each sample was loaded onto the column. SDS-PAGE was performed using NuPAGE 4-12% Bis-Tris gels (InVitrogen NP0321BOX), NuPAGE MES SDS running buffer (InVItrogen NP000202) according to the manufacturer's instructions. Samples were run under either reducing or non-reducing conditions. Bands were visualized with SimplyBlue (Invitrogen LC6065) and quantified using a densitometric scanner (Oddyssey, Li-cor). 5 μg of each sample was loaded into each well.

Nano-DSF (differential scanning fluorimetry) was performed using a Prometheus instrument (NanoTemper) using a temperature gradient from 20 to 95 °C with a ramp of 1 degree per minute. Fluorescence emission at 300 nm and 350 nm and backscatter signal were recorded. For each assay, 10 μl of 5 mg/ml sample were loaded. Data were analyzed using PR.Stability analysis v1.02 software from Nanotemper.

Dynamic light scattering was performed using Zetasizer Pro (Malvern). Scattered light was recorded and analyzed using ZS Explorer v 1.0.0.436 software and the built-in algorithm. Samples were analyzed at a concentration of 5 mg/mL.

Results

Size exclusion chromatography (SEC)

Chromatograms were obtained for each sample according to the graph presented in Table 14. It was noted that additional peaks appearing over time were of lower molecular weight, indicating degradation of the material. A plot of the total integrated area over time demonstrated that no material was lost in the prefilter or on the column.

The size exclusion chromatography data are shown in Fig. 7. Looking at the percentage of total area consisting of the main peak, only small changes can be observed at +4 °C. At +40 °C the effects were more pronounced.

The greatest reduction was observed for S-SL048-46 hIgG4 S228P and anti-NP hIgG1 LALA-PG. S-SL048-118 hIgG4 S228P, AS20 hIgG4 S228P and AS20 hIgG1 LALA-PG showed intermediate reduction, while CDR graft showed virtually no reduction.

SDS PAGE

Samples were run under reducing and non-reducing conditions as listed in Table 14. The -80 °C samples showed no additional bands except for S-SL048-46 hIgG4 S228P and anti-NP hIgG1 LALA-PG, which showed one weak band, each less than 1% under reducing conditions. This was nearly identical to what was observed for the day 0 samples. In fact, the day 0 sample showed additional bands that were not observed at -80 °C when run on day 30, reflecting assay variability. Non-reduced samples were nearly identical to the day 0 samples. No additional significant band was observed at +4 °C on day 9 or day 30 under reducing or non-reducing conditions. At +40 °C, all samples showed additional bands appearing on day 9 and day 30. Under reducing conditions, all candidates showed lower molecular weight bands, i.e., less than 25 kDa, whereas AS20 hIgG4 S228P, AS20 hIgG1 LALA-PG and anti-NP hIgG1 LALA-PG showed higher molecular weight bands (i.e., greater than 50 kDa on day 9 and day 30). In both cases, these bands became more pronounced over time. Under non-reducing conditions, clear differences could be seen with the appearance of additional bands in all samples. These results indicate that candidates S-SL048-46 and S-SL048-106 may be more susceptible to degradation.

Nano-DSF

The samples were analyzed according to the schedule given in Table 14 and the results are presented in Fig. 8. The main observations based on the nano-DSF data were the changes occurring when using the AS20 hIgG4 S228P, AS20 hIgG1 LALA-PG and anti-NP hIgG1 LALA-PG formats, where the main shifts in melting temperature T _m could be observed under storage conditions at +40 °C.

For the candidates, the main observation was a reduced amplitude of the second T _m (data not shown), which may reflect the major contribution of the Fab fragment of the antibody. Candidates S-SL048-46 and S-SL048-106 showed a slightly larger change, and 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 was the most stable of the samples tested.

Dynamic Light Scattering (DLS)

Samples were analyzed by DLS on day 30. The results at three different temperatures for each sample are shown in Fig. 9. At -80 and +4 °C, all samples showed a single peak with an apparent diameter of about 10-11 nm, consistent with the expected results for monomeric antibodies. No larger particles were observed in any of the samples, i.e. only protein molecules could be detected.

At +40 °C, larger particles are clearly visible to varying degrees. Using the mean Z value and polydispersity number as indicators of size uniformity (data not shown), it was determined that S-SL048-106 exhibited the lowest number of particles with higher Mw. In contrast, AS20 hIgG1 LALA-PG and anti-NP hIgG1 LALA-PG exhibited the highest number of particles with higher Mw, indicating that these two molecules were more prone to aggregation.

Conclusion

Thus, when combining the data into a general ranking, the most stable candidates in the hIgG4 S228P format are: S-SL048-106, S-SL048-118 and S-SL048-116 in that order.

Example 13 - Mapping of the AS20 epitope using HDX-MS

In this example, hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed on the chimeric AS20 IgG4 antibody together with human BSSL to map its epitope.

Methods and materials

40 μl of 2 mg/ml human BSSL (SEQ ID NO: 138) in PBS were mixed with 92.5 μl of 1.7 mg/ml AS20 hIgG4 chimeric antibody (containing HC SEQ ID NO: 139 and LC SEQ ID NO: 140) at a molar ratio of 1:1. The BSSL/antibody complex was concentrated to 40 μl using a 10K centrifugal filter unit (Amicon Ultra, Merck). In parallel, a BSSL-only sample without added antibody was prepared in a similar manner. Samples were analyzed on an automated HDX-MS system (CTC PAL/Biomotif HDX), in which samples were automatically labeled, quenched, digested, cleaned, and separated at 2 °C. More specifically, samples were labeled by mixing 3 µl BSSL (or BSSL/antibody complex) with 22 µl 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 lowering the pH to ~2.3 and the temperature to ~4 °C by adding 20 µl of a solution containing 6 M urea, 100 mM TCEP, and 0.5% TFA. Samples were digested on a pepsin immobilized column (2.1 × 30 mm column) at 250 μL/min followed by an in-line desalting step using a 2 mm i.d. x 10 mm long C-18 precolumn (ACE HPLC column, Aberdeen, UK) using 0.05% TFA at 350 μL/min for 3 min. Peptides were then separated with an 18-min 8-55% linear gradient of ACN in 0.1% formic acid using a HALO C18/1.8 μm 2 mm i.d. x 50 mm long analytical column running at 95 μL/min. An Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific) operating at 70,000 resolution was used for analysis. and m/z 400. Mascot software was used to identify peptides, and HDExaminer software (Sierra Analytics, USA) was used to process all HDX-MS data. Statistical analysis was performed using a 95% confidence interval.

Results

The deuteration kinetics of 66 peptides were monitored by HDX-MS, covering 57.9% of the protein. Deuterium labeling (5, 30, 90 and 180 min) and differential deuteration uptake kinetics between BSSL alone and in the presence of AS20 were calculated. Several peptides close to the N-terminus showed statistically lower deuterium uptake in the presence of the AS20 antibody, with clear epitope mapping to this region. By considering overlapping peptides, the spatial resolution can be reduced, resulting in the two peptide regions shown in Table 15.

Table 15 - List of AS20 epitope regions predicted in the HDX-MS experiment

Epitope regions Start-End Amino acid (aa) Subsequence SEQ ID NO: 1 aa 7-12 YTEGGF 1 2 aa 42-55 LENPQPHPGWQGTL 2 3 aa 172-180 AWVKRNIAA 13

Conclusion

In summary, the AS20 epitope was successfully mapped to the N-terminal region of BSSL. A cluster of three discontinuous peptide regions with significantly lower deuterium uptake in the presence of AS20 was identified. These regions map together to the three-dimensional structure of BSSL, indicating that these peptides together constitute the conformational epitope of AS20. In Example 21, the authors report the crystal structure of the AS20 Fab fragment in complex with hBSSL. The three-dimensional structure analysis confirms the results of the HDX-MS data and can specifically identify the amino acids involved in the interaction.

Example 14 - Epitope mapping of previously identified candidate antibodies using HDX-MS

In this example, hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed to map the BSSL epitopes of five previously reported candidate antibodies S-SL048-11 hIgG4 S228P (mAb11, HC SEQ ID NO: 119, LC SEQ ID NO: 120), S-SL048-46 hIgG4 S228P (mAb46, HC SEQ ID NO: 121, LC SEQ ID NO: 122), S-SL048-106 hIgG4 S228P (mAb106, HC SEQ ID NO: 123, LC SEQ ID NO: 124), S-SL048-116 hIgG4 S228P (mAb116, HC SEQ ID NO: 125, LC SEQ ID NO: 126) and S-SL048-118 hIgG4 S228P (mAb11.8 HC SEQ ID NO: 127, LC SEQ ID NO: 128).

Methods and materials.

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

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

Results

The deuteration kinetics of 54 peptides were monitored by HDX-MS, covering 64% of the protein. Deuterium labeling (5, 30, 90, and 180 min) and differential deuteration uptake kinetics between BSSL alone and in the presence of any of the five antibodies were calculated. It was observed that several peptides near the N-terminus of BSSL showed statistically lower deuterium uptake in the presence of antibody for each of the previously mentioned candidates. By considering overlapping peptides, the spatial resolution can be reduced, resulting in the two peptide regions shown in Table 16.

Table 16 - List of the most frequent epitope regions proposed in the HDX-MS experiment

Epitope regions Start-End Amino acid (aa) Subsequence SEQ ID NO: 1 aa 1-12 AKLGAVYTEGGF 3 2 aa 42-55 LENPQPHPGWQGTL 2

In addition to the epitope regions common to all the antibodies previously reported, one additional peptide was identified as having statistically lower deuterium uptake in three of the antibodies, namely peptide 10 (aa 84-101; NIWVPQGRKQVSRDLPVM (SEQ ID NO: 4)) for S-SL048-46, peptide 24 for S-SL048-116 (aa 174-180; VKRNIAA (SEQ ID NO: 5)) and peptide 39 (aa 283-295; HYVGFVPVIDGDF (SEQ ID NO: 6)) for S-SL048-11 (Table 17). However, their signals were relatively weak.

It can be noted that the two epitope regions common to all five antibodies (aa 1-12 and aa 42-55), although dispersed throughout the primary sequence, are grouped together in the three-dimensional structure (Fig. 10). Peptide 24 (174-180 amino acid residues) is located relatively close to this core region, whereas peptides 10 (aa 84-101) and 39 (aa 283-295) are more dispersed.

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

Peptide, No. Start End Subsequence
(SEQ ID NO:) Statistical significance mAb46 mAb116 mAb106 mAb118 mAb11 1 1 7 AKLGAVY
(148) ++ ++ + 2 1 11 AKLGAVYTEGG (149) ++ ++ + ++ 3 1 12 AKLGAVYTEGGF (3) ++ ++ + + ++ 4 1 25 AKLGAVYTEGGFVEGVNKKLGLLGD (150) ++ ++ ++ 5 1 26 AKLGAVYTEGGFVEGVNKKLGLLGDS (151) + + ++ 6 1 28 AKLGAVYTEGGFVEGVNKKLGLLGDSVD (152) ++ ++ + + ++ 9 42 55 LENPQPHPGWQGTL (2) ++ ++ +++ +++ +++ 10 84 101 NIWVPQGRKQVSRDLPVM
(4) + 24 174 180 VKRNIAA
(5) + 39 283 295 HYVGFVPVIDGDF (6) +

Conclusion

Epitopes of the five previously reported candidate antibodies were successfully mapped to the N-terminal region of BSSL. Two discontinuous peptide regions with significantly lower deuterium uptake in the presence of all five antibodies were identified.

Overlapping peptides may contribute to a decrease in spatial resolution. For example, in the AS20 experiment (Example 13), it was found that the peptide corresponding to aa 1-6 did not alter the deuterium exchange, whereas a longer overlapping peptide did alter this exchange (AKLGAVYTEGGF, aa 1-12, SEQ ID NO: 3). In other words, the epitope residues can be restored to YTEGGF (aa 7-12) (SEQ ID NO: 1). In contrast, such restoration is not possible in the current example, since the peptide corresponding to amino acid aa 1-6 was not detected.

In addition to the two common epitope regions, an additional peptide was each identified as a potential epitope region in three of the previously reported candidates (S-SL048-46, S-SL048-116, and S-SL048-11). Although statistically significant, the magnitude of signal change for these peptides was relatively low and may be of lesser importance for the interaction. However, given that the VKRNIAA peptide (aa 174-180, SEQ ID NO: 5) clusters in the central region in three-dimensional space, it is conceivable that this region is also part of the epitope. It should be noted that this peptide also overlaps with one peptide found in the original AS20 mapping. The signal change of two more peripheral peptides 10 (aa 84-101, SEQ ID NO: 4) and peptide 39 (aa 283-295, SEQ ID NO: 6) can be explained by stabilization upon antibody binding (“allosteric” effects).

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

Example 15 - Assessment of immunogenicity

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

Methods and materials.

Using Abzena's iTope™ MHC class II in silico prediction algorithm, sequences provided by Lipum were analyzed for immunogenicity potential. The iTope™ software predicts beneficial interactions between peptide amino acid side chains and specific binding pockets (pocket positions; p1, p4, p6, p7, and p9) within the open binding grooves of 34 human MHC class II alleles. These alleles represent the most common HLA-DR alleles found worldwide, without assessing those that are most common in any ethnic population. Twenty alleles contain the "open" p1 configuration, and 14 contain a "closed" configuration in which glycine at position 83 is replaced by valine. The locations of key binding residues are achieved in silico by generating 9-mer peptides that overlap the eight amino acids spanning the test protein sequence.

The results should be evaluated in light of the fact that all methods for predicting MHC class II binding inherently overpredict the number of T cell epitopes because they do not take into account other important processes during antigen presentation such as protein/peptide processing, T cell receptor recognition, or T cell tolerance to the peptide.

The positions of the p1 anchor residues (including the first residue of the 9-mer MHC class II core ligand) are highlighted in S-SL048-11, S-SL048-46, S-SL048-106, S-SL048-116, S-SL048-118 and AS20 in the hIgG4 S228P format.

If ≥50% of MHC class II binding peptides (i.e. ≥17 of 34 alleles) had high binding affinity (score >0.6), such peptides were defined as “non-selective high affinity” MHC class II binding peptides.

Peptides binding ≥50% of MHC class II alleles with a binding index >0.55 (but not the vast majority >0.6) were defined as “non-selective moderate affinity” peptides.

These criteria were modified when a large aromatic amino acid (i.e., F, W, Y) was present at the p1 anchor position, with the open p1 pocket in 20 of the 34 alleles allowing binding to a large aromatic residue. When this occurred, a non-selective peptide was defined as binding to 10 or more of the subset of 20 alleles.

The anchor position of p1 germline binding peptides with moderate and high affinity were also analyzed and were not expected to be problematic in healthy individuals due to T cell tolerance.

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

Results

TCED™ analysis of peptides previously tested in the EpiScreen™ ex vivo T cell assays revealed strong homology to a number of iTope™ MHC binding peptides. In Table 18, “+” indicates mismatched residues where the substitutions represent amino acids with similar physicochemical properties, and “-” indicates other mismatched residues. The positions of key MHC class II pocket positions for the iTope™ and TCED™ sequences are listed in the bottom row of Table 18.

Table 18 - TCED ^™ Analysis Results

HC sequence Anchor residue iTope ^TM residues (SEQ ID NO:) TCED ^TM homologous match AS20 V12 VKPGASVKM (153) VKPGASVKM S-SL048-11, -46, -106, -118 Y32 YNMHWVRQA (154) +-MHWVRQA S-SL048-11, -116, -118 Y59 YAQKFKGRV (155) YAQK++GRV AS20 W103 WGQGTTLTV (156) WGQGT-+TV MHC class II, pocket position P1 4 67 9 P1 4 67 9 HC sequence Anchor residue iTope ^TM residues (SEQ ID NO:) TCED ^TM homologous match AS20 V19 VTMTCSASS(157) VTMTC-ASS S-SL048-46 V19 VTITCRASS (158) VT-TCRASS S-SL048-106 V19 VTITCSASS (159) VTITC-AS- S-SL048-106 L46 LLIYATSKL (160) LLIYA-SKL S-SL048-11, -116 I48 IYATSSLAS (161) IYATS-LAS S-SL048-46 I48 IYAASSLAS (162) IYA+S-LAS AS20 F98 FGGGTKLEI (163) FG-GTKLEI MHC class II pocket position P1 4 67 9 P1 4 67 9

Table 19 shows the total number of iTope™ mixed MHC class II peptides with medium and high affinity and TCED™ hits for each candidate sequence (AS20 is shown for information only).

Table 19 - Summary of immunogenicity analysis results

Subsequence Non-germline MHC class II non-selective binding peptides with moderate affinity Non-germline MHC class II non-selective high affinity binding peptides TCED ^TM hits AS20 hIgG4 S228P 8 13 4 S-SL048-116 hIgG4 S228P 5 7 3 S-SL048-11 hIgG4 S228P 6 6 3 S-SL048-118 hIgG4 S228P 6 6 2 S-SL048-46 hIgG4 S228P 3 6 3 S-SL048-106 hIgG4 S228P 5 5 3

All five humanized candidate clones contained fewer non-germline peptides that indiscriminately bind to MHC class II compared to AS20 hIgG4 S228P. All variant sequences contained at least two TCED™ hits with partial (minimum 6 of 9 positions) homology to peptides known to induce T cell activation in ex vivo EpiScreen™ assays. Of the five variants, S-SL048-106 hIgG4 S228P presented the lowest risk of immunogenicity based on the number of non-germline peptides that indiscriminately bind to MHC class II with high to moderate affinity. Candidates were ranked as follows: fewer indiscriminate hits, 106 < 46 < 11 = 118 < 116, more indiscriminate hits.

It should be noted that in silico immunogenicity analysis is inherently over-predictive.

Example 16 - Manufacturing Feasibility Assessment

This example describes the feasibility assessment of in silico manufacturing 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) in the hIgG4 S228P format, performed using the Abzena algorithm.

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 hIgG4 S228P format were analyzed using the Abzena in silico susceptibility prediction algorithm. The identified susceptibilities were subsequently analyzed in a structural context. Briefly, the following were analyzed for each V domain sequence:

• Presence of deamidation sites;

• Presence of isomerization sites;

• Potential oxidation sites;

• Presence of N-linked glycosylation sites

• Presence of free cysteines.

In this document, the IMGT CDR definitions and numbering apply unless otherwise noted.

Presence of deamidation sites

Deamidation of asparagine residues may result in structural changes, alterations in pharmacokinetics, potency and potential immunogenicity. Asparagine residues as potential deamidation sites were analyzed in the context of both amino- and carboxy-adjacent amino acids using a method based on [14].

Presence of isomerization sites

The presence of aspartate isomerization sites was predicted by sequence analysis of known isomerization motifs (DG, DS, DT, or DD) centered in the CDR regions.

Potential oxidation sites

Structural models of both the heavy and light chain variable domains were generated, and methionine and tryptophan residues were identified and assessed to determine whether they were surface-exposed and therefore candidates for oxidation. Oxidation of individual residues may affect the biological activity of antibodies and may have biological consequences such as decreased efficacy or altered pharmacokinetics.

Presence of N-linked glycosylation sites

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

Presence of free cysteines

Sequences were analyzed to identify unpaired cysteine residues. The VH and Vκ domains each typically contain two canonical cysteines that form an intrachain disulfide bond in the folded molecule. Additional cysteines are expected to negatively affect folding and could potentially cause problems such as aggregation.

Results

Analysis was performed and no potential N-linked glycosylation sites were identified in either the VH or Vκ sequence for the five candidate antibodies; no unpaired cysteines were identified in either the VH or Vκ sequence for the five candidate antibodies; no potential oxidation sites were identified in either the VH or Vκ sequence for the five candidate antibodies, and no sites with high (t _1/2 < 10 days) or intermediate (t _1/2 < 25 days) deamidation potential were identified in either the VH or Vκ sequence for any of the five candidate antibodies.

Within the VH for all five clones, two potential isomerization sites were identified in or near the CDR regions.

- Asp 54 (DG) is located within VH CDR2, so isomerization may affect antigen binding;

- Asp72 is located next to the CDR in a region sometimes called "CDR4", and therefore isomerization may affect antigen binding.

Table 20 - Summary of sequence predisposition information

Candidate Identified features S-SL048-11 hIgG4 S228P Two potential aspartate isomerization sites have been identified:
- Asp 54 (VH CDR2) and Asp 72 (FW2 adjacent to the CDR region) S-SL048-46 hIgG4 S228P Two potential aspartate isomerization sites have been identified:
- Asp 54 (VH CDR2) and Asp 72 (FW2 adjacent to the CDR region)
One potential deamidation site was identified at Asn 60 in VH CDR2. S-SL048-106 hIgG4 S228P Two potential aspartate isomerization sites have been identified:
- Asp 54 (VH CDR2) and Asp 72 (FW2 adjacent to the CDR region)
One potential deamidation site was identified at Asn 60 in VH CDR2. S-SL048-116 hIgG4 S228P Two potential aspartate isomerization sites have been identified:
- Asp 54 (VH CDR2) and Asp 72 (FW2 adjacent to the CDR region) S-SL048-118 hIgG4 S228P Two potential aspartate isomerization sites have been identified:
- Asp 54 (VH CDR2) and Asp 72 (FW2 adjacent to the CDR region)

Figure 11 shows S-SL048-106 Fv with potential post-translational biases highlighted.

Thus, no free cysteines, oxidation sites, or N-linked glycosylation sites were identified in any of the five top candidates. Sites with high deamidation potential were not identified in S-SL048-11, S-SL048-116, or S-SL048-118. S-SL048-46 and S-SL048-106 contain a potential deamidation site in the VH CDR2. Two potential isomerization sites were identified in the heavy chain of each of the five top variants.

Example 17 - Characterization of AS20 hIgG1 LALA-PG and anti-NP hIgG1 LALA-PG production and binding

This example shows the production and binding characteristics of AS20 and anti-NP (clone B1-8) antibodies of the human IgG1-LALA-PG subclass, hereinafter referred to as AS20 hIgG1 LALA-PG (containing HC SEQ ID NO: 115 and LC SEQ ID NO: 116) and anti-NP hIgG1 LALA-PG (containing HC SEQ ID NO: 117 and LC SEQ ID NO: 118). The anti-NP antibody was used as an isotype control.

Material and methods

Expression and purification

IgG4 is the most Fc inert natural subclass in humans. However, several publications have demonstrated that IgG4 can interact with FcRs as well as complement in both mice [15] and humans. Therefore, human IgG1 with mutations at three positions, namely L234A, L235A and P329G (hIgG1 LALA-PG for short), was chosen in this study as it is reported to be the most Fc silent of the available variants [7, 16], i.e., lacking immune effector functions.

Absolute Antibody (Oxford, UK) was engaged to produce the material. AS20 VH and VL sequence information was sent to the company, where the genes were synthesized and cloned into vectors encoding the human IgG1-LALA-PG subclass. The antibodies were transiently expressed in mammalian HEK293 cells and then purified by protein A affinity chromatography. Purity and integrity were assessed by SDS-PAGE, and endotoxin levels were determined by the LAL endotoxin chromogenic assay.

Surface Plasmon Resonance (SPR)

Single cycle kinetics were used to measure antibody affinity. AS20 hIgG1 LALA-PG and anti-NP hIgG1 LALA-PG were immobilized on the CM5 S sensor chip via primary amine coupling using NHS-EDC chemistry. Five different concentrations of antigen were spotted on the surface (1:3 dilutions starting at 50 nM for hBSSL and 1:2 dilutions starting at 800 nM for mBSSL). The sensor chip surface was regenerated with 10 mM glycine-HCl pH 2.1. The resulting single cycle kinetic data were fitted to a 1:1 Langmuir binding model and kinetic parameters were obtained using BIAevaluation software. For mouse BSSL, steady state analysis was also performed by plotting the response rate at equilibrium against each concentration and K _D values were obtained using BIAevalution software.

Results

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

Single cycle SPR kinetics were used to determine IgG affinity. For AS20 hIgG1 LALA-PG binding to hBSSL, the equilibrium dissociation constant (K _D ) was calculated to be 0.91 nM. For mBSSL, the K _D was determined to be 361 nM. As expected, no binding of anti-NP hIgG1 LALA-PG to BSSL was observed.

Conclusion

This Example describes the quality check performed to verify that AS20 hIgG1 LALA-PG is functional, i.e. that binding to BSSL is comparable to AS20 binding in the hIgG4 S228P format.

SPR analysis of LALA-PG AS20 hIgG1 demonstrated that binding to BSSL was very similar to that observed for AS20 in other IgG formats. The _KD for hBSSL was determined by SPR to be 0.91 nM, which is comparable to the _KD values of 0.6-3 nM that have been determined for other AS20 IgG formats (as described in Examples 1, 2 and 4). The binding affinity of AS20 hIgG1 LALA-PG to mBSSL was assessed in a steady state affinity assay to a _KD value of 361 nM, which is in the same order of magnitude as the _KD previously estimated by the same assay (155 nM, Example 1), and the sensorgram appearance was similar (not shown).

Thus, these data demonstrated that AS20 hIgG1 LALA-PG binding was not affected by the subclass change. These results demonstrated that the isotype control antibody anti-NP hIgG1 LALA-PG does not bind to BSSL and may therefore be suitable for use as a negative control in future assays.

Example 18 - Validation of the 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 examined in a mouse model of rheumatoid arthritis (RA) in vivo, i.e., collagen antibody-induced arthritis (CAIA). Anti-NP hIgG1 LALA-PG antibody (heavy chain SEQ ID NO: 117 and light chain SEQ ID NO: 118) was included in the study as an isotype control.

Model Description - CAIA in Mice

Collagen antibody-induced arthritis (CAIA) in mice is a model of arthritis independent of both B and T cells. The disease was induced by intravenous (i.v.) administration of anti-collagen type II (CII) antibodies followed by intraperitoneal (i.p.) administration of LPS 3–5 days later to accelerate disease development. The injected antibodies bind to cartilage, thereby activating the immune system and recruiting macrophages and granulocytes to the joints. A booster injection of LPS is required to achieve significant disease severity and incidence. The disease course is very predictable and begins after a booster dose of LPS. The disease reaches its maximum severity around day 15, after which its severity decreases until complete recovery. The study was approved by the local animal ethics committee of Malmö/Lund, Sweden (M118-15).

Material and methods

Disease induction

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

Experimental groups and management of study products

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

Table 21 - Experimental groups

Group Treatment Dose (mg/kg) Route of administration Time of therm. Number of animals 1 Isotypic control anti-NP hIgG1 LALA-PG 90 v/br 96 hours after last dose 14 2 AS20
hIgG1 LALA-PG 90 v/br 96 hours after last dose 16* 3 AS20
hIgG1 LALA-PG 30 v/br 96 hours after last dose 16* 4 AS20
hIgG1 LALA-PG 10 v/br 96 hours after last dose 16*

*Two satellite animals per group.

Disease assessment

Disease was assessed daily starting on day 3 in a blinded manner using a macroscopic four-limb score ranging from 0 to 15 (1 point for each swollen or hyperemic toe, 1 point for a swollen or hyperemic middle toe or foot joint, 5 points for a swollen ankle) with a maximum score of 60 for each mouse. Due to ethical restrictions, animals with a score above 45 were excluded from the experiment.

Blood collection

Blood was collected from all mice on study termination day 19 by cardiac puncture under deep anesthesia (96 hours after the last injection on day 15) into microtubes containing LiHeparin. Samples were immediately placed on ice and centrifuged (2000 × g for 5 min, at 4°C) within 20 min of collection. Plasma samples were aliquoted and frozen on dry ice. Aliquots were stored at -80°C until assay for AS20 IgG1 LALA-PG and anti-drug antibody (ADA) exposure (all groups, n = 70) and safety biomarker panel assay (groups 1-3, n = 42).

Blood sampling for PK studies in satellite animals

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

Fabric collection

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

Drug exposure in plasma

AS20 IgG1 LALA-PG exposure was analyzed in plasma samples collected from satellite animals (see above) and from all animals at study termination (day 19) by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Anti-drug antibodies (ADA)

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

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

Measuring 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 analysed by clinical biochemistry 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). Analyses were performed at the Chemical and Pharmaceutical Safety Department, RISE, Södertälje, Sweden using the Abaxis Vetscan system with cassette no. 500-0038.

Results

Arthritis severity

Animals induced with CAIA and receiving vehicle developed moderate to severe disease at a rate of 100%. Similar results were observed in animals receiving isotype control. The efficacy of AS20 hIgG1 LALA-PG administered intraperitoneally every fourth day from day -1 until treatment cessation was assessed at three doses (10, 30, and 90 mg/kg). AS20 hIgG1 LALA-PG at 90 mg/kg and 30 mg/kg demonstrated significant beneficial effects on disease progression compared to isotype control on days 7-14, 16, 18, and 7-12, respectively. A slight reduction in disease severity was observed with AS20 hIgG1 LALA-PG at 90 mg/kg when compared to vehicle, although this was not statistically significant (Figs. 18 and 19).

Two animals, one in the AS20 hIgG1 LALA-PG 10 mg/kg group and one in the isotype control group, were excluded from the study early (Day 12) for ethical reasons (high score). These animals were included in the analysis of the maximum score but excluded from the analysis of mean, AUC, and % inhibition (Fig. 19, Table 22).

Statistics of disease parameters are presented in Table 22.

Table 22 - CAIA Severity Parameter Statistics

Group Frequency ¹ MES ² Max. AUC ³ % inhibition ⁴ Isot. control hIgG1 LALA-PG90 mg/kg 100% 21.8±1.6 35.1±8.2 355.0±26.5 0.0±7.5 AS20 hIgG1 LALA-PG 90 mg/kg 100% 13.9±2.1** 23.1±3.2** 226.0±34.7* 36.4±9.8** AS20 hIgG1 LALA-PG 30 mg/kg 100% 16.5±2.3 27.5±3.2 267.0±36.9 24.8±10.4 AS20 hIgG1 LALA-PG 10 mg/kg 100% 16.7±2.3 29.1±3.4 270.8±37.3 23.71±10.5

¹ Cumulative frequency. A mouse was considered to be sick if it scored 1 or higher for two consecutive days.

² Average CAIA score for all time points assessed.

³ Area under the curve.

⁴ Percentage change in overall disease burden for each animal compared to the isotype control group. Calculated by taking the difference between the mean isotype control group AUC and the AUC for each individual animal, divided by the isotype control group AUC, and multiplied by 100*(-1). *p<0.05;**p<0.01 compared to isotype control.

Health assessment

General health assessments were performed daily along with disease assessments from day 3 until study termination. Mice were weighed twice weekly during general health assessments. No adverse effects were observed during treatment (data not shown).

Drug exposure in plasma

Plasma exposure to AS20 hIgG1 LALA-PG at the end of the study on Day 19 was generally within the expected concentration range based on the previous single dose pharmacokinetic evaluation. The mean concentration in the 10 mg/kg group was 199 μg/mL (~1.3 μM, 58 - 309 μg/mL), in the 30 mg/kg group it was up to 614 μg/mL (~4.1 μM, 229 - 970 μg/mL), and in the 90 mg/mL group the corresponding mean concentration was 1408 μg/mL (~9.4 μM, 520 - 2557 μg/mL). Thus, a somewhat non-linear dose-exposure relationship was observed. There was no evidence of altered plasma exposure due to immunogenicity during treatment. The mean hIgG1 LALA-PG concentration in plasma samples from mice receiving isotype control was 1815 (~12 μM, 1224-2511 μg/mL).

Anti-drug antibodies (ADA)

No ADAs of concern were detected in the study samples. Although the majority of samples tested positive, titers in the AS20 hIgG1 LALA-PG groups were not higher than in the control groups. Given the early onset of response (~day 7) and unchanged drug exposure over time, it is possible that the anti-drug signal detected in the samples was primarily due to non-specific/low-affinity IgM.

Safety biomarkers

Plasma glucose levels increased after administration of 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 toward increased plasma creatinine in the AS20 hIgG1 LALA-PG group compared to the isotype control group, although this was not statistically significant (p<0.06). Other markers of renal injury, i.e. blood urea, electrolytes, or total protein, did not differ between AS20 hIgG1 LALA-PG and isotype control treated mice.

Conclusion

Animals induced with CAIA and given isotype control developed moderate to severe disease at a rate of 100%, as did vehicle-treated mice.

Administration of AS20 hIgG1 LALA-PG at 90 mg/kg and 30 mg/kg demonstrated significant beneficial effects on disease progression compared to isotype control on days 7-14, 16-18, and 7-12, respectively. A slight reduction in disease severity was observed with AS20 hIgG1 LALA-PG at 90 mg/kg compared to vehicle, although this was not statistically significant.

Plasma exposure to AS20 IgG1 LALA-PG at the end of the study was generally within the expected concentration range. There was no evidence of changes in plasma exposure during treatment due to immunogenicity. No ADA of concern were detected in the study samples.

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

Example 19 - FACS analysis of spleen from CAIA experiment

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 subpopulations in the spleen of mice with collagen antibody-induced arthritis (CAIA) was examined by fluorescence-activated cell sorting (FACS). 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.

Material and methods

This study is a subset of the in vivo pilot study described above (Example 18). Briefly, CAIA was induced in DBA/1 mice (male, 8-9 weeks old) by intravenous (i.v.) administration of anti-collagen type II antibodies followed by intraperitoneal (i.p.) administration of LPS to accelerate disease development. Mice were injected i.p. with AS20 hIgG1 LALA-PG or an isotype control antibody (anti-NP hIgG1 LALA-PG) every 4 days beginning the day before disease induction (day -1). At study termination (day 19), spleens from animals receiving the highest dose of AS20 hIgG1 LALA-PG (90 mg/kg) or the isotype control (90 mg/kg) were dissected, weighed, and homogenized as described below. A FACS panel was developed to identify T cells, B cells, NKT cells, neutrophils, eosinophils, NK cells, dendritic cells, monocytes and macrophages.

Single cell suspensions

Spleens were passed through a 70-μm mesh filter in RPMI-1640 culture medium (Thermo Fisher, HyClone) to obtain single-cell suspensions. Cells were pelleted and resuspended in 1 ml MilliQ water to lyse red blood cells (10 sec), 1 ml 2× BS was added, followed by 10 ml 1× PBS. Cells were washed with 10 ml HBSS (Thermo Fisher, HyClone) and finally resuspended in an appropriate volume of HBSS to obtain 10× ¹⁰⁶ cells/ml.

FACS protocol

Cells at 1× ¹⁰⁶ were seeded into each well of a 96-well round-bottom plate and centrifuged for 1 min at 850×g, 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 the cells were incubated for 15 min, after which they were washed again with FACS buffer. An antibody mixture was prepared by adding the following antibodies to an 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 I-A/I-E (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 Regenerable Viability Dye (1:400).

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

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 ⁺

Neutrophils: CD45.2 ⁺ , CD11b ⁺ , Ly6G ⁺

Eosinophils: CD45.2 ⁺ , CD11b ⁺ , Ly6G ^- , Siglec F ⁺

NK cells: CD45.2 ⁺ , CD11b ⁺ , Ly6G ^- , Siglec F ^- , CD49b ⁺

Dendritic cells: CD45.2 ⁺ , CD11b ⁺ , Ly6G ^- , Siglec F ^- , CD11c ⁺ , MHCII ⁺

Monocytes: CD45.2 ⁺ , CD11b ⁺ , Ly6G ^- , Siglec F ^- , CD11C ^- , Ly6C ⁺

Macrophages: CD45.2 ⁺ , CD11b ⁺ , Ly6G ^- , Siglec F ^- , CD11C ^- , Ly6C ^- , MHCII ⁺

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

Results

There was no significant difference in body weight between the AS20 hIgG1 LALA-PG group (Group 3, Example 18) and the isotype control group (Group 2, Example 18), either two days before disease induction (AS20, 23.9 ± 1.4 g; isotype control, 23.8 ± 1.2 g) or on day 19 of study termination (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 group compared with the isotype control group (110 ± 23 mg vs. 88 ± 21 mg; p = 0.02), and total CD45 ⁺ leukocyte count was also higher in the AS20 group compared with the isotype control group (44.5×10 ⁶ vs. 39.0×10 ⁶ ; p = 0.008).

Total splenic B cell counts were higher in AS20 hIgG1 LALA-PG-treated mice compared with isotype control-treated mice (27.1 ± 3.5 × 10 ⁶ vs. 21.0 ± 5.8 × 10 ⁶ ; p = 0.004). In contrast, total NK cell counts were lower in AS20 hIgG1 LALA-PG-treated mice compared with isotype control-treated mice (0.44 ± 0.09 × 10 ⁶ vs. 0.54 ± 0.09 × 10 ⁶ ; p = 0.01). Total NKT cell counts were also lower in AS20 hIgG1 LALA-PG-treated mice compared to isotype control-treated mice, although not statistically significant (0.14 ± 0.03 × 10 ⁶ vs. 0.16 ± 0.03 × 10 ⁶ ; p = 0.08). No significant difference was found in total neutrophils, eosinophils, monocytes, macrophages, dendritic cells, or T cells between AS20 IgG1 LALA-PG-treated mice and isotype control-treated mice (Fig. 20).

The proportion of B cells from CD45 ⁺ cells in the spleen was higher in mice receiving AS20 IgG1 LALA-PG compared with mice receiving isotype control (60.9 ± 5.4% vs. 53.0 ± 10.0%, p = 0.02). In contrast, the proportion of T cells, NKT cells, and NK cells from CD45 ⁺ cells was lower in mice receiving AS20 hIgG1 LALA-PG compared with 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 no significant difference in the proportion of neutrophils, eosinophils, monocytes, macrophages, or dendritic cells from CD45 ⁺ leukocytes between AS20 IgG1 LALA-PG-treated mice and isotype control-treated mice (Fig. 21). There was no significant difference between AS20 IgG1 LALA-PG-treated mice and isotype control-treated mice in cells expressing CD80 or CD86, either in the total number or percentage of CD45 ⁺ cells (data not shown).

Conclusion

Administration of AS20 hIgG1 LALA-PG (90 mg/kg dose) significantly reduced the total number of NK cells and the proportion of T cells, NKT cells, and CD45 ⁺ NK cells in the spleen of CAIA mice.

Example 20 - Analysis of the effect of previously identified candidate antibodies on BSSL enzyme activity

In this example, the effect of five previously reported candidate antibodies, namely S-SL048-11 (heavy chain SEQ ID NO: 119 and light chain SEQ ID NO: 120), S-SL048-46 (heavy chain SEQ ID NO: 121 and light chain SEQ ID NO: 122), S-SL048-106 (heavy chain SEQ ID NO: 123 and light chain SEQ ID NO: 124), S-SL048-116 (heavy chain SEQ ID NO: 125 and light chain SEQ ID NO: 126) and S-SL048-118 (heavy chain SEQ ID NO: 127 and light chain SEQ ID NO: 128) in the hIgG4 S228P format, on the enzymatic activity of BSSL was studied. Comparisons were 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), and an anti-NP isotype control antibody (heavy chain SEQ ID NO: 133 and light chain SEQ ID NO: 134) in hIgG4 S228P format.

Methods and materials

Preincubation of BSSL with previously indicated candidate antibodies

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

Antibodies were diluted to a stock concentration of 0.3 μg/μL in MQ-H ₂ O, followed by serial 1:1 dilutions in MQ-H ₂ O to yield 6-point concentrations ranging from 0.3 μg/μL to 0.009 μg/μL. From each antibody dilution, 20 μL was added to 2.4 μL BSSL (1 μg), and the antibody/BSSL mixtures were incubated at +4°C for 1 hour. Following incubation, 20 μL MQ-H ₂ O was added to each BSSL/antibody reaction.

Triglyceride hydrolysis analysis

The triglyceride (TG) emulsion was prepared by mixing 25 mg of unlabeled triolein (Sigma, Cat. No. 92860) with 50 μL of ³ H-labeled triolein (triolein [9,10-3H(N)], 91 Ci/mmol) NET431001MC Perkin Elmer (Waltham, MA) in a 30 mm round-bottomed glass sonicator; evaporating the solvent under nitrogen (N ₂ ) at room temperature; adding to the vessel 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-H ₂ O; cooling the vessel in ice water and sonicating for 10 min at 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 liquid surface until an emulsion was formed; and adding 2.5 ml 18.7% BSA (Sigma, Cat# A7906), 2.5 ml 1.0 M NaCl and 3.25 ml MQ- _H2O to the above emulsion. The emulsion was used on the same day it was prepared. Next, 10 μl of the pre-incubated BSSL/antibody solution (see above) was mixed with 150 μl of TG emulsion and 10 mM sodium cholate (Sigma, cat. no. C-1254) and MQ- _H2O in a total volume of 200 μl in 13 × 100 mm glass tubes. Samples were prepared in duplicate. Tubes were incubated at 37°C for 15 min, and then reactions were stopped by adding 3.25 ml of methanol/chloroform/heptane (v/v/v, 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 water-soluble phase containing hydrolyzed free fatty acids, 1400 μl were recovered and mixed with 2.0 ml of Optiphase Hisafe 3 scintillation cocktail (Perkin Elmer, Waltham, MA) in 6 ml polyethylene vials (Perkin Elmer) and the amount of hydrolyzed ³ H-labeled free fatty acids was measured by liquid scintillation spectrometry on a WinSpectral 1414 instrument (Wallac, Turku, Finland).

Cholesterol Ester Hydrolysis Analysis

Cholesterol ester (CE) emulsion was prepared by adding 40 μL of ^14C -labeled cholesteryl oleate (oleate-1-14C, NEC6380050UC, Perkin Elmer) to a 30 mm round bottom glass sonicator and evaporating the solvent under nitrogen at room temperature; adding 2.0 mL of 0.2 M Tris-HCl pH 7.5 and 0.85 mL of MQ-H ₂ O to the vessel; cooling the vessel in ice water and sonicating for 10 min at 50% pulse mode using a Soniprep 150 (MSE) with a 9 mm flat tip probe at medium setting placed a few mm below the liquid surface until an emulsion was formed; and adding 1.65 ml of 200 mM sodium cholate and 1.0 ml of MQ- _H2O to the emulsion. The emulsion was used on the same day it was prepared. Then 10 μl of the pre-incubated BSSL/antibody solution (see above) was mixed with 100 μl of the CE emulsion and MQ- _H2O in a total volume of 200 μl in 13 × 100 mm glass tubes. Samples were prepared in duplicate. The tubes were incubated at 37°C for 30 min and then the reaction was stopped by adding 3.25 ml of methanol/chloroform/heptane (v/v/v, 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 water-soluble phase containing hydrolyzed free fatty acids, 400 μl were recovered and mixed with 2.0 ml of Optiphase Hisafe 3 scintillation cocktail (Perkin Elmer) in 6 ml polyethylene vials (Perkin Elmer), and the amount of hydrolyzed ¹⁴ C-labeled free fatty acids was measured by liquid scintillation spectrometry on a WinSpectral 1414 instrument (Wallac).

Results

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

Table 23 - Effect of five BSSL-specific hIgG4 S228P and control antibodies of the hIgG4 S228P subtype on the enzymatic activity of BSSL (triglyceride hydrolysis)

Antibody Kit Effect of the previously mentioned antibodies on the hydrolysis of BSSL triglycerides
(relative values; values obtained without antibodies in the reaction are taken as 100%)   0.19 mcg 0.38 mcg 0.75 mcg 1.5 mcg 3.0 mcg 6.0 mcg S-SL048-11 1 109.4±4.0 114.3±1.1 125.0±8.8 111.6±6.8 119.8±4.4 112.9±0.2 S-SL048-46 1 104.9±5.0 123.0±1.6 120.8±4.2 112.7±0.9 119.3±2.8 126.3±4.6 S-SL048-106 1 108.7±1.0 123.2±4.0 129.4±0.0 120.5±0.8 99.2±2.4 104.3±7.4 S-SL048-116 2 104.7±6.3 111.8±0.7 100.4±3.2 96.2±0.5 105.6±2.1 108.1±3.2 S-SL048-118 2 107.3±1.4 106.5±4.1 102.2±1.0 88.0±0.1 93.9±2.8 119.4±0.2 AS20 (set 1) 1 112.4±3.4 120.6±2.9 128.6±2.5 120.4±9.8 128.0±5.1 123.0±3.4 AS20 (set 2) 2 105.0±0.1 116.4±0.9 133.5±0.3 127.1±1.4 127.4±1.5 133.8±4.9 AS20 CDR graft 2 98.0±0.4 96.0±0.6 76.7±3.6 87.8±0.1 85.6±3.8 106.0±1.6 Anti-NP (set 1) 1 106.3±0.9 104.5±3.2 105.5±2.7 99.8±4.1 107.2±2.0 103.1±4.5 Anti-NP (set 2) 2 99.8±2.4 102.2±0.8 105.5±2.3 92.5±2.8 101.1±2.2 102.6±7.1

Table 24 - Effect of five BSSL-specific hIgG4 S228P and control antibodies of the hIgG4 S228P subtype on the enzymatic activity of BSSL (hydrolysis of cholesterol esters)

Antibody Kit Effect of the previously mentioned antibodies on the hydrolysis of BSSL cholesterol esters (relative values; values obtained without antibodies in the reaction are set to 100%)   0.19 mcg 0.38 mcg 0.75 mcg 1.5 mcg 3.0 mcg 6.0 mcg S-SL048-11 1 96.2±3.0 98.2±4.4 92.2±1.3 95.2±0.0 93.7±2.1 90.2±1.3 S-SL048-46 1 95.7±2.5 99.0±0.5 97.6±0.4 88.3±1.3 93.5±0.2 97.7±0.3 S-SL048-106 1 94.8±2.0 97.2±6.1 93.9±2.4 95.3±0.4 99.0±2.6 90.5±0.2 S-SL048-116 2 104.1±3.2 107.2±3.9 102.4±0.9 102.8±2.5 107.0±1.3 107.1±1.2 S-SL048-118 2 104.6±4.5 102.6±7.4 110.3±2.5 104.8±5.8 105.6±1.4 102.0±3.1 AS20 (set 1) 1 96.0±1.4 94.7±0.9 95.4±0.1 94.3±0.5 96.1±1.3 93.5±2.9 AS20 (set 2) 2 101.7±2.1 108.2±4.1 106.6±0.7 103.0±7.0 101.5±1.8 108.3±0.7 AS20 CDR graft 2 99.8±0.9 108.4±1.6 103.7±0.4 106.7±0.9 107.7±0.3 109.6±1.8 Anti-NP (set 1) 1 98.5±5.4 100.4±3.4 101.5±0.5 102.0±0.15 102.8±0.0 95.7±2.5 Anti-NP (set 2) 2 100.7±1.5 101.8±2.7 103.2±1.2 97.2±1.6 97.2±0.7 99.2±4.4

Conclusion

None of the antibodies tested, i.e. the five previously reported drug candidates, AS20, AS20 CDR graft and anti-NP in hIgG4 S228P format, showed any significant effect on the enzymatic activity of BSSL.

Example 21 - Mapping of the AS20 epitope using X-ray crystallography

X-ray crystallography was used to determine the three-dimensional structure of AS20 in complex with hBSSL. For this experiment, the antibody was digested to form Fab fragments and a new hBSSL construct (t-hBSSL) truncated at the C-terminus was generated corresponding to amino acids 1-530 of hBSSL followed by AHHHHHH (SEQ ID NO: 146).

Material and methods

Cloning, expression and purification of t-hBSSL

Human BSSL was previously crystallized in a truncated form without the flexible C-terminal portion. Therefore, a new truncated BSSL construct incorporating a C-terminal his tag for purification was generated to obtain AS20 Fab and hBSSL crystals. The gp67-BSSL-6×H construct consisted of amino acids 1-530 + AHHHHHH (BSSL numbering based on sequence after removal of the signal peptide) was ordered from GeneArt and cloned into the pFastBac tGFP Dual vector, which had been prepared for ligation-independent cloning (LIC). LIC was performed using the InFusion Cloning Kit and transformed into Stellar competent cells.

Recombinant bacmid DNA was generated in E. coli DH10Bac cells. Bacmid was transfected into Sf9 cells for 120 h at 27 °C. P1 virus was collected from the growth medium and subsequently used to generate P2 virus stock. After 96 h, P2 virus stock was collected by centrifugation. 400 ml Sf9 cells (1.5 × 10 ⁶ cells/ml) were infected with 2 ml P2 virus stock and grown for 72 h post infection. The medium (P3 stock) was collected by centrifugation and filtered. For large-scale expression, 35 ml P3 virus stock was added to 2130 ml Sf9 cells (1.6 × 10 ⁶ cells/ml) in a Thomson Optimum Growth Flask (5 L). Expression was performed at 27 °C for 72 h. At harvest, the cell density was 2.34 x 10 ⁶ cells/mL with 75% GFP expression and 89% viability. The culture was 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. Further purification was performed by size exclusion chromatography on a Superdex 200 16/60 column in 50 mM Hepes pH 7.0, 500 mM NaCl. The protein eluted as a single monomeric peak, which was pooled and concentrated.

Complex formation of Fab fragment AS20 and t-hBSSL

AS20 antibody production was carried out by Absolute Antibody (Oxford, UK). Fabs were generated following the papain digestion protocol from the supplier of immobilized papain (Thermo Scientific, product #20341). AS20, 15 mg total, was concentrated to 17 mg/ml and the buffer was exchanged to 20 mM sodium phosphate, 10 mM EDTA, pH 7, 20 mM cysteine. The antibody was incubated with immobilized papain (0.6 ml suspension) at 37 °C for 3 h followed by incubation at 4 °C overnight. Since digestion was not complete, the sample was incubated at 37 °C for an additional 3 h and then at room temperature over the weekend. The antibody was eluted with 10 mM Tris, pH 7.5, and purified t-hBSSL was added. The mixture was incubated for about 30 min at room temperature and then loaded onto a Superdex 200 column in 50 mM Hepes pH 7.0, 500 mM NaCl. The first major peak at about 60 ml elution volume contained the complex of t-hBSSL and the AS20 Fab fragment. The relevant fractions were concentrated (10K MWCO) and the salt concentration was reduced to 250 mM by dilution with 50 mM Hepes buffer pH 7.0.

Crystallization of AS20 Fab in complex with t-hBSSL and solution structuring

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

Crystallization experiments were performed using a Mosquito liquid handling robot in Swiss CI XTAL SD-3 3-well plates with 35 µl reservoir solution and droplets of 150 + 50, 100 + 100, 50 + 150 nl protein + reservoir. A Morpheus screen from Molecular Dimensions yielded crystals at 20 degrees under conditions B9 and B10. Rod-shaped crystals appeared in the first 12 h and enlarged over the next 24 h. After rapid transfer to reservoir solution containing an additional 10% glycerol, they were cryo-cooled by immersion in liquid nitrogen. Data were collected in the Biomax synchrotron radiation beamline in the MaxIV. The automatically processed data file was used to solve the structure by molecular replacement in Phaser with 1f6w.pdb (hBSSL) and 4n0y.pdb (Fab fragment) as search models. Water molecules were removed, as was the C-terminal-most part of BSSL, and for 4n0y.pdb only the Fab chains converted to the polyalanine model were retained. Manual model building was performed in Coot, and the model was optimized with Refmac 5 (all for the CCP4 suite).

Results

Crystallization and structure determination of AS20 Fab t-hBSSL

The AS20 Fab fragment could be co-crystallized with t-hBSSL under Morpheus B9 conditions consisting of 30 mM NaBr, 30 mM NaFl, 30 mM NaI, 0.1 M Tris (base), 0.1 M Bicine (buffer adjusted to pH 8.5 with these two buffers), 20% PEG550 MME, and 10% PEG 20K. The crystals belonged to space group C2 with unit cell parameters of 323, 67, 123, 90, 101.7, 90 and diffracted to 2.5 Å. The structure was formed by molecular substitution and two complete complexes were present in the asymmetric unit. The crystal packing around the two copies differs, resulting in slight variations in the t-hBSSL structures. Especially the last residues, including the 6-His tag, are visible in molecule A but not in molecule B due to B having fewer crystal contacts in this region and more room for C-terminal flexibility. The region between residues 272 and 283 is also highly disordered in B and cannot be modeled. Apart from these discrepancies, the two t-hBSSL models superimpose very well.

Interaction surface analysis of AS20 Fab and t-hBSSL

The structure of BSSL has been described as having a large core region consisting of a twisted 11-stranded beta-sheet surrounded by alpha helices and connecting loops [9]. At the N-terminus there is a smaller 3-stranded beta-sheet. The structure can be compared to a left-handed glove in which the palm contains the active site triad near the "thumb". In this similarity, the small N-terminal beta-sheet is located on the back of the hand near the "little finger", see Fig. 12. The portion of the BSSL structure that interacts with the Fab molecule is located on the small N-terminal beta-sheet and in the C-terminal portion of the alpha-C [17], the third alpha helix in the structure. In other words, the antibody binding region is not near the active site, but on the opposite side of the antigen. In Fig. 13 shows how the variable chains of AS20 bind to t-hBSSL with the epitope sequence highlighted in light grey.

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

Table 25 - List of BSSL residues involved in the interaction between BSSL and Fab AS20, ranked with the most important interactions in the top row. BSSL residues that are important for the folding of BSSL into the epitope region are also listed.

Minimum of basic interactions Y7, E9, F12
P47, Q52, T54
R176 Key interactions and residues important for BSSL folding A5, Y7, E9, G10, G11, F12, E14
P47, H48, P49, G50, W51, Q52, G53, T54, K56
W173, V174, K175, R176, N177, A180 Residues important for BSSL interactions and structure A5, V6, Y7, T8, E9, G10, G11, F12, V13, E14
Q46, P47, H48, P49, G50, W51, Q52, G53, T54, L55, K56
W173, V174, K175, R176, N177, I178, A179, A180 Extended Important Balances A5, V6, Y7, T8, E9, G10, G11, F12, V13, E14
Q46, P47, H48, P49, G50, W51, Q52, G53, T54, L55, K56
H168, M169, I171, A172, W173, V174, K175, R176, N177, I178, A179, A180

Conclusion

This Example describes the crystallization and patterning of a solution of AS20 Fab and t-hBSSL. The structure demonstrates that the epitope region is located in the same region as that previously identified by HDX-MS described in Example 13. It is a three-dimensional epitope consisting of a small beta-sheet, a well-ordered loop region leading to the third strand of the sheet, and the C-terminal portion of the adjacent helix. The epitope sequences are 7-12 (YTEGGF, SEQ ID NO: 1), 42-55 (LENPQPHPGWQGTL, SEQ ID NO: 2), and 174-180 (VKRNIAA, SEQ ID NO: 5); they are spread out in sequence but close together in structure. The most defining residues are Tyr7, Phe12, and Gln52, which protrude above the surface level.

The sequences of the five antibodies were analyzed in the context of the AS20 Fab t-hBSSL structure. The sequence differences, which are largely conserved, and the results of HDX-MS mapping (Example 14) demonstrate that all five antibodies will bind the same epitope on BSSL.

Example 22 - Complexation, crystallization, structure determination and epitope analysis of the S-SL048-116 Fab-BSSL complex

Materials and methods

Cleavage of S-SL048-116 using FabRICATOR

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

Recovery of F(ab') ₂ fragment and purification of Fab' fragment

Large-scale reconstitution of the purified F(ab') ₂ fragment was performed using cysteamine at a final concentration of 50 mM at room temperature for 2 h in PBS, pH 7.2, containing 5 mM EDTA. Four-fifths of the resulting sample was purified on a HiLoad 26/60 Superdex 200 instrument (GE Healthcare) with 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 subsequent uses. The overall yield was 7.2 mg.

Alkylation of the Fab fragment and subsequent purification

The remaining one-fifth of the recovered 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 in the same manner as the unalkylated sample. The overall yield was 2 mg.

Expression and purification of BSSL

The P2 virus stock used for expression was obtained from SciLifeLab (Stockholm, Sweden). The insect cell line ExpiSf9 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 viral load recommended by SciLife Lab.

EDTA-free protease inhibitor tablets (Merck Millipore), _NiSO4, and imidazole (final concentration 15 mM) were added to the 2 L expression culture supernatant. Any particulate matter was removed by centrifugation at 13,000 × g. The supernatant was loaded onto a 5 mL HisTrap Excel column (GE Healthcare) overnight at 4 °C with a flow rate of 2 mL/min. The column was washed with 6 column volumes (CV) of 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. The bound protein was eluted with a linear gradient of 20 CV 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 run on a HiLoad 26/600 Superdex 200 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 overall yield was 6.2 mg and the sample purity was approximately 85%.

Formation and purification of the BSSL:Fab’ complex

Prior to complex generation, BSSL was assessed by SEC on a HiLoad 26/600 Superdex 200 prep grade semi-preparative column to ensure absence of concentration/storage induced oligomerization. The complex was mixed at a molar ratio of 1:1.3 BSSL:Fab’ and incubated on ice for 1 h. 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 to 50 mM HEPES, 250 mM NaCl, 2 mM EDTA; pH 7. The buffer exchanged sample was concentrated to 17 mg/mL, snap frozen in liquid nitrogen and stored at -80 °C for crystallization. The sample purity was >90% according to SDS-PAGE analysis.

Formation and purification of the BSSL:Alk-Fab’ complex

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

Crystallization and freezing of the Fab-BSSL complex

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

150 nl complex + 37 nl seeds (crushed crystals in 0.1 M sodium citrate pH 5.0, 20% (w/v) PEG 4000 and 0.2 M ammonium sulfate) + 113 nl reservoir

The sessile drop was pipetted onto an MRC plate with a 40 µl reservoir. Crystals appeared over several days and were frozen in a cryo-solution containing 20% (v/v) glycerol, 16% (w/v) PEG 4000, 0.1 M sodium citrate, pH 5.0, and 3% (v/v) ethanol.

Data collection

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

Results

Definition of structure

The structure of Fab-BSSL was solved using molecular replacement with Phaser software with the 2.3 Å structure of the catalytic domain of human bile salt-activated BSSL lipase (from PDB: 1F6W) and the homologous 2.86 Å Fab structure (from PDB: 3NFP) as templates. One complex was found in an asymmetric block. The structure was refined in Refmac5, then in Buster, and model building was performed in Coot. The final model included three protein chains with BSSL (chain A) and the heavy chain (H) and light chain (L) of Fab S-SL048-116 (Fig. 22). The final model included amino acids 1-531 in chain A except for a flexible loop not visible in the electron density (amino acids 117-123). The model included amino acids 1–227 in chain H and amino acids 1–211 in chain L. The first amino acid in chain H, Gln 1, was modeled as PCA, pyroglutamic acid, to best fit the electron density. In chain H, the two free cysteines (Cys 133 and Cys 225) were modeled as unalkylated cysteines, although treatment with S-SL048-116 Fab was necessary for crystallization. No additional density was provided for the methylamide group presumably attached to the sulfur atom of cysteine. The cysteine residues were likely at least partially alkylated, but the attached atoms were so flexible that they were not clearly visible in the electron density map. In addition, 87 water molecules were modeled.

Epitope analysis

Epitope analysis was performed using the coordinates of the Fab-BSSL complex. The analysis was performed using CONTACT software in the CCP4 software package. S-SL048-116-Fab binds to BSSL via both the heavy and light chains. In the heavy chain, all three CDR loops (CDR1, CDR2, and CDR3) are involved, while in the light chain, only CDR1 and CDR3 contact BSSL (Fig. 23, Table 26). Also, Ile 2 close to the N-terminus of the light chain provides a hydrophobic van der Waals interaction with BSSL. The hydrogen bond network stabilizes the interaction between the heavy chain and BSSL (7 hydrogen bonds in total) and BSSL and the light chain (5 hydrogen bonds in total) calculated by PISA (QT) analysis. PISA(QT) analysis shows that 426 Å ² of the solvent accessible region is buried at the BSSL-heavy chain interface, and 468 Å ² is buried at the BSSL-light chain interface.

Table 26 - Summary of all interacting residues between S-SL048-116-Fab and BSSL between 0 and 4 Å

CDR Fab remainder BSSL remainder HC-CDR1 Ser 31 Gln 52 Tyr 32 Gln 52 Asn 33 Gln 52, Gly 53 His 35 Tyr 7 HC-CDR2 Val 50 Tyr 7, Phe 12 Asn 52 Phe 12, Gly 53, Thr 54 Asp 55 Thr 54 Ala 57 Ala 5, Phe 12, Thr 54 Thr 58 Phe 12 Ser 59 Tyr 7 HC-CDR3 Asp 99 Gln 52 Gly 102 Pro 49 Ser 103 Pro 49, Gly 50, Trp 51, Gln 52 LC Number 2 Arg 176 LC-CDR1 Pro 27 Lys 175 Ser 28 Ala 172, Arg 176 Number 29 Arg 176 Ser 30 Glu 9, Pro 47, Arg 176 Tyr 31 Gln 46, Pro 47, His 48 LC-CDR3 Arg 90 Tyr 7, Glu 9 Ser 91 Tyr 7, Thr 8, Glu 9 Ser 92 Tyr 7, Arg 176, Asn 177, Ala 180

Conclusion

The results obtained in this Example for S-SCL048-116 are consistent with the results obtained in the HDX-MS analysis study in Example 14.

Example 23 - Confirmation of the efficacy of S-SL048-116 in a mouse model of rheumatoid arthritis

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

Material and methods

Disease induction

DBA/1 mice (male, 8 weeks old) were injected intravenously on day 0 with a mixture of anti-CII monoclonal antibodies at a dose of 2 mg/mouse (CIA-MAB-50, MD Bioproducts). On day 5, mice were injected intraperitoneally with LPS (50 μg/mouse) to enhance disease.

Experimental groups and management of study products

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

Table 27 - Experimental groups

Group Treatment Dose (mg/kg) Route of administration Day of termination Number of animals 1 Carrier n.d. v/br 19 15 2 S-SL048-116 90 v/br 19 15 3 S-SL048-116 30 v/br 19 15 4 S-SL048-116 10 v/br 19 15 5 Naive n.d. n.d. 20 15

Disease assessment

Disease was assessed daily starting on day 3 in a blinded manner using a macroscopic scoring system for four limbs ranging from 0 to 15 (1 point for each swollen or hyperemic toe, 1 point for a swollen or hyperemic middle toe or foot joint, 5 points for a swollen ankle) with a maximum score of 60 for each mouse. Due to ethical restrictions, animals with a score above 45 were excluded from the experiment.

Results

Arthritis severity

CAIA-induced animals treated with vehicle developed moderate to severe disease at a rate of 100%. The efficacy of S-SL048-116 (SOL-116) administered intraperitoneally every fourth day from day -1 until treatment cessation was assessed at three doses (10, 30, and 90 mg/kg). S-SL048-116 administered at a dose of 90 mg/kg demonstrated a beneficial effect in reducing disease severity compared to vehicle (Figs. 24 and 25).

Two animals were withdrawn from the study early for ethical reasons (high score), one in the vehicle group (day 15) and one in the 90 mg/kg group (day 12). These animals were included in the analysis of the maximum score but were excluded from the analysis of mean, AUC, and % inhibition (Fig. 25, Table 28). One mouse in the vehicle group did not recover from the LPS booster dose, i.e., was hypothermic, kyphotic, and very underweight. This mouse was removed from the experimental group prior to disease onset (day 7) and therefore its data were excluded from all results.

Statistics of disease parameters are presented in Table 28.

Table 28 - CAIA Severity Parameter Statistics

Group Frequency ¹ MES ² Max. AUC ³ % inhibition ⁴ Carrier 100% 19.5±2.1 33.2±3.6 317.9±34.9 n.d. S-SL048-116, 90 mg/kg 80% 15.3±3.4 28.4±4.9 249.0±54.5 21.2±17.0 S-SL048-116, 30 mg/kg 93% 21.5±2.1 35.1±2.7 349.8±33.4 -10.0±10.5 S-SL048-116, 10 mg/kg 87% 19.8±2.9 34.4±4.5 321.8±47.7 -1.23±15.0

¹ Cumulative frequency. A mouse was considered to be sick if it scored 1 or higher for two consecutive days.

² Average CAIA score for all time points assessed.

³ Area under the curve.

⁴ Percentage change in overall disease burden in each animal compared to the vehicle control group. Calculated by taking the difference between the mean AUC of the vehicle control group and the AUC for each individual animal, divided by the AUC of the vehicle control group, and multiplied by 100 *(-1).

Health assessment

General health assessments were performed daily along with disease assessments from day 3 until study termination. Mice were weighed twice weekly during general health assessments. No adverse effects were observed in the animals during treatment.

Conclusion

Animals induced with CAIA and treated with vehicle developed moderate to severe disease with 100% frequency. The efficacy of S-SL048-116 at three different doses, i.e., 10, 30, and 90 mg/kg intraperitoneally, was evaluated with an initial treatment day of -1. S-SL048-116 administered at a dose of 90 mg/kg demonstrated a positive effect in reducing the severity of disease compared to vehicle. No adverse effects were observed in animals during treatment with S-SL048-116 or vehicle alone.

Example 24 - Analysis of cellularity in blood, spleen and mesenteric lymph nodes of 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) collected from BSSL knockout (KO) mice and wild-type littermates was examined using fluorescence-activated cell sorting (FACS).

Material 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 the spleen and MLN by 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 application of two different antibody cocktails, Stain A and Stain B.

Coloring A

Antibodies/clones/fluorochromes used for Staining A were as follows: CD19 (ID3/BB515), γδTCR (GL3/PE), CD8a (53-6.7/PerCP-Cy5.5), CD45.2 (104/PE-Cy7), NK1.×(PK136/APC), CD4 (GK1.5/APC-H7), TCRβ (H57-597/BV421), and FVD (Horizon 510).

The cell populations identified by A staining were as follows:

αβ T cells: CD45.2 ⁺ , TCRβ ⁺

CD4 αβ T cells: CD45.2 ⁺ , TCRβ ⁺ , CD4 ⁺

CD8 αβ T cells: CD45.2 ⁺ , TCRβ ⁺ , CD8 ⁺

γδ T cells: CD45.2 ⁺ , TCRγδ ⁺

NKT cells: CD45.2 ⁺ , TCRβ ⁺ , NK1.1 ⁺

B cells: CD45.2 ⁺ , CD19 ⁺

NK cells: CD45.2 ⁺ , TCRβ ^- , TCRγδ ^- , NK1.1 ⁺

Coloring B

Antibodies/clones/fluorochromes used for Staining B were as follows: 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 FVD (Horizon 510).

The cell populations identified by B staining were as follows:

Myeloid cells: CD45.2 ⁺ , CD11b ⁺

Neutrophils: CD45.2 ⁺ , CD11b ⁺ , Ly6G ⁺

Eosinophils: CD45.2 ⁺ , CD11b ⁺ , Ly6G ^- , Siglec F ⁺ , SSC ^high

Monocytes: CD45.2 ⁺ , CD11b ⁺ , Ly6G ^- , Siglec F ^- , SSC ^low , Ly6C ⁺ , MHCII ^-

Macrophages: CD45.2 ⁺ , CD11b ⁺ , Ly6G ^- , Siglec F ^- , SSC ^low , Ly6C ^- , MHCII ⁺

Mast cells: CD45.2 ⁺ , CD11b ⁺ , FcεRlα ⁺ , CD117 ⁺

Basophils: CD45.2 ⁺ , CD11b ⁺ , FcεRlα ⁺ , CD117 ^-

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

Results

Total spleen white blood cell count and MLN

It was found that the number of leukocytes in the spleen of KO mice was significantly reduced to approximately 50% of that in WT mice. No significant difference in the number of leukocytes in the MLN was observed between KO and WT mice (Fig. 26).

αβT cells

Total αβT cells were found to be reduced in the spleen in KO mice compared to WT mice. The reduction was similar to that observed for total CD45 ⁺ leukocytes. No significant difference in αβ T cell numbers was observed in the MLN. The percentage of αβT cells to total CD45 ⁺ cells was found to be higher in the spleen and blood of KO mice compared to WT mice, but with no observable difference in the MLN. In the spleen, the CD4 ⁺ /CD8 ⁺ ratio was higher in KO mice compared to WT mice.

γδT cells

It was found that the total number of γδT cells in the spleen was reduced in KO mice compared to WT mice. The reduction was similar to that observed for total CD45 ⁺ leukocytes. In the MLN, this reduction was even more pronounced than in the spleen. The proportion of γδT cells from CD45 ⁺ cells in the MLN was also lower in KO mice compared to WT mice. No such difference was observed in the spleen and blood.

NKT cells

Total NKT cell counts were found to be reduced in the spleen in KO mice compared to WT mice. The reduction was similar to that observed for total CD45 ⁺ leukocytes. No significant difference in NKT cell counts was observed in the MLN. The proportion of NKT cells from CD45 ⁺ cells in the blood was also lower in KO mice compared to WT mice. No such difference was observed in the spleen and MLN.

B cells

Total B cells in the spleen were found to be reduced in KO mice compared to WT mice. The reduction was similar to that observed for total CD45 ⁺ leukocytes. No significant difference in B cell numbers was observed in the MLN. The proportion of B cells from CD45 ⁺ cells in the spleen and blood was also lower in KO mice compared to WT mice. No such difference was observed in the MLN.

NK cells

The total number of NK cells in the spleen was found to be reduced in KO mice compared to WT mice. The reduction was found to be more pronounced for NK cells compared to that observed for total CD45 ⁺ leukocytes and other lymphoid subsets. No significant difference was observed in the number of NK cells in the MLN. The proportion of NK cells from CD45 ⁺ cells in the spleen and blood was also lower in KO mice compared to WT mice. No such difference was observed in the MLN (Fig. 27).

Myeloid cells

Total myeloid cell counts were reduced in KO mice compared to WT mice in both the spleen and MLN. The proportion of myeloid cells from CD45 ⁺ cells in the spleen and MLN was also found to be lower in KO mice compared to WT mice. No such difference was observed in the blood.

Neutrophils

There was no significant difference in the neutrophil counts in the spleen and MLN between KO and WT mice. The proportion of neutrophils in the blood was higher in KO mice, but no significant differences were observed in the spleen and MLN.

Eosinophils

There was no significant difference in the number of eosinophils in the spleen and MLN between KO and WT mice. The proportion of eosinophils in the blood was found to be higher in KO mice, but no significant difference was observed in the spleen and MLN.

Monocytes

Total monocyte counts were found to be lower in KO mice compared to WT mice in both the spleen and MLN. No significant difference in monocyte proportion was observed between KO and WT mice in any of the organs examined.

Macrophages

Total macrophage numbers were found to be lower in KO mice compared to WT mice in both the spleen and MLN. No significant difference in the proportion of macrophages was observed between KO and WT mice in any of the organs examined.

Mast cells

There was no significant difference in the number of mast cells in the spleen and MLN between KO and WT mice. The proportion of mast cells in the spleen was higher in KO mice compared to WT mice. No such difference was observed in the blood and MLN.

Basophils

The total number of basophils in the spleen was found to be reduced in KO mice compared to WT mice. No difference in the number of basophils in the MLN was observed. The proportion of basophils in the spleen was found to be higher in KO mice compared to WT mice. No such difference was observed in the spleen or MLN.

Conclusion

In addition to the general effect on CD45 ⁺ leukocytes, these data indicate an additional effect specifically on NK cells, such that both the total number and the proportion of NK cells from CD45 ⁺ cells were lower in the blood and spleen of naïve BSSL-deficient mice compared to their wild-type littermates. These data support the conclusion that AS20 hIgG1 LALA-PG (90 mg/kg dose) significantly reduced the total number of NK cells and the proportion of T cells, NKT cells, and NK cells from CD45 ⁺ cells in the spleen of CAIA mice (Example 19).

Table 29 - List of sequences and SEQ ID NO:s used in the present invention

SEQ ID NO: Nomenclature Subsequence 1 BSSL Epitope 7-12 YTEGGF 2 BSSL Epitope 42-55 LENPQPHPGWQGTL 3 BSSL Epitope 1-12 AKLGAVYTEGGF 4 BSSL Epitope 84-101 (clone 46) NIWVPQGRKQVSRDLPVM 5 BSSL Epitope 174-180 (clone 116) VKRNIAA 6 BSSL Epitope 283-295 (clone 11) HYVGFVPVIDGDF 7 HCDR1 (clone 11, 46, 106, 116, 118 + AS20) GYTFTSYN 8 HCDR2 (clone 46, 116, 118) INPGDGAT 9 HCDR3 (clone 11, 46, 106, 116, 118 + AS20) ARDYYGSSPLGY 10 LCDR1 (clone 11, 116, 118) PSIS 11 LCDR3 (clone 46, 116, 118) HQRSSSPT 12 eHCDR2 (clone 116 och 118) MGVINPGDGATSYAQKFK 13 BSSL Epitope 172-180 (AS20) AWVKRNIAA 14 eLCDR1 (clone 116) RASPSISSYMN 15 eLCDR2 (clone 11, 116) ATSSLA 16 eLCDR1 (clone 11, 118) SASPSISYMN 17 eLCDR2 (clone 118) ATSSLP 18 HCDR2 (clone 11) IYPGDGAT 19 HCDR2 (clone 106) IYPGDGST 20 LCDR1 (clone 46, 106) SSISY 21 LCDR3 (clone 11 + AS20) HQRSSYPT 22 LCDR3 (clone 106) HQRSSTPT 23 eHCDR2 (clone 11) MGVIYPGDGATSYAQKFK 24 eHCDR2 (clone 46) MGVINPGDGATSYNQKFQ 25 eHCDR2 (clone 106) IGVIYPGDGSTSYNQKFQ 26 eLCDR1 (clone 106) SASSSISYMN 27 eLCDR1 (clone 46) RASSSISYLN 28 eLCDR2 (clone 106) ATSKLP 29 eLCDR2 (clone 46) AASSLA 30 S-SL048-11HCVR QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYPGDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 31 S-SL048-11 LCVR DIQMTQSPSSLSASVGDRVTITTCSASPSISYMNWYQQKPGKAPKLLIYATSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSYPTFGQGTKLEIK 32 S-SL048-46HCVR QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 33 S-SL048-46 LCVR DIQMTQSPSSLSASVGDRVTITCRASSSISYLNWYQQKPGKAPKLLIYAASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 34 S-SL048-106HCVR QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVIYPGDGSTSYNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 35 S-SL048-106 LCVR DIQMTQSPSSLSASVGDRVTITCSASSSISYMNWYQQKPGKAPKLLIYATSKLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSTPTFGQGTKLEIK 36 S-SL048-116 and 118 HCVR QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 37 S-SL048-116LCVR DIQMTQSPSSLSASVGDRVTITCRASPSISYMNWYQQKPGKAPKLLIYATSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 38 S-SL048-118LCVR DIQMTQSPSSLSASVGDRVTITTCSASPSISYMNWYQQKPGKAPKLLIYATSSLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 39 ZH1 QVQLVQSGAEVKKPGASVKVSCKAS 40 ZH2 MHWVRQAPGQGLEW 41 ZH3 GRVTMTRDTSTSTVYMELSSLRSEDTAVYYC 42 ZH4 WGQGTLVTVSS 43 ZL1 DIQMTQSPSSLSASVGDRVTITC 44 ZL2 WYQQKPGKAPKLLIY 45 ZL3 SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 46 ZL4 FGQGTKLEIK 47 S-SL048-10 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVINPSDGYTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 48 S-SL048-12 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPSDGATSYTQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 49 S-SL048-13 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVINPSDGATSYTQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 50 S-SL048-14 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 51 S-SL048-17 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGYTSYTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 52 S-SL048-18 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPSDGATSYTQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 53 S-SL048-38 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 54 S-SL048-40 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVINPSDGATSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 55 S-SL048-41 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVIYPGDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 56 S-SL048-43 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYTQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 57 S-SL048-45 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPSDGATSYTQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 58 S-SL048-47 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVINPGGGYTSYTQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 59 S-SL048-48 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVINPGDGATSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 60 S-SL048-65 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVINPGDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 61 S-SL048-66 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 62 S-SL048-74 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVINPGDGATSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 63 S-SL048-75 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYPGGGYTSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 64 S-SL048-79 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPSDGATSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 65 S-SL048-81 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVINPGDGATSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 66 S-SL048-86 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 67 S-SL048-89 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPSSGSTSYTQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 68 S-SL048-103 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 69 S-SL048-104 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYDQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 70 S-SL048-105 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPSDGATSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 71 S-SL048-107 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 72 S-SL048-108 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVINPSDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 73 S-SL048-109 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPSDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 74 S-SL048-110 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 75 S-SL048-112 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGIIYPGDGATSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 76 S-SL048-115 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPSDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 77 S-SL048-125 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVINPGDGATSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 78 S-SL048-131 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVINPGDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 79 S-SL048-134 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPSDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 80 AS20 VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGVIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDYYGSSPLGYWGQGTTLTVSS 81 S-SL048-10 VL DIQMTQSPSSLSASVGDRVTITCRASPSISYLNWYQQKPGKAPKLLIYATSRLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSSPTFGQGTKLEIK 82 S-SL048-12 VL DIQMTQSPSSLSASVGDRVTITCSASSSISYMHWYQQKPGKAPKLLIYAASRLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSTSTFGQGTKLEIK 83 S-SL048-13 VL DIQMTQSPSSLSASVGDRVTTITCSASPSISYLNWYQQKPGKAPKLLIYATSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSSPTFGQGTKLEIK 84 S-SL048-14 VL DIQMTQSPSSLSASVGDRVTITCRASPSISYLNWYQQKPGKAPKLLIYATSRLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSSPTFGQGTKLEIK 85 S-SL048-17 VL DIQMTQSPSSLSASVGDRVTITCRASQSISYLNWYQQKPGKAPKLLIYAASKLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSSPTFGQGTKLEIK 86 S-SL048-18 VL DIQMTQSPSSLSASVGDRVTITCRASSSISYLNWYQQKPGKAPKLLIYATSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSTPTFGQGTKLEIK 87 S-SL048-38 VL DIQMTQSPSSLSASVGDRVTITCRASPSISYLNWYQQKPGKAPKLLIYATSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSSPTFGQGTKLEIK 88 S-SL048-40 VL DIQMTQSPSSLSASVGDRVTITCRASSSISYLNWYQQKPGKAPKLLIYAASRLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSSPTFGQGTKLEIK 89 S-SL048-41 VL DIQMTQSPSSLSASVGDRVTITCRASSSISYMNWYQQKPGKAPKLLIYATSRLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 90 S-SL048-43 VL DIQMTQSPSSLSASVGDRVTITCRASPSISYMNWYQQKPGKAPKLLIYAASSLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSSPTFGQGTKLEIK 91 S-SL048-45 VL DIQMTQSPSSLSASVGDRVTITCRASQSISYLHWYQQKPGKAPKLLIYAASRLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSTPTFGQGTKLEIK 92 S-SL048-47 VL DIQMTQSPSSLSASVGDRVTTITCSASPSISYMNWYQQKPGKAPKLLIYATSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSTPTFGQGTKLEIK 93 S-SL048-48 VL DIQMTQSPSSLSASVGDRVTITCRASPSISYMNWYQQKPGKAPKLLIYATSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 94 S-SL048-65 VL DIQMTQSPSSLSASVGDRVTITCSASQSISYLNWYQQKPGKAPKLLIYDASSLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 95 S-SL048-66 VL DIQMTQSPSSLSASVGDRVTITCSASQSISYMNWYQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSTPTFGQGTKLEIK 96 S-SL048-74 VL DIQMTQSPSSLSASVGDRVTITCRASQSISYLNWYQQKPGKAPKLLIYAASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 97 S-SL048-75 VL DIQMTQSPSSLSSASVGDRVTITCRASQSISYLNWYQQKPGKAPKLLIYATSNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 98 S-SL048-79 VL DIQMTQSPSSLSASVGDRVTITCSASQSISYLNWYQQKPGKAPKLLIYAASRLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSYPTFGQGTKLEIK 99 S-SL048-81 VL DIQMTQSPSSLSASVGDRVTITCRASSSISYMHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 100 S-SL048-86 VL DIQMTQSPSSLSSASVGDRVTITCRASQSISYLHWYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSYPTFGQGTKLEIK 101 S-SL048-89 VL DIQMTQSPSSLSASVGDRVTITCRASPSISYLNWYQQKPGKAPKLLIYATSRLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSNPTFGQGTKLEIK 102 S-SL048-103 VL DIQMTQSPSSLSASVGDRVTITCRASSSISYMHWYQQKPGKAPKLLIYATSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 103 S-SL048-104 VL DIQMTQSPSSLSASVGDRVTITCSASSSISYLHWYQQKPGKAPKLLIYAASRLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSTPTFGQGTKLEIK 104 S-SL048-105 VL DIQMTQSPSSLSASVGDRVTITCSASQSISYLHWYQQKPGKAPKLLIYAASKLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSYPTFGQGTKLEIK 105 S-SL048-107 VL DIQMTQSPSSLSASVGDRVTITCRASPSISYLNWYQQKPGKAPKLLIYATSSLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSSPTFGQGTKLEIK 106 S-SL048-108 VL DIQMTQSPSSLSASVGDRVTTITCSASPSISYLNWYQQKPGKAPKLLIYATSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSSPTFGQGTKLEIK 107 S-SL048-109 VL DIQMTQSPSSLSASVGDRVTITCRASPSISYLNWYQQKPGKAPKLLIYATSRLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSSPTFGQGTKLEIK 108 S-SL048-110 VL DIQMTQSPSSLSASVGDRVTITCSASQSISYLHWYQQKPGKAPKLLIYAASRLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSTPTFGQGTKLEIK 109 S-SL048-112 VL DIQMTQSPSSLSASVGDRVTITCRASSSISYMHWYQQKPGKAPKLLIYATSRLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSTPTFGQGTKLEIK 110 S-SL048-115 VL DIQMTQSPSSLSASVGDRVTITCRASQSISYMHWYQQKPGKAPKLLIYAASKLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 111 S-SL048-125 VL DIQMTQSPSSLSASVGDRVTITCRASSSISYLHWYQQKPGKAPKLLIYAASRLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSYPTFGQGTKLEIK 112 S-SL048-131 VL DIQMTQSPSSLSASVGDRVTITCRASPSISYLNWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIK 113 S-SL048-134 VL DIQMTQSPSSSLSASVGDRVTITCRASPSISYLNWYQQKPGKAPKLLIYATSKLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRSSYPTFGQGTKLEIK 114 AS20 VL QIVLTQSPAVMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPTFGGGTKLEIK 115 AS20 hIgG1 LALA-PG HC QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGVIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDYYGSSPLGYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 116 AS20 hIgG1 LALA-PG LC QIVLTQSPAVMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 117 Anti-NP hIgG1 LALA-PG HC QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGRGLEWIGRIDPNSGGTKYNEKFKSKATLTVDKPSSTAYMQLSSLTSEDSAVYYCAR YDYYGSSYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 118 Anti-NP hIgG1 LALA-PG LC QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGNKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPTEC 119 S-SL048-11 hIgG4 S228P HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYPGDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DYYGSSPLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 120 S-SL048-11 hIgG4 S228P LC DIQMTQSPSSLSASVGDRVTITTCSASPSISYMNWYQQKPGKAPKLLIYATSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSYPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 121 S-SL048-46 hIgG4 S228P HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DYYGSSPLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 122 S-SL048-46 hIgG4 S228P LC DIQMTQSPSSLSASVGDRVTITCRASSSISYLNWYQQKPGKAPKLLIYAASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 123 S-SL048-106 hIgG4 S228P HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVIYPGDGSTSYNQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DYYGSSPLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 124 S-SL048-106 hIgG4 S228P LC DIQMTQSPSSLSASVGDRVTITCSASSSISYMNWYQQKPGKAPKLLIYATSKLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSTPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 125 S-SL048-116 hIgG4 S228P HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DYYGSSPLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 126 S-SL048-116 hIgG4 S228P LC DIQMTQSPSSLSASVGDRVTITCRASPSISYMNWYQQKPGKAPKLLIYATSSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 127 S-SL048-118 hIgG4 S228P HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVINPGDGATSYAQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DYYGSSPLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 128 S-SL048-118 hIgG4 S228P LC DIQMTQSPSSLSASVGDRVTITCSASPSISYMNWYQQKPGKAPKLLIYATSSLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSSPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 129 AS20 hIgG4 S228P HC QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGVIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARDYYGSSPLGYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 130 AS20 hIgG4 S228P LC QIVLTQSPAVMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 131 CDR graft hIgG4 S228P HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVIYPGNGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARD YYGSSPLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 132 CDR graft hIgG4 S228P LC DIQMTQSPSSLSASVGDRVTITCSASSSISYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSYPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 133 Anti-NP hIgG4 S228P HC QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGRGLEWIGRIDPNSGGTKYNEKFKSKATLTVDKPSSTAYMQLSSLTSEDSAVYYCARYDYYGSSYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 134 Anti-NP hIgG4 S228P LC QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGNKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 135 AS20 IgG HC MGWSCIILFLVATATGVHSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGVIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARDYYGSSPLGYWGQGTTLTVSS 136 AS20 IgG LC MDFQVQIFSFLLISASVILSRGQIVLTQSPAVMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPTFGGGTKLEIK 137 mBSSL AKLGAVYTEGGFVEGVNKKLSLLGGDSVDIFKGIPFATAKTLENPQRHPGWQGTLKATNFKKRCLQATITQDNTYGQEDCLYLNIWVPQGRKQVSHNLPVMVWIYGGAFLMGSGQGANFLKNYLYDGEEIATRGNVIVVTFNYRVGPLGFLSTGDANLPGNFGLRDQHMAIAWVKRNIAAFGGDPDNITIFGESAGA ASVSLQTLSPYNKGLIRRAISQSGMALSPWAIQKNPLFWAKTIAKKVGCPTEDTGKMAACLKITDPRALTLAYKLPVKKQEYPVVHYLAFIP VIDGDFIPDDPINLYNNTADIDYIAGINNMDGHLFATIDVPAVDKTKQTVTEEDFYRLVSGHTVAKGLKGAQATFDIYTESWAQDPSQENMKKTVVAFETDVLFLIPTEIALAQHKAHAKSAKTYSYLFSHPSRMPIYPKWMGADHADDLQYVFGKPFATPLGYRPQDRAVSKAMIAYWTNFARSGDPNMGNSPVPTH WYPYTLENGNYLDITKTITSASMKEHLREKFLKFWAVTFEVLPTVTGDQDTLTPPEDDSEVAPPDPPSDDSQVVPVPPTDDSVEAQMPATIGF 138 hBSSL AKLGAVYTEG GFVEGVNKKL GLLGDSVDIF KGIPFAAPTK ALENPQPHPG WQGTLKAKNF KKRCLQATIT QDSTYGDEDC LYLNIWVPQG RKQVSRDLPV MIWIYGGAFL MGSGHGANFL NNYLYDGEEI ATRGNVIVVT FNYRVGPLGF LSTGDANLPG NYGLRDQHMA IAWVKRNIAA NNITL FGESAGGASV SLQTLSPYNK GLIRRAISQS GVALSPWVIQ KNPLFWAKKV AEKVGCPVGD AARMAQCLKV TDPRALTLAY KVPLAGLEYP MLHYVGFVPV IDGDFIPADP INLYANAADI DYIAGTNNMD GHIFASIDMP AINKGNKKVT EEDFYKLVSE FTITKGLRGA KTTFDVYTES WAQDPSQENK KKTVVDFETD VLFLVPTEIA LAQHRANAKS AKTYAYLFSH PSRMPVYPKW VGADHADDIQ YVFGKPFATP TGYRPQDRTV SKAMIAYWTN FAKTGDPNMG DSAVPTHWEP YTTENSGYLE ITKKMGSSSM KRSLRTNFLR YWTLTYLALP TVTDQEATPV PPTGDSEATP VPP TGDSETA PVPPTGDSGA PPVPPTGDSG APPVPPTGDS GAPPVPPTGD SGAPPVPPTG DSGAPPVPPT GDSGAPPVPP TGDSGAPPVP PTGDAGPPPV PPTGDSGAPP VPPTGDSGAP PVTPTGDSET APVPPTGDSG APPVPPTGDS EAAPVPPTDD SKEAQMPAVIRF 139 chimeric AS20 IgG4 HC QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGVIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARDYYGSSPLGYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 140 chimeric AS20 IgG4 LC QIVLTQSPAVMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 141 eHCDR2 AS20 IGVIYPGNGDTSYNQKFK 142 eLCDR1 AS20 SASSSISYMH 143 eLCDR2 AS20 DTSKLA 144 CDR graft HCVR QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWIGVIYPGNGDTSYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYYGSSPLGYWGQGTLVTVSS 145 CDR graft LCVR DIQMTQSPSSLSASVGDRVTITCSASSSISYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQRSSYPTFGQGTKLEIK 146 t-hBSSL AKLGAVYTEGGFVEGVNKKLGLLGDSVDIFKGIPFAAPTKALENPQPHPGWQGTLKAKNFKKRCLQATITQDSTYGDEDCLYLNIWVPQGRKQVSRDLPVMIWIYGGAFLMGSGHGANFLNNYLYDGEEIATRGNVIVVTFNYRVGPLGFLSTGDANLPGNYGLRDQHMAIAWVKRNIAAFGGDPNNITLFGESAGGA SVSLQTLSPYNKGLIRRAISQSGVALSPWVIQKNPLFWAKKVAEKVGCPVGDAARMAQCLKVTDPRALTL AYKVPLAGLEYPMLHYVGFVPVIDGDFIPADPINLYANAADIDYIAGTNNMDGHIFASIDMPAINKGNKKVTEEDFYKLVSEFTITKGLRGAKTTFDVYTESWAQDPSQENKKKTVVDFETDVLFLVPTEIALAQHRANAKSAKTYAYLFSHPSRMPVYPKWVGADHADDIQYVFGKPFATPTGYRPQDRTVSKA MIAYWTNFAKTGDPNMGDSAVPTHWEPYTTENSGYLEITKKMGSSSMKRSLRTNFLRYWTLTYLALPAHHHHHH 147 BSSL epitope aa 7-11 YTEGG 148 BSSL epitope 1-7 AKLGAVY 149 BSSL epitope 1-11 AKLGAVYTEGG 150 BSSL epitope 1-25 AKLGAVYTEGGFVEGVNKKLGLLGD 151 BSSL epitope 1-26 AKLGAVYTEGGFVEGVNKKLGLLGDS 152 BSSL epitope 1-28 AKLGAVYTEGGFVEGVNKKLGLLGDSVD 153 iTope ^TM AS20 Remains VKPGASVKM 154 iTopeTM remnants S-SL048-11, -46, -106, -118 YNMHWVRQA 155 iTopeTM remnants S-SL048-11, -116, -118 YAQKFKGRV 156 iTopeTM AS20 Remains WGQGTTLTV 157 iTopeTM AS20 Remains VTMTCSASS 158 iTopeTM Remains S-SL048-46 VTITCRASS 159 iTopeTM Remains S-SL048-106 VTITCSASS 160 iTopeTM Remains S-SL048-106 LLIYATSKL 161 iTopeTM remnants S-SL048-11, -116 IYATSSLAS 162 iTopeTM Remains S-SL048-46 IYAASSLAS 163 iTopeTM AS20 Remains FGGGTKLEI 164 SL048_hum_AS20_H1 GATACACCTTCACCAGCTACWATATGCACTGGGTGCG 165 SL048_hum_AS20_H2 GACAAGGGCTTGAGTGGATRGGARTAATCWACCCTRGTRRTGGTKMCACAAGCTACRMTCAGAAGTTCMAGGGCCGCGTCACC 166 SL048_hum_AS20_L1 CGTCACCATCACCTGCAGKGCAAGTYMGAGCATTAGCTATWTGMATTGGTATCAGCAGAAAC 167 SL048_hum_AS20_L2 CCTAAGCTCCTGATCTATGMTRCATCCARSTTGSMAAGTGGGGTCCCATCAC 168 SL048_hum_AS20_L3 GATTTTGCAACTTATTACTGTCASCAGAGKTMTAGTWMTYHCACTTTTGGCCAGGGG 169 HVCR part 2 XGVIXPGDGXTSYXQKFX 170 LVCR part 1 XASXSISYXN 171 LVCR part 2 AXSXLX 172 LVCR part 3 HQRSSXPT 173 consensus sequence QQSYSTPT 174 nt S-SL048-11HC CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACAATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGTAATCTACCCTGGTGATGGTGCCACAAGCTACGCTCAGAAGTTCAAGGGCCGCGTCACCATGACCGGCGACACGTCCACGAGCACAGTC TACATGGAGCTGAGCAGCCTGCGCTCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATTACTACGGTAGTAGCCCCCTTGGCTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA 175 nt S-SL048-11 LC GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACCTGCAGTGCAAGTCCGAGCATTAGCTATATGAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTACATCCAGCTTGGCAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTT TGCAACTTATTACTGTCACCAGAGGTCTAGTTATCCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 176 nt S-SL048-46HC CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACAATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGTAATCAACCCTGGTGATGGTGCCACAAGCTACAATCAGAAGTTCCAGGGCCGCGTCACCATGACCGGCGACACGTCCACGAGCACAGTC TACATGGAGCTGAGCAGCCTGCGCTCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATTACTACGGTAGTAGCCCCCTTGGCTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA 177 nt S-SL048-46 LC GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACCTGCAGGGCAAGTTCGAGCATTAGCTATTTGAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGCTTGGCAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTT TGCAACTTATTACTGTCACCAGAGGTCTAGTTCTCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 178 nt S-SL048-106HC CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACAATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATAGGAGTAATCTACCCCTGGTGATGGTTCCACAAGCTACAATCAGAAGTTCCAGGGCCGCGTCACCATGACCGGCGACACGTCCACGAGCACAGTC TACATGGAGCTGAGCAGCCTGCGCTCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATTACTACGGTAGTAGCCCCCTTGGCTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA 179 nt S-SL048-106 LC GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACCTGCAGTGCAAGTTCGAGCATTAGCTATATGAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTACATCCAAGTTGCCAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGAT TTTGCAACTTATTACTGTCACCAGAGGTCTAGTACTCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 180 nt S-SL048-116 and 118 HC CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACAATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGTAATCAACCCTGGTGATGGTGCCACAAGCTACGCTCAGAAGTTCAAGGGCCGCGTCACCATGACCGGCGACACGTCCACGAGCACAGTC TACATGGAGCTGAGCAGCCTGCGCTCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATTACTACGGTAGTAGCCCCCTTGGCTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA 181 nt S-SL048-116 LC GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACCTGCAGGGCAAGTCCGAGCATTAGCTATATGAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTACATCCAGCTTGGCAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTT TGCAACTTATTACTGTCACCAGAGGTCTAGTTCTCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 181 nt S-SL048-118 LC GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACCTGCAGTGCAAGTCCGAGCATTAGCTATATGAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTACATCCAGCTTGCCAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTT TGCAACTTATTACTGTCACCAGAGGTCTAGTTCTCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 183 nt AS20 HC CAAGTTCAGCTGCAGCAGCCCGGTGCCGAGCTGGTGAAACCCGGTGCCTCTGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTTACCAGCTACAACATGCACTGGGTGAAGCAGACACCCGGACAAGGTTTAGAGTGGATCGGCGTGATCTACCCCGGCAACGGCGACACCTCTTACAACCAGAAGTTCAAGGGCAAGGCCACACTGACCGCCGACAAGAGCAGCAGCACCGCCTACAT GCAGCTGAGCTCTTTAACCAGCGAGGACTCCGCCGTGTACTACTGCGCTCGTGATTACTACGGCAGCAGCCCTTTAGGCTATTGGGGACAAG GTACCACTTTAACCGTCTCGAGCGCCTCAACCAAAGGACCCTCCGTGTTTCCCCTCGCCCCCTGTTCCCGCTCCACATCCGAGTCAACCGCGGCGCTGGGCTGCCTCGTGAAGGACTACTTCCCTGAGCCCGTCACTGTGTCGTGGAACTCCGGGGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTTCAATCCTCCGGACTGTACTCCCTGTCCTCGGTGGTCACCGTGCC GTCGAGCTCGTTGGGAACCAAGACTTACACTTGCAACGTGGACCACAAGCCAAGCAACACCAAAGTGGACAAGAGAGTCGAATCTAAGTACGGACCG CCCTGCCCGCCTTGCCCCGCCCCTGAGTTTCTCGGCGGTCCTAGCGTGTTCCTGTTCCCACCCAAGCCCAAGGACACTCTGATGATCTCCCGGACCCCTGAAGTGACCTGTGTGGTCGTGGACGTGTCGCAGGAAGATCCGGAGGTCCAGTTCAATTGGTACGTGGATGGGGTGGAGGTCCACAACGCCAAGACGAAGCCGAGAGAAGAACAGTTCAACTCAACTTACCGGGTGG TGTCCGTGCTGACCGTGCTGCATCAGGATTGGCTCAACGGAAAGGAGTACAAGTGCAAAGTGTCCAACAAGGGCCTGCCTAGCTCAATCGAAAAGACCA TTTCCAAGGCCAAGGGCCAGCCGAGGGAACCACAGGTCTATACTCTGCCACCGAGCCAAGAAGAGATGACCAAGAACCAAGTGTCCCTGACTTGCCTGGTCAAGGGGTTCTACCCGTCGGACATCGCAGTGGAGTGGGAGAGCAACGGACAGCCTGAAAACAATTACAAGACCACCCCGCCCGTGCTGGATAGCGACGGTTCCTTCTTCCTTTACTCGCGCCTCACCGTCGACAAGAGCC GGTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAAGCTCTGCATAACCACTACACTCAGAAGTCCTTGTCGCTGAGCCTCGGAAAG 184 nt AS20 LC CAGATCGTGCTGACCCAGAGCCCCGCTGTGATGAGCGCCTCTCCCGGTGAGAAGGTGACCATGACTTGTAGCGCCAGCAGCAGCATCAGCTACATGCACTGGTACCAGCAGAAGCCCGGCACCAGCCCTAAGAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTGCCCGCTAGGTTCAGCGGAAGCGGCAGCGGCACCAGCTACTCTTTAACCATCAGCAGCATGGAGGCCGAGGAT GCCGCCACCTACTACTGCCACCAGAGAAGCAGCTACCCCACCTTCGGCGGCGGCACCAAGCTCGAGATCAAGAGA ACTGTGGCCGCGCCGTCAGTGTTTATCTTCCTCCATCGGATGAACAGCTTAAGTCCGGCACGGCGTCTGTGGTCTGCCTGCTCAATAACTTTTACCCTAGGGAAGCTAAAGTCCAATGGAAAGTGGATAACGCCCTGCAGTCAGGAAACAGCCAGGAATCGGTTACCGAACAGGACAGCAAGGACAGCACTTACTCCTTGTCGTCGACTCTTACTCTGAGCAAGGCCGATTACGAGAAG CACAAGGTCTACGCCTGCGAGGTCACCCATCAGGGACTCTCGTCCCCGGTGACCAAATCCTTCAATAGAGGCGAATGC 185 nt S-SL048-11HC CTCAAGTTCAGCTCGTGCAGAGCGGTGCTGAAGTGAAGAAGCCCGGTGCCTCTGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGAGACAAGCTCCCGGTCAAGGTTTAGAGTGGATGGGCGTGATCTACCCCGGTGATGGTGCTACCAGCTACGCCCAGAAGTTCAAGGGTCGTGTGACCATGACCAGAGACACCAGCACCAGCACCGTG TACATGGAGCTGAGCTCTTTAAGGAGCGAGGACACCGCCGTGTACTACTGCGCTCGTGACTACTACGGCAGCAGCCCTTTAGGCTATTGGGGACAA GGTACTTTAGTGACCGTCTCGAGCGCCTCAACCAAAGGACCCTCCGTGTTTCCCCTCGCCCCCTGTTCCCGCTCCACATCCGAGTCAACCGCGGCGCTGGGCTGCCTCGTGAAGGACTACTTCCCTGAGCCCGTCACTGTGTCGTGGAACTCCGGGGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTTCAATCCTCCGGACTGTACTCCCTGTCCTCGGTGGTCACCGTGCC GTCGAGCTCGTTGGGAACCAAGACTTACACTTGCAACGTGGACCACAAGCCAAGCAACACCAAAGTGGACAAGAGAGTCGAATCTAAGTACGGACC GCCCTGCCCGCCTTGCCCCGCCCCTGAGTTTCTCGGCGGTCCTAGCGTGTTCCTGTTCCCACCCAAGCCCAAGGACACTCTGATGATCTCCCGGACCCCTGAAGTGACCTGTGTGGTCGTGGACGTGTCGCAGGAAGATCCGGAGGTCCAGTTCAATTGGTACGTGGATGGGGTGGAGGTCCACAACGCCAAGACGAAGCCGAGAGAAGAACAGTTCAACTCAACTTACCGGGTG GTGTCCGTGCTGACCGTGCTGCATCAGGATTGGCTCAACGGAAAGGAGTACAAGTGCAAAGTGTCCAACAAGGGCCTGCCTAGCTCAATCGAAAAGACCA TTTCCAAGGCCAAGGGCCAGCCGAGGGAACCACAGGTCTATACTCTGCCACCGAGCCAAGAAGAGATGACCAAGAACCAAGTGTCCCTGACTTGCCTGGTCAAGGGGTTCTACCCGTCGGACATCGCAGTGGAGTGGGAGAGCAACGGACAGCCTGAAAACAATTACAAGACCACCCCGCCCGTGCTGGATAGCGACGGTTCCTTCTTCCTTTACTCGCGCCTCACCGTCGACAAGAGCC GGTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAAGCTCTGCATAACCACTACACTCAGAAGTCCTTGTCGCTGAGCCTCGGAAAG 186 nt S-SL048-11 LC GACATCCAGATGACCCAGAGCCCTAGCTCTTTAAGCGCCTCTGTGGGCGATCGTGTGACCATCACTTGTAGCGCCAGCCCCAGCATCAGCTACATGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCACAAGCTCTTTAGCCAGCGGCGTGCCTAGCAGATTTAGCGGCAGCGGTAGCGGCACAGACTTCACTTTAACCATCAGCTCTTTACAGCCAGAAGACTTC GCCACCTACTACTGCCACCAGAGGAGCAGCTACCCCACCTTCGGCCAAGGTACCAAGCTCGAGATCAAGAGA ACTGTGGCCGCGCCGTCAGTGTTTATCTTCCTCCATCGGATGAACAGCTTAAGTCCGGCACGGCGTCTGTGGTCTGCCTGCTCAATAACTTTTACCCTAGGGAAGCTAAAGTCCAATGGAAAGTGGATAACGCCCTGCAGTCAGGAAACAGCCAGGAATCGGTTACCGAACAGGACAGCAAGGACAGCACTTACTCCTTGTCGTCGACTCTTACTCTGAGCAAGGCCGATTACGAGAAG CACAAGGTCTACGCCTGCGAGGTCACCCATCAGGGACTCTCGTCCCCGGTGACCAAATCCTTCAATAGAGGCGAATGC 187 nt S-SL048-46HC CAAGTTCAGCTCGTGCAGAGCGGTGCTGAAGTGAAGAAGCCCGGTGCCTCTGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGAGACAAGCTCCCGGTCAAGGTTTAGAGTGGATGGGCGTGATCAACCCCCGGTGATGGTGCTACCAGCTACAACCAGAAGTTCCAGGGTCGTGTGACCATGACCAGAGACACCAGCACCAGCACCGTGTACAT GGAGCTGAGCTCTTTAAGGAGCGAGGACACCGCCGTGTACTACTGCGCTCGTGACTACTACGGCAGCAGCCCTTTAGGCTATTGGGGACAAG GTACTTTAGTGACCGTCTCGAGCGCCTCAACCAAAGGACCCTCCGTGTTTCCCCTCGCCCCCTGTTCCCGCTCCACATCCGAGTCAACCGCGGCGCTGGGCTGCCTCGTGAAGGACTACTTCCCTGAGCCCGTCACTGTGTCGTGGAACTCCGGGGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTTCAATCCTCCGGACTGTACTCCCTGTCCTCGGTGGTCACCGTGCCG TCGAGCTCGTTGGGAACCAAGACTTACACTTGCAACGTGGACCACAAGCCAAGCAACACCAAAGTGGACAAGAGAGTCGAATCTAAGTACGGACCG CCCTGCCCGCCTTGCCCCGCCCCTGAGTTTCTCGGCGGTCCTAGCGTGTTCCTGTTCCCACCCAAGCCCAAGGACACTCTGATGATCTCCCGGACCCCTGAAGTGACCTGTGTGGTCGTGGACGTGTCGCAGGAAGATCCGGAGGTCCAGTTCAATTGGTACGTGGATGGGGTGGAGGTCCACAACGCCAAGACGAAGCCGAGAGAAGAACAGTTCAACTCAACTTACCGGGTGG TGTCCGTGCTGACCGTGCTGCATCAGGATTGGCTCAACGGAAAGGAGTACAAGTGCAAAGTGTCCAACAAGGGCCTGCCTAGCTCAATCGAAAAGACCA TTTCCAAGGCCAAGGGCCAGCCGAGGGAACCACAGGTCTATACTCTGCCACCGAGCCAAGAAGAGATGACCAAGAACCAAGTGTCCCTGACTTGCCTGGTCAAGGGGTTCTACCCGTCGGACATCGCAGTGGAGTGGGAGAGCAACGGACAGCCTGAAAACAATTACAAGACCACCCCGCCCGTGCTGGATAGCGACGGTTCCTTCTTCCTTTACTCGCGCCTCACCGTCGACAAGAGCC GGTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAAGCTCTGCATAACCACTACACTCAGAAGTCCTTGTCGCTGAGCCTCGGAAAG 188 nt S-SL048-46 LC GACATCCAGATGACCCAGAGCCCTAGCTCTTTAAGCGCCTCTGTGGGCGATCGTGTGACCATCACTTGTCGTGCCAGCAGCAGCATCAGCTATTTAAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCTCTTCTTTAGCCTCTGGCGTGCCTTCTCGTTTCAGCGGAAGCGGCAGCGGCACCGACTTCACTTTAACCATCAGCTCTTTACAGCCAGAAGACTTC GCCACCTACTACTGCCACCAGAGGAGCAGCAGCCCCACCTTCGGACAAGGTACCAAGCTCGAGATCAAGAGA ACTGTGGCCGCGCCGTCAGTGTTTATCTTCCTCCATCGGATGAACAGCTTAAGTCCGGCACGGCGTCTGTGGTCTGCCTGCTCAATAACTTTTACCCTAGGGAAGCTAAAGTCCAATGGAAAGTGGATAACGCCCTGCAGTCAGGAAACAGCCAGGAATCGGTTACCGAACAGGACAGCAAGGACAGCACTTACTCCTTGTCGTCGACTCTTACTCTGAGCAAGGCCGATTACGAGAAG CACAAGGTCTACGCCTGCGAGGTCACCCATCAGGGACTCTCGTCCCCGGTGACCAAATCCTTCAATAGAGGCGAATGC 189 nt S-SL048-106HC CAAGTTCAGCTGGTGCAGAGCGGAGCCGAGGTGAAAAAGCCCGGTGCCTCTGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTTACCAGCTACAACATGCACTGGGTGAGGCAAGCTCCCGGTCAAGGTCTGGAGTGGATCGGCGTGATCTACCCCGGCGACGGCAGCACCTCTTACAACCAGAAGTTCCAAGGTCGTGTGACCATGACTCGTGACACCAGCACCAGCACCGTGTACATGGAG CTGAGCTCTTTAAGGAGCGAGGATACCGCCGTGTACTACTGCGCTCGTGACTACTACGGCAGCAGCCCTCTGGGCTATTGGGGCCAAG GTACTTTAGTGACCGTCTCGAGCGCCTCAACCAAAGGACCCTCCGTGTTTCCCCTCGCCCCCTGTTCCCGCTCCACATCCGAGTCAACCGCGGCGCTGGGCTGCCTCGTGAAGGACTACTTCCCTGAGCCCGTCACTGTGTCGTGGAACTCCGGGGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTTCAATCCTCCGGACTGTACTCCCTGTCCTCGGTGGTCACCGTGCCG TCGAGCTCGTTGGGAACCAAGACTTACACTTGCAACGTGGACCACAAGCCAAGCAACACCAAAGTGGACAAGAGAGTCGAATCTAAGTACGGACCG CCCTGCCCGCCTTGCCCCGCCCCTGAGTTTCTCGGCGGTCCTAGCGTGTTCCTGTTCCCACCCAAGCCCAAGGACACTCTGATGATCTCCCGGACCCCTGAAGTGACCTGTGTGGTCGTGGACGTGTCGCAGGAAGATCCGGAGGTCCAGTTCAATTGGTACGTGGATGGGGTGGAGGTCCACAACGCCAAGACGAAGCCGAGAGAAGAACAGTTCAACTCAACTTACCGGGTGG TGTCCGTGCTGACCGTGCTGCATCAGGATTGGCTCAACGGAAAGGAGTACAAGTGCAAAGTGTCCAACAAGGGCCTGCCTAGCTCAATCGAAAAGACCA TTTCCAAGGCCAAGGGCCAGCCGAGGGAACCACAGGTCTATACTCTGCCACCGAGCCAAGAAGAGATGACCAAGAACCAAGTGTCCCTGACTTGCCTGGTCAAGGGGTTCTACCCGTCGGACATCGCAGTGGAGTGGGAGAGCAACGGACAGCCTGAAAACAATTACAAGACCACCCCGCCCGTGCTGGATAGCGACGGTTCCTTCTTCCTTTACTCGCGCCTCACCGTCGACAAGAGCC GGTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAAGCTCTGCATAACCACTACACTCAGAAGTCCTTGTCGCTGAGCCTCGGAAAG 190 nt S-SL048-106 LC GACATCCAGATGACCCAGAGCCCTAGCTCTTTAAGCGCCTCTGTGGGCGATCGTGTGACCATCACTTGTAGCGCCAGCAGCAGCATCAGCTACATGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCACCAGCAAGCTGCCTAGCGGCGTGCCCTCCAGATTTTCTGGCAGCGGCTCTGGCACCGACTTTACTTTAACCATCAGCTCTTTACAGCCAGAAGACTTCGCC ACCTACTACTGCCACCAGAGGAGCAGCACCCCTACCTTCGGCCAAGGTACCAAGCTCGAGATCAAGAGA ACTGTGGCCGCGCCGTCAGTGTTTATCTTCCTCCATCGGATGAACAGCTTAAGTCCGGCACGGCGTCTGTGGTCTGCCTGCTCAATAACTTTTACCCTAGGGAAGCTAAAGTCCAATGGAAAGTGGATAACGCCCTGCAGTCAGGAAACAGCCAGGAATCGGTTACCGAACAGGACAGCAAGGACAGCACTTACTCCTTGTCGTCGACTCTTACTCTGAGCAAGGCCGATTACGAGAAG CACAAGGTCTACGCCTGCGAGGTCACCCATCAGGGACTCTCGTCCCCGGTGACCAAATCCTTCAATAGAGGCGAATGC 191 nt S-SL048-116 and 118 HC CAAGTTCAGCTCGTGCAGAGCGGTGCTGAAGTGAAGAAGCCCGGTGCCTCTGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGAGACAAGCTCCCGGTCAAGGTTTAGAGTGGATGGGCGTGATCAACCCCCGGTGATGGTGCTACCAGCTACGCCCAGAAGTTCAAGGGTCGTGTGACCATGACCAGAGACACCAGCACCAGCACCGTGTACAT GGAGCTGAGCTCTTTAAGGAGCGAGGACACCGCCGTGTACTACTGCGCTCGTGACTACTACGGCAGCAGCCCTTTAGGCTATTGGGGACAAG GTACTTTAGTGACCGTCTCGAGCGCCTCAACCAAAGGACCCTCCGTGTTTCCCCTCGCCCCCTGTTCCCGCTCCACATCCGAGTCAACCGCGGCGCTGGGCTGCCTCGTGAAGGACTACTTCCCTGAGCCCGTCACTGTGTCGTGGAACTCCGGGGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTTCAATCCTCCGGACTGTACTCCCTGTCCTCGGTGGTCACCGTGCCG TCGAGCTCGTTGGGAACCAAGACTTACACTTGCAACGTGGACCACAAGCCAAGCAACACCAAAGTGGACAAGAGAGTCGAATCTAAGTACGGACCG CCCTGCCCGCCTTGCCCCGCCCCTGAGTTTCTCGGCGGTCCTAGCGTGTTCCTGTTCCCACCCAAGCCCAAGGACACTCTGATGATCTCCCGGACCCCTGAAGTGACCTGTGTGGTCGTGGACGTGTCGCAGGAAGATCCGGAGGTCCAGTTCAATTGGTACGTGGATGGGGTGGAGGTCCACAACGCCAAGACGAAGCCGAGAGAAGAACAGTTCAACTCAACTTACCGGGTGG TGTCCGTGCTGACCGTGCTGCATCAGGATTGGCTCAACGGAAAGGAGTACAAGTGCAAAGTGTCCAACAAGGGCCTGCCTAGCTCAATCGAAAAGACCA TTTCCAAGGCCAAGGGCCAGCCGAGGGAACCACAGGTCTATACTCTGCCACCGAGCCAAGAAGAGATGACCAAGAACCAAGTGTCCCTGACTTGCCTGGTCAAGGGGTTCTACCCGTCGGACATCGCAGTGGAGTGGGAGAGCAACGGACAGCCTGAAAACAATTACAAGACCACCCCGCCCGTGCTGGATAGCGACGGTTCCTTCTTCCTTTACTCGCGCCTCACCGTCGACAAGAGCC GGTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAAGCTCTGCATAACCACTACACTCAGAAGTCCTTGTCGCTGAGCCTCGGAAAG 192 nt S-SL048-116 LC GACATCCAGATGACCCAGAGCCCTAGCTCTTTAAGCGCCAGCGTGGGAGATCGTGTGACCATCACTTGTCGTGCCAGCCCCAGCATCAGCTACATGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCACCAGCTCTTTAGCCTCTGGCGTGCCTAGCAGATTCAGCGGCAGCGGAAGCGGCACCGACTTCACTTTAACCATCAGCTCTTTACAGCCAGAAGACTTCG CCACCTACTACTGCCACCAGAGGAGCAGCAGCAGCCCTACCTTCGGCCAAGGTACCAAGCTCGAGATCAAGAGA ACTGTGGCCGCGCCGTCAGTGTTTATCTTCCTCCATCGGATGAACAGCTTAAGTCCGGCACGGCGTCTGTGGTCTGCCTGCTCAATAACTTTTACCCTAGGGAAGCTAAAGTCCAATGGAAAGTGGATAACGCCCTGCAGTCAGGAAACAGCCAGGAATCGGTTACCGAACAGGACAGCAAGGACAGCACTTACTCCTTGTCGTCGACTCTTACTCTGAGCAAGGCCGATTACGAGAAG CACAAGGTCTACGCCTGCGAGGTCACCCATCAGGGACTCTCGTCCCCGGTGACCAAATCCTTCAATAGAGGCGAATGC 193 nt S-SL048-118 LC GACATCCAGATGACCCAGAGCCCTAGCTCTTTAAGCGCCTCTGTGGGCGATCGTGTGACCATCACTTGTAGCGCCAGCCCCAGCATCAGCTACATGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCACAAGCTCTTTACCCAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGAAGCGGCACCGACTTCACTTTAACCATCAGCTCTTTACAGCCAGAAGACTTCGCC ACCTACTACTGCCACCAGGAAGCAGCAGCCCCACCTTCGGCCAAGGTACAAAGCTCGAGATCAAGAGA ACTGTGGCCGCGCCGTCAGTGTTTATCTTCCTCCATCGGATGAACAGCTTAAGTCCGGCACGGCGTCTGTGGTCTGCCTGCTCAATAACTTTTACCCTAGGGAAGCTAAAGTCCAATGGAAAGTGGATAACGCCCTGCAGTCAGGAAACAGCCAGGAATCGGTTACCGAACAGGACAGCAAGGACAGCACTTACTCCTTGTCGTCGACTCTTACTCTGAGCAAGGCCGATTACGAGAAG CACAAGGTCTACGCCTGCGAGGTCACCCATCAGGGACTCTCGTCCCCGGTGACCAAATCCTTCAATAGAGGCGAATGC 194 nt CDR graft HC CAAGTTCAGCTGGTGCAGAGCGGTGCTGAGGTGAAAAAACCCGGTGCTTCCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTTACCAGCTACAACATGCACTGGGTGAGACAAGCCCCCGGTCAAGGTTTAGAGTGGATCGGCGTGATCTACCCCGGCAACGGCGACACCTCTTACAACCAGAAGTTCAAGGGTCGTGTGACCATGACTCGTGACACCTCCACCAGCACCGTGTACATGG AGCTGAGCTCTTTAAGGAGCGAGGACACAGCCGTGTACTACTGCGCTCGTGACTACTACGGCAGCAGCCCTCTGGGCTATTGGGGCCAAG GTACTTTAGTGACCGTCTCGAGCGCCTCAACCAAAGGACCCTCCGTGTTTCCCCTCGCCCCCTGTTCCCGCTCCACATCCGAGTCAACCGCGGCGCTGGGCTGCCTCGTGAAGGACTACTTCCCTGAGCCCGTCACTGTGTCGTGGAACTCCGGGGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTTCAATCCTCCGGACTGTACTCCCTGTCCTCGGTGGTCACCGTGCCG TCGAGCTCGTTGGGAACCAAGACTTACACTTGCAACGTGGACCACAAGCCAAGCAACACCAAAGTGGACAAGAGAGTCGAATCTAAGTACGGACCG CCCTGCCCGCCTTGCCCCGCCCCTGAGTTTCTCGGCGGTCCTAGCGTGTTCCTGTTCCCACCCAAGCCCAAGGACACTCTGATGATCTCCCGGACCCCTGAAGTGACCTGTGTGGTCGTGGACGTGTCGCAGGAAGATCCGGAGGTCCAGTTCAATTGGTACGTGGATGGGGTGGAGGTCCACAACGCCAAGACGAAGCCGAGAGAAGAACAGTTCAACTCAACTTACCGGGTGG TGTCCGTGCTGACCGTGCTGCATCAGGATTGGCTCAACGGAAAGGAGTACAAGTGCAAAGTGTCCAACAAGGGCCTGCCTAGCTCAATCGAAAAGACCA TTTCCAAGGCCAAGGGCCAGCCGAGGGAACCACAGGTCTATACTCTGCCACCGAGCCAAGAAGAGATGACCAAGAACCAAGTGTCCCTGACTTGCCTGGTCAAGGGGTTCTACCCGTCGGACATCGCAGTGGAGTGGGAGAGCAACGGACAGCCTGAAAACAATTACAAGACCACCCCGCCCGTGCTGGATAGCGACGGTTCCTTCTTCCTTTACTCGCGCCTCACCGTCGACAAGAGCC GGTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAAGCTCTGCATAACCACTACACTCAGAAGTCCTTGTCGCTGAGCCTCGGAAAG 195 nt CDR graft LC GACATCCAGATGACCCAGAGCCCTAGCTCTTTAAGCGCCTCTGTGGGCGATCGTGTGACCATCACTTGTAGCGCCAGCAGCAGCATCAGCTACATGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACACCAGCAAGCTGGCCAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGAAGCGGCACCGACTTCACTTTAACCATCAGCTCTTTACAGCCAGAAGACTTCG CCACCTACTACTGCCACCAGAGGAGCAGCAGCTACCCCACCTTCGGCCAAGGTACCAAGCTCGAGATCAAGAGA ACTGTGGCCGCGCCGTCAGTGTTTATCTTCCTCCATCGGATGAACAGCTTAAGTCCGGCACGGCGTCTGTGGTCTGCCTGCTCAATAACTTTTACCCTAGGGAAGCTAAAGTCCAATGGAAAGTGGATAACGCCCTGCAGTCAGGAAACAGCCAGGAATCGGTTACCGAACAGGACAGCAAGGACAGCACTTACTCCTTGTCGTCGACTCTTACTCTGAGCAAGGCCGATTACGAGAAG CACAAGGTCTACGCCTGCGAGGTCACCCATCAGGGACTCTCGTCCCCGGTGACCAAATCCTTCAATAGAGGCGAATGC 196 nt S-SL048-11 HC codon-optimized CAAGTGCAGCTCGTGCAGTCCGGAGCCGAAGTCAAGAAGCCCGGAGCGTCAGTGAAAGTGTCCTGCAAGGCCTCGGGCTACACTTTCACAAGCTACAACATGCACTGGGTCAGACAGGCACCTGGGCAGGGTCTGGAGTGGATGGGAGTGATCTACCCGGGCGACGGCGCCACTTCCTACGCCCAAAAGTTCAAGGGCCGCGTGACCATGACTAGGGACACCTCGACCTCAACCGTGTACAT GGAACTGAGCTCCCTGCGGTCCGAGGATACCGCCGTGTACTATATTGTGCTCGGGACTACTACGGGTCCAGCCCACTGGGATACTGGGGACAGG GTACCCTTGTCACGGTGTCGTCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACGAAGACCTACACCTGCAATGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAATTGGTCCC CCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTG GCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA 197 nt S-SL048-11 LC codon-optimized GACATCCAAATGACTCAGTCCCCGTCATCCCTGTCGGCATCCGTGGGAGACAGAGTCACCATTACGTGCAGCGCGAGCCCGAGCATTTCCTATATGAACTGGTACCAGCAGAAGCCCGGGAAAGCCCCTAAGCTGTTGATCTACGCCACTTCCTCACTGGCTTCCGGCGTGCCATCCCGGTTCTCGGGTTCCGGCTCGGGAACCGATTTTACCCTTACTATCTCGTCCCTGCAACCCGAGGACTTC GCCACCTACTACTGTCACCAGCGCTCTAGCTACCCTACATTCGGACAGGGCACCAAGCTCGAAATCAAACGA ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 198 nt S-SL048-106 HC codon-optimized CAAGTGCAGCTCGTGCAGTCCGGAGCCGAAGTCAAGAAGCCCGGAGCGTCAGTGAAAGTGTCCTGCAAGGCCTCGGGCTACACTTTCACAAGCTACAACATGCACTGGGTCAGACAGGCACCTGGGCAGGGTCTGGAGTGGATTGGAGTGATCTACCCGGGCGACGGCTCCACTTCCTACAACCAAAAGTTCCAGGGCCGCGTGACCATGACTAGGGACACCTCGACCTCAACCGTGTACAT GGAACTGAGCTCCCTGCGGTCCGAGGATACCGCCGTGTACTATATTGTGCTCGGGACTACTACGGGTCCAGCCCACTGGGATACTGGGGACAGG GTACCCTTGTCACGGTGTCGTCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACGAAGACCTACACCTGCAATGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAATTGGTCCC CCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTG GCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA 199 nt S-SL048-106 LC codon-optimized GACATCCAAATGACTCAGTCCCCGTCATCCCTGTCGGCATCCGTGGGAGACAGAGTCACCATTACGTGCAGCGCGAGCTCAAGCATTTCCTATATGAACTGGTACCAGCAGAAGCCCGGGAAAGCCCCTAAGCTGTTGATCTACGCCACTTCCAAGCTGCCGTCCGGCGTGCCATCCCGGTTCTCGGGTTCCGGCTCGGGAACCGATTTTACCCTTACTATCTCGTCCCTGCAACCCGAGGACTTC GCCACCTACTACTGTCACCAGCGCTCTAGCACCCCTACATTCGGACAGGGCACCAAGCTCGAAATCAAACGA ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 200 nt S-SL048-116 and 118 HC codon-optimized CAAGTGCAGCTCGTGCAGTCCGGAGCCGAAGTCAAGAAGCCCGGAGCGTCAGTGAAAGTGTCCTGCAAGGCCTCGGGCTACACTTTCACAAGCTACAACATGCACTGGGTCAGACAGGCACCTGGGCAGGGTCTGGAGTGGATGGGAGTGATCAACCCGGGCGACGGCGCCACTTCCTACGCCCAAAAGTTCAAGGGCCGCGTGACCATGACTAGGGACACCTCGACCTCAACCGTGTACAT GGAACTGAGCTCCCTGCGGTCCGAGGATACCGCCGTGTACTATATTGTGCTCGGGACTACTACGGGTCCAGCCCACTGGGATACTGGGGACAGG GTACCCTTGTCACGGTGTCGTCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACGAAGACCTACACCTGCAATGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAATTGGTCCC CCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTG GCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA 201 nt S-SL048-116 LC codon-optimized GACATCCAAATGACTCAGTCCCCGTCATCCCTGTCGGCATCCGTGGGAGACAGAGTCACCATTACGTGCCGCGCGAGCCCGAGCATTTCCTATATGAACTGGTACCAGCAGAAGCCCGGGAAAGCCCCTAAGCTGTTGATCTACGCCACTTCCTCACTGGCTTCCGGCGTGCCATCCCGGTTCTCGGGTTCCGGCTCGGGAACCGATTTTACCCTTACTATCTCGTCCCTGCAACCCGAGGACTTC GCCACCTACTACTGTCACCAGCGCCTCTAGCAGCCCTACATTCGGACAGGGCACCAAGCTCGAAATCAAACGA ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 202 nt S-SL048-118 LC codon-optimized GACATCCAAATGACTCAGTCCCCGTCATCCCTGTCGGCATCCGTGGGAGACAGAGTCACCATTACGTGCAGCGCGAGCCCGAGCATTTCCTATATGAACTGGTACCAGCAGAAGCCCGGGAAAGCCCCTAAGCTGTTGATCTACGCCACTTCCTCACTGCCGTCCGGCGTGCCATCCCGGTTCTCGGGTTCCGGCTCGGGAACCGATTTTACCCTTACTATCTCGTCCCTGCAACCCGAGGACTTC GCCACCTACTACTGTCACCAGCGCCTCTAGCAGCCCTACATTCGGACAGGGCACCAAGCTCGAAATCAAACGA ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

LINKS

1 European Journal of Biochemistry (1981) 116(2): 221-225

2 US Patent Application No. 2010/0124555

3 U.S. Patent Application No. 8,597,650

4 U.S. Patent Application No. 9,168,299

5 Developmental and Comparative Immunology (2003) 27(1): 55-77

6 Nucleic Acids Research (1994) 22:4673-4680

7 Protein Engineering, Design and Selection (2016) 29(10): 457-466

8 The Journal of Biological Chemistry (2015) 290(9): 5462–5469

9 Journal of Molecular Biology (2001) 312:511-523

10 MAbs (2013) 5(3): 445-470

11 Journal of Molecular Biology (2007) 373:924–940

12 Protein Engineering, Design and Selection (2016) 29: 427–437

13 Molecular Immunoogy.(1993) 30(1): 105–1058

14 PNAS (2001) 98(3): 944–949

15 The Journal of Immunology (2012) 189(7): 3430-3438

16 The Journal of Biological Chemistry (2017) 292(9): 3900-3908

17 Protein Science (2000) 9:1783–1790.

--->

SEQUENCE LIST

<110> Lipum AB

<120> NEW ANTIBODIES TO BSSL

<130>HSJ106660P.WOP

<150> SE 1950888-6

<151> 2019-07-12

<160> 202

<170> PatentIn version 3.5

<210> 1

<211> 6

<212> PROTEIN

<213> Homo sapiens

<400> 1

Tyr Thr Glu Gly Gly Phe

1 5

<210> 2

<211> 14

<212> PROTEIN

<213> Homo sapiens

<400> 2

Leu Glu Asn Pro Gln Pro His Pro Gly Trp Gln Gly Thr Leu

1 5 10

<210> 3

<211> 12

<212> PROTEIN

<213> Homo sapiens

<400> 3

Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe

1 5 10

<210> 4

<211> 18

<212> PROTEIN

<213> Homo sapiens

<400> 4

Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg Asp Leu Pro

1 5 10 15

Val Met

<210> 5

<211> 7

<212> PROTEIN

<213> Homo sapiens

<400> 5

Val Lys Arg Asn Ile Ala Ala

1 5

<210> 6

<211> 13

<212> PROTEIN

<213> Homo sapiens

<400> 6

His Tyr Val Gly Phe Val Pro Val Ile Asp Gly Asp Phe

1 5 10

<210> 7

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223>HCDR1

<400> 7

Gly Tyr Thr Phe Thr Ser Tyr Asn

1 5

<210> 8

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223>HCDR2

<400> 8

Ile Asn Pro Gly Asp Gly Ala Thr

1 5

<210> 9

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223>HCDR3

<400> 9

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr

1 5 10

<210> 10

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR1

<400> 10

Pro Ser Ile Ser Tyr

1 5

<210> 11

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR3

<400> 11

His Gln Arg Ser Ser Ser Pro Thr

1 5

<210> 12

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended HCDR2

<400> 12

Met Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys

1 5 10 15

Phe Lys

<210> 13

<211> 9

<212> PROTEIN

<213> Homo sapiens

<400> 13

Ala Trp Val Lys Arg Asn Ile Ala Ala

1 5

<210> 14

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended LCDR1

<400> 14

Arg Ala Ser Pro Ser Ile Ser Tyr Met Asn

1 5 10

<210> 15

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended LCDR2

<400> 15

Ala Thr Ser Ser Leu Ala

1 5

<210> 16

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended LCDR1

<400> 16

Ser Ala Ser Pro Ser Ile Ser Tyr Met Asn

1 5 10

<210> 17

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended LCDR2

<400> 17

Ala Thr Ser Ser Leu Pro

1 5

<210> 18

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223>HCDR2

<400> 18

Ile Tyr Pro Gly Asp Gly Ala Thr

1 5

<210> 19

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223>HCDR2

<400> 19

Ile Tyr Pro Gly Asp Gly Ser Thr

1 5

<210> 20

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR1

<400> 20

Ser Ser Ile Ser Tyr

1 5

<210> 21

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR3

<400> 21

His Gln Arg Ser Ser Tyr Pro Thr

1 5

<210> 22

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LCDR3

<400> 22

His Gln Arg Ser Ser Thr Pro Thr

1 5

<210> 23

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended HCDR2

<400> 23

Met Gly Val Ile Tyr Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys

1 5 10 15

Phe Lys

<210> 24

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended HCDR2

<400> 24

Met Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Asn Gln Lys

1 5 10 15

Phe Gln

<210> 25

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended HCDR2

<400> 25

Ile Gly Val Ile Tyr Pro Gly Asp Gly Ser Thr Ser Tyr Asn Gln Lys

1 5 10 15

Phe Gln

<210> 26

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended LCDR1

<400> 26

Ser Ala Ser Ser Ser Ile Ser Tyr Met Asn

1 5 10

<210> 27

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended LCDR1

<400> 27

Arg Ala Ser Ser Ser Ile Ser Tyr Leu Asn

1 5 10

<210> 28

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended LCDR2

<400> 28

Ala Thr Ser Lys Leu Pro

1 5

<210> 29

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended LCDR2

<400> 29

Ala Ala Ser Ser Leu Ala

1 5

<210> 30

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HVCR

<400> 30

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Tyr Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 31

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223>LVCR

<400> 31

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Pro Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 32

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HVCR

<400> 32

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 33

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223>LCVR

<400> 33

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 34

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HVCR

<400> 34

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Tyr Pro Gly Asp Gly Ser Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 35

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223>LCVR

<400> 35

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Lys Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Thr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 36

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HVCR

<400> 36

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 37

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223>LCVR

<400> 37

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 38

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223>LCVR

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Pro Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 39

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> ZH1

<400> 39

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210> 40

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223>ZH2

<400> 40

Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

1 5 10

<210> 41

<211> 31

<212> PROTEIN

<213> Artificial sequence

<220>

<223>ZH3

<400> 41

Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met

1 5 10 15

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

20 25 30

<210> 42

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223>ZH4

<400> 42

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 43

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> ZL1

<400> 43

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys

20

<210> 44

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> ZL2

<400> 44

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210> 45

<211> 33

<212> PROTEIN

<213> Artificial sequence

<220>

<223> ZL3

<400> 45

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

1 5 10 15

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

20 25 30

Cys

<210> 46

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> ZL4

<400> 46

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 47

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Tyr Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 48

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 48

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Ala Thr Ser Tyr Thr Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 49

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 49

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Ala Thr Ser Tyr Thr Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 50

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 50

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 51

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 51

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Tyr Thr Ser Tyr Thr Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 52

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 52

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Ala Thr Ser Tyr Thr Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 53

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 53

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 54

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 54

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 55

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 55

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Tyr Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 56

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 56

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Thr Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 57

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 57

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Ala Thr Ser Tyr Thr Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 58

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 58

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Gly Gly Tyr Thr Ser Tyr Thr Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 59

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 59

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 60

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 60

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 61

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 61

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 62

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 62

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 63

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 63

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Tyr Pro Gly Gly Gly Tyr Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 64

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 64

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 65

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 65

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 66

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 66

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 67

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 67

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Ser Gly Ser Thr Ser Tyr Thr Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 68

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 68

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 69

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 69

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Asp Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 70

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 70

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 71

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 71

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 72

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 72

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 73

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 73

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 74

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 74

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 75

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 75

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Gly Ala Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 76

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 76

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 77

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 77

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 78

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 78

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 79

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 79

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 80

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH

<400> 80

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 81

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 81

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Arg Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 82

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 82

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Thr Ser Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 83

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 83

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Pro Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 84

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 84

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Arg Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 85

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 85

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Lys Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 86

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 86

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Thr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 87

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 87

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 88

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 88

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Arg Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 89

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 89

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Arg Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 90

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 90

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Ser Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 91

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 91

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Tyr Leu

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Thr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 92

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 92

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Pro Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Thr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 93

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 93

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 94

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 94

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Ala Ser Ser Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 95

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 95

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Thr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 96

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 96

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 97

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 97

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 98

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 98

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Arg Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 99

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 99

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 100

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 100

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Tyr Leu

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 101

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 101

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Arg Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Asn Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 102

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 102

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 103

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 103

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Leu

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Thr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 104

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 104

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Ser Ile Ser Tyr Leu

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Lys Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 105

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 105

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 106

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 106

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Pro Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 107

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 107

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 108

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 108

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Ser Ile Ser Tyr Leu

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Thr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 109

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 109

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Thr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 110

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 110

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Lys Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 111

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 111

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Tyr Leu

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Arg Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 112

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 112

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 113

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 113

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Lys Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 114

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL

<400> 114

Gln Ile Val Leu Thr Gln Ser Pro Ala Val Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 115

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IgG1 LALA-PG HC

<400> 115

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 116

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG1 LALA-PG LC

<400> 116

Gln Ile Val Leu Thr Gln Ser Pro Ala Val Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

ArgGlyGluCys

210

<210> 117

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG1 LALA-PG HC

<400> 117

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Tyr Tyr Gly Ser Ser Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 118

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG1 LALA-PG LC

<400> 118

Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn

85 90 95

His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys

210

<210> 119

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P HC

<400> 119

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Tyr Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 120

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P LC

<400> 120

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Pro Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

ArgGlyGluCys

210

<210> 121

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P HC

<400> 121

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 122

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P LC

<400> 122

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Ile Ser Tyr Leu

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Ala Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

ArgGlyGluCys

210

<210> 123

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P HC

<400> 123

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Tyr Pro Gly Asp Gly Ser Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 124

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P LC

<400> 124

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Lys Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Thr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

ArgGlyGluCys

210

<210> 125

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P HC

<400> 125

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 126

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P LC

<400> 126

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

ArgGlyGluCys

210

<210> 127

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P HC

<400> 127

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Gly Asp Gly Ala Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 128

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P LC

<400> 128

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Pro Ser Ile Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Ser Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Ser Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

ArgGlyGluCys

210

<210> 129

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P HC

<400> 129

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 130

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P LC

<400> 130

Gln Ile Val Leu Thr Gln Ser Pro Ala Val Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

ArgGlyGluCys

210

<210> 131

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P HC

<400> 131

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 132

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P LC

<400> 132

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

ArgGlyGluCys

210

<210> 133

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P HC

<400> 133

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Tyr Tyr Gly Ser Ser Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 134

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<223> hIgG4 S228P LC

<400> 134

Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn

85 90 95

His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 135

<211> 138

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IgG HC

<400> 135

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Val Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135

<210> 136

<211> 127

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IgG LC

<400> 136

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Leu Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Val

20 25 30

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser

35 40 45

Ser Ser Ile Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Thr Ser

50 55 60

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

85 90 95

Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg

100 105 110

Ser Ser Tyr Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210> 137

<211> 579

<212> PROTEIN

<213> Mus musculus

<400> 137

Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val

1 5 10 15

Asn Lys Lys Leu Ser Leu Leu Gly Gly Asp Ser Val Asp Ile Phe Lys

20 25 30

Gly Ile Pro Phe Ala Thr Ala Lys Thr Leu Glu Asn Pro Gln Arg His

35 40 45

Pro Gly Trp Gln Gly Thr Leu Lys Ala Thr Asn Phe Lys Lys Arg Cys

50 55 60

Leu Gln Ala Thr Ile Thr Gln Asp Asn Thr Tyr Gly Gln Glu Asp Cys

65 70 75 80

Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser His

85 90 95

Asn Leu Pro Val Met Val Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly

100 105 110

Ser Gly Gln Gly Ala Asn Phe Leu Lys Asn Tyr Leu Tyr Asp Gly Glu

115 120 125

Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg

130 135 140

Val Gly Pro Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly

145 150 155 160

Asn Phe Gly Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg

165 170 175

Asn Ile Ala Ala Phe Gly Gly Asp Pro Asp Asn Ile Thr Ile Phe Gly

180 185 190

Glu Ser Ala Gly Ala Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr

195 200 205

Asn Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Met Ala Leu

210 215 220

Ser Pro Trp Ala Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Thr Ile

225 230 235 240

Ala Lys Lys Val Gly Cys Pro Thr Glu Asp Thr Gly Lys Met Ala Ala

245 250 255

Cys Leu Lys Ile Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Leu

260 265 270

Pro Val Lys Lys Gln Glu Tyr Pro Val Val His Tyr Leu Ala Phe Ile

275 280 285

Pro Val Ile Asp Gly Asp Phe Ile Pro Asp Asp Pro Ile Asn Leu Tyr

290 295 300

Asn Asn Thr Ala Asp Ile Asp Tyr Ile Ala Gly Ile Asn Asn Met Asp

305 310 315 320

Gly His Leu Phe Ala Thr Ile Asp Val Pro Ala Val Asp Lys Thr Lys

325 330 335

Gln Thr Val Thr Glu Glu Asp Phe Tyr Arg Leu Val Ser Gly His Thr

340 345 350

Val Ala Lys Gly Leu Lys Gly Ala Gln Ala Thr Phe Asp Ile Tyr Thr

355 360 365

Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Met Lys Lys Thr Val

370 375 380

Val Ala Phe Glu Thr Asp Val Leu Phe Leu Ile Pro Thr Glu Ile Ala

385 390 395 400

Leu Ala Gln His Lys Ala His Ala Lys Ser Ala Lys Thr Tyr Ser Tyr

405 410 415

Leu Phe Ser His Pro Ser Arg Met Pro Ile Tyr Pro Lys Trp Met Gly

420 425 430

Ala Asp His Ala Asp Asp Leu Gln Tyr Val Phe Gly Lys Pro Phe Ala

435 440 445

Thr Pro Leu Gly Tyr Arg Pro Gln Asp Arg Ala Val Ser Lys Ala Met

450 455 460

Ile Ala Tyr Trp Thr Asn Phe Ala Arg Ser Gly Asp Pro Asn Met Gly

465 470 475 480

Asn Ser Pro Val Pro Thr His Trp Tyr Pro Tyr Thr Leu Glu Asn Gly

485 490 495

Asn Tyr Leu Asp Ile Thr Lys Thr Ile Thr Ser Ala Ser Met Lys Glu

500 505 510

His Leu Arg Glu Lys Phe Leu Lys Phe Trp Ala Val Thr Phe Glu Val

515 520 525

Leu Pro Thr Val Thr Gly Asp Gln Asp Thr Leu Thr Pro Pro Glu Asp

530 535 540

Asp Ser Glu Val Ala Pro Asp Pro Pro Ser Asp Asp Ser Gln Val Val

545 550 555 560

Pro Val Pro Pro Thr Asp Asp Ser Val Glu Ala Gln Met Pro Ala Thr

565 570 575

Ile Gly Phe

<210> 138

<211> 722

<212> PROTEIN

<213> Homo sapiens

<400> 138

Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val

1 5 10 15

Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly

20 25 30

Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His

35 40 45

Pro Gly Trp Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys

50 55 60

Leu Gln Ala Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys

65 70 75 80

Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg

85 90 95

Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly

100 105 110

Ser Gly His Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu

115 120 125

Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg

130 135 140

Val Gly Pro Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly

145 150 155 160

Asn Tyr Gly Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg

165 170 175

Asn Ile Ala Ala Phe Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly

180 185 190

Glu Ser Ala Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr

195 200 205

Asn Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu

210 215 220

Ser Pro Trp Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val

225 230 235 240

Ala Glu Lys Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln

245 250 255

Cys Leu Lys Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val

260 265 270

Pro Leu Ala Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val

275 280 285

Pro Val Ile Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr

290 295 300

Ala Asn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp

305 310 315 320

Gly His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn

325 330 335

Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr

340 345 350

Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr

355 360 365

Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys Lys Thr Val

370 375 380

Val Asp Phe Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala

385 390 395 400

Leu Ala Gln His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr

405 410 415

Leu Phe Ser His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly

420 425 430

Ala Asp His Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala

435 440 445

Thr Pro Thr Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met

450 455 460

Ile Ala Tyr Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly

465 470 475 480

Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser

485 490 495

Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg

500 505 510

Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala

515 520 525

Leu Pro Thr Val Thr Asp Gln Glu Ala Thr Pro Val Pro Pro Thr Gly

530 535 540

Asp Ser Glu Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Thr Ala

545 550 555 560

Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr

565 570 575

Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala

580 585 590

Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro

595 600 605

Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly

610 615 620

Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro

625 630 635 640

Pro Thr Gly Asp Ala Gly Pro Pro Pro Val Pro Pro Thr Gly Asp Ser

645 650 655

Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val

660 665 670

Thr Pro Thr Gly Asp Ser Glu Thr Ala Pro Val Pro Pro Thr Gly Asp

675 680 685

Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Glu Ala Ala Pro

690 695 700

Val Pro Pro Thr Asp Asp Ser Lys Glu Ala Gln Met Pro Ala Val Ile

705 710 715 720

Arg Phe

<210> 139

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IgG4HC

<400> 139

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 140

<211> 212

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IgG4 LC

<400> 140

Gln Ile Val Leu Thr Gln Ser Pro Ala Val Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

145 150 155 160

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

165 170 175

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

180 185 190

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

195 200 205

ArgGlyGluCys

210

<210> 141

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended HCDR2

<400> 141

Ile Gly Val Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys

1 5 10 15

Phe Lys

<210> 142

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended LCDR1

<400> 142

Ser Ala Ser Ser Ser Ile Ser Tyr Met His

1 5 10

<210> 143

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Extended LCDR2

<400> 143

Asp Thr Ser Lys Leu Ala

1 5

<210> 144

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223>HCVR

<400> 144

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Leu Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 145

<211> 105

<212> PROTEIN

<213> Artificial sequence

<220>

<223>LCVR

<400> 145

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Thr Phe

85 90 95

Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 146

<211> 537

<212> PROTEIN

<213> Artificial sequence

<220>

<223> t-hBSSL

<400> 146

Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val

1 5 10 15

Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly

20 25 30

Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His

35 40 45

Pro Gly Trp Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys

50 55 60

Leu Gln Ala Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys

65 70 75 80

Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg

85 90 95

Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly

100 105 110

Ser Gly His Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu

115 120 125

Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg

130 135 140

Val Gly Pro Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly

145 150 155 160

Asn Tyr Gly Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg

165 170 175

Asn Ile Ala Ala Phe Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly

180 185 190

Glu Ser Ala Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr

195 200 205

Asn Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu

210 215 220

Ser Pro Trp Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val

225 230 235 240

Ala Glu Lys Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln

245 250 255

Cys Leu Lys Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val

260 265 270

Pro Leu Ala Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val

275 280 285

Pro Val Ile Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr

290 295 300

Ala Asn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp

305 310 315 320

Gly His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn

325 330 335

Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr

340 345 350

Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr

355 360 365

Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys Lys Thr Val

370 375 380

Val Asp Phe Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala

385 390 395 400

Leu Ala Gln His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr

405 410 415

Leu Phe Ser His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly

420 425 430

Ala Asp His Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala

435 440 445

Thr Pro Thr Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met

450 455 460

Ile Ala Tyr Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly

465 470 475 480

Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser

485 490 495

Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg

500 505 510

Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala

515 520 525

Leu Pro Ala His His His His His

530 535

<210> 147

<211> 5

<212> PROTEIN

<213> Homo sapiens

<400> 147

Tyr Thr Glu Gly Gly

1 5

<210> 148

<211> 7

<212> PROTEIN

<213> Homo sapiens

<400> 148

Ala Lys Leu Gly Ala Val Tyr

1 5

<210> 149

<211> 11

<212> PROTEIN

<213> Homo sapiens

<400> 149

Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly

1 5 10

<210> 150

<211> 25

<212> PROTEIN

<213> Homo sapiens

<400> 150

Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val

1 5 10 15

Asn Lys Lys Leu Gly Leu Leu Gly Asp

20 25

<210> 151

<211> 26

<212> PROTEIN

<213> Homo sapiens

<400> 151

Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val

1 5 10 15

Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser

20 25

<210> 152

<211> 28

<212> PROTEIN

<213> Homo sapiens

<400> 152

Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val

1 5 10 15

Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp

20 25

<210> 153

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> iTope leftovers

<400> 153

Val Lys Pro Gly Ala Ser Val Lys Met

1 5

<210> 154

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> iTope leftovers

<400> 154

Tyr Asn Met His Trp Val Arg Gln Ala

1 5

<210> 155

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> iTope leftovers

<400> 155

Tyr Ala Gln Lys Phe Lys Gly Arg Val

1 5

<210> 156

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> iTope leftovers

<400> 156

Trp Gly Gln Gly Thr Thr Leu Thr Val

1 5

<210> 157

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> iTope leftovers

<400> 157

Val Thr Met Thr Cys Ser Ala Ser Ser

1 5

<210> 158

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> iTope leftovers

<400> 158

Val Thr Ile Thr Cys Arg Ala Ser Ser

1 5

<210> 159

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> iTope leftovers

<400> 159

Val Thr Ile Thr Cys Ser Ala Ser Ser

1 5

<210> 160

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> iTope leftovers

<400> 160

Leu Leu Ile Tyr Ala Thr Ser Lys Leu

1 5

<210> 161

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> iTope leftovers

<400> 161

Ile Tyr Ala Thr Ser Ser Leu Ala Ser

1 5

<210> 162

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> iTope leftovers

<400> 162

Ile Tyr Ala Ala Ser Ser Leu Ala Ser

1 5

<210> 163

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> iTope leftovers

<400> 163

Phe Gly Gly Gly Thr Lys Leu Glu Ile

1 5

<210> 164

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Oligonucleotide primer

<400> 164

gatacacctt caccagctac watatgcact gggtgcg 37

<210> 165

<211> 83

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Oligonucleotide primer

<400> 165

Gly Ala Cys Ala Ala Gly Gly Gly Cys Thr Thr Gly Ala Gly Thr Gly

1 5 10 15

Gly Ala Thr Arg Gly Gly Ala Arg Thr Ala Ala Thr Cys Trp Ala Cys

20 25 30

Cys Cys Thr Arg Gly Thr Arg Arg Thr Gly Gly Thr Lys Met Cys Ala

35 40 45

Cys Ala Ala Gly Cys Thr Ala Cys Arg Met Thr Cys Ala Gly Ala Ala

50 55 60

Gly Thr Thr Cys Met Ala Gly Gly Gly Cys Cys Gly Cys Gly Thr Cys

65 70 75 80

Ala Cys Cys

<210> 166

<211> 62

<212> DNA

<213> Artificial sequence

<220>

<223> Oligonucleotide primer

<400> 166

cgtcaccatc acctgcagkg caagtymgag cattagctat wtgmattggt atcagcagaa 60

ac 62

<210> 167

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> Oligonucleotide primer

<400> 167

cctaagctcc tgatctatgm trcatccars ttgsmaagtg gggtcccatc ac 52

<210> 168

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Oligonucleotide primer

<400> 168

gattttgcaa cttattactg tcascagagk tmtagtwmty hcacttttgg ccagggg 57

<210> 169

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> HVCR part 2

<220>

<221> Other_feature

<222> (1)..(1)

<223> X=I or M

<220>

<221> Other_feature

<222> (5)..(5)

<223> X=N or Y

<220>

<221> Other_feature

<222> (10)..(10)

<223> X=A or S

<220>

<221> Other_feature

<222> (14)..(14)

<223> X=A or N

<220>

<221> Other_feature

<222> (18)..(18)

<223> X=K or Q

<400> 169

Xaa Gly Val Ile Xaa Pro Gly Asp Gly Xaa Thr Ser Tyr Xaa Gln Lys

1 5 10 15

Phe Xaa

<210> 170

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LVCR part 1

<220>

<221> Other_feature

<222> (1)..(1)

<223> X=S or R

<220>

<221> Other_feature

<222> (4)..(4)

<223> X=S or P

<220>

<221> Other_feature

<222> (9)..(9)

<223> X=M or L

<400> 170

Xaa Ala Ser Xaa Ser Ile Ser Tyr Xaa Asn

1 5 10

<210> 171

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LVCR part 2

<220>

<221> Other_feature

<222> (2)..(2)

<223> X=A or T

<220>

<221> Other_feature

<222> (4)..(4)

<223> X=K or S

<220>

<221> Other_feature

<222> (6)..(6)

<223> X=A or P

<400> 171

Ala Xaa Ser Xaa Leu Xaa

1 5

<210> 172

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> LVCR part 3

<220>

<221> Other_feature

<222> (6)..(6)

<223> X=S, T or Y

<400> 172

His Gln Arg Ser Ser Xaa Pro Thr

1 5

<210> 173

<211> 8

<212> PROTEIN

<213> Homo sapiens

<400> 173

Gln Gln Ser Tyr Ser Thr Pro Thr

1 5

<210> 174

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 174

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctacaata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggagta atctaccctg gtgatggtgc cacaagctac 180

gctcagaagt tcaagggccg cgtcaccatg acccgcgaca cgtccacgag cacagtctac 240

atggagctga gcagcctgcg ctctgaggac acggctgtgt attactgtgc gagagattac 300

tacggtagta gcccccttgg ctactggggc caaggaaccc tggtcaccgt ctcctca 357

<210> 175

<211> 315

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 175

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacctgca gtgcaagtcc gagcattagc tatatgaatt ggtatcagca gaaaccaggg 120

aaagccccta agctcctgat ctatgctaca tccagcttgg caagtggggt cccatcacgt 180

ttcagtggca gtggaagcgg gacagatttc actctcacca tcagcagtct gcaacctgaa 240

gattttgcaa cttattactg tcaccagagg tctagttatc ccacttttgg ccaggggacc 300

aagctggaga tcaaa 315

<210> 176

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 176

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctacaata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggagta atcaaccctg gtgatggtgc cacaagctac 180

aatcagaagt tccagggccg cgtcaccatg acccgcgaca cgtccacgag cacagtctac 240

atggagctga gcagcctgcg ctctgaggac acggctgtgt attactgtgc gagagattac 300

tacggtagta gcccccttgg ctactggggc caaggaaccc tggtcaccgt ctcctca 357

<210> 177

<211> 315

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 177

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacctgca gggcaagttc gagcattagc tatttgaatt ggtatcagca gaaaccaggg 120

aaagccccta agctcctgat ctatgctgca tccagcttgg caagtggggt cccatcacgt 180

ttcagtggca gtggaagcgg gacagatttc actctcacca tcagcagtct gcaacctgaa 240

gattttgcaa cttattactg tcaccagagg tctagttctc ccacttttgg ccaggggacc 300

aagctggaga tcaaa 315

<210> 178

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 178

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctacaata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gataggagta atctaccctg gtgatggttc cacaagctac 180

aatcagaagt tccagggccg cgtcaccatg acccgcgaca cgtccacgag cacagtctac 240

atggagctga gcagcctgcg ctctgaggac acggctgtgt attactgtgc gagagattac 300

tacggtagta gcccccttgg ctactggggc caaggaaccc tggtcaccgt ctcctca 357

<210> 179

<211> 315

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 179

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacctgca gtgcaagttc gagcattagc tatatgaatt ggtatcagca gaaaccaggg 120

aaagccccta agctcctgat ctatgctaca tccaagttgc caagtggggt cccatcacgt 180

ttcagtggca gtggaagcgg gacagatttc actctcacca tcagcagtct gcaacctgaa 240

gattttgcaa cttattactg tcaccagagg tctagtactc ccacttttgg ccaggggacc 300

aagctggaga tcaaa 315

<210> 180

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 180

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctacaata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggagta atcaaccctg gtgatggtgc cacaagctac 180

gctcagaagt tcaagggccg cgtcaccatg acccgcgaca cgtccacgag cacagtctac 240

atggagctga gcagcctgcg ctctgaggac acggctgtgt attactgtgc gagagattac 300

tacggtagta gcccccttgg ctactggggc caaggaaccc tggtcaccgt ctcctca 357

<210> 181

<211> 315

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 181

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacctgca gggcaagtcc gagcattagc tatatgaatt ggtatcagca gaaaccaggg 120

aaagccccta agctcctgat ctatgctaca tccagcttgg caagtggggt cccatcacgt 180

ttcagtggca gtggaagcgg gacagatttc actctcacca tcagcagtct gcaacctgaa 240

gattttgcaa cttattactg tcaccagagg tctagttctc ccacttttgg ccaggggacc 300

aagctggaga tcaaa 315

<210> 182

<211> 315

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 182

gacatccaga tgacccagtc tccatcctcc ctgagcgcat ctgtaggaga ccgcgtcacc 60

atcacctgca gtgcaagtcc gagcattagc tatatgaatt ggtatcagca gaaaccaggg 120

aaagccccta agctcctgat ctatgctaca tccagcttgc caagtggggt cccatcacgt 180

ttcagtggca gtggaagcgg gacagatttc actctcacca tcagcagtct gcaacctgaa 240

gattttgcaa cttattactg tcaccagagg tctagttctc ccacttttgg ccaggggacc 300

aagctggaga tcaaa 315

<210> 183

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 183

caagttcagc tgcagcagcc cggtgccgag ctggtgaaac ccggtgcctc tgtgaagatg 60

agctgcaagg ccagcggcta cacctttacc agctacaaca tgcactgggt gaagcagaca 120

cccggacaag gtttagagtg gatcggcgtg atctaccccg gcaacggcga cacctcttac 180

aaccagaagt tcaagggcaa ggccacactg accgccgaca agagcagcag caccgcctac 240

atgcagctga gctctttaac cagcgaggac tccgccgtgt actactgcgc tcgtgattac 300

tacggcagca gccctttagg ctattgggga caaggtacca ctttaaccgt ctcgagcgcc 360

tcaaccaaag gaccctccgt gtttcccctc gccccctgtt cccgctccac atccgagtca 420

accgcggcgc tgggctgcct cgtgaaggac tacttccctg agcccgtcac tgtgtcgtgg 480

aactccgggg ccctgacctc cggcgtgcac accttccctg ccgtgcttca atcctccgga 540

ctgtactccc tgtcctcggt ggtcaccgtg ccgtcgagct cgttgggaac caagacttac 600

acttgcaacg tggaccacaa gccaagcaac accaaagtgg acaagagagt cgaatctaag 660

tacggaccgc cctgcccgcc ttgccccgcc cctgagtttc tcggcggtcc tagcgtgttc 720

ctgttcccac ccaagcccaa ggacactctg atgatctccc ggacccctga agtgacctgt 780

gtggtcgtgg acgtgtcgca ggaagatccg gaggtccagt tcaattggta cgtggatggg 840

gtggaggtcc acaacgccaa gacgaagccg agagaagaac agttcaactc aacttaccgg 900

gtggtgtccg tgctgaccgt gctgcatcag gattggctca acggaaagga gtacaagtgc 960

aaagtgtcca acaagggcct gcctagctca atcgaaaaga ccatttccaa ggccaagggc 1020

cagccgaggg aaccacaggt ctatactctg ccaccgagcc aagaagagat gaccaagaac 1080

caagtgtccc tgacttgcct ggtcaagggg ttctacccgt cggacatcgc agtggagtgg 1140

gagagcaacg gacagcctga aaacaattac aagaccaccc cgcccgtgct ggatagcgac 1200

ggttccttct tcctttactc gcgcctcacc gtcgacaaga gccggtggca ggagggcaac 1260

gtgttctcct gctccgtgat gcacgaagct ctgcataacc actacactca gaagtccttg 1320

tcgctgagcc tcggaaag 1338

<210> 184

<211> 636

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 184

cagatcgtgc tgacccagag ccccgctgtg atgagcgcct ctcccggtga gaaggtgacc 60

atgacttgta gcgccagcag cagcatcagc tacatgcact ggtaccagca gaagcccggc 120

accagcccta agaggtggat ctacgacacc agcaagctgg ccagcggcgt gcccgctagg 180

ttcagcggaa gcggcagcgg caccagctac tctttaacca tcagcagcat ggaggccgag 240

gatgccgcca cctactactg ccaccagaga agcagctacc ccaccttcgg cggcggcacc 300

aagctcgaga tcaagagaac tgtggccgcg ccgtcagtgt ttatcttccc tccatcggat 360

gaacagctta agtccggcac ggcgtctgtg gtctgcctgc tcaataactt ttaccctagg 420

gaagctaaag tccaatggaa agtggataac gccctgcagt caggaaacag cccaggaatcg 480

gttaccgaac aggacagcaa ggacagcact tactccttgt cgtcgactct tactctgagc 540

aaggccgatt acgagaagca caaggtctac gcctgcgagg tcacccatca gggactctcg 600

tccccggtga ccaaatcctt caatagaggc gaatgc 636

<210> 185

<211> 1340

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 185

ctcaagttca gctcgtgcag agcggtgctg aagtgaagaa gcccggtgcc tctgtgaagg 60

tgagctgcaa ggccagcggc tacaccttca ccagctacaa catgcactgg gtgagacaag 120

ctcccggtca aggtttagag tggatgggcg tgatctaccc cggtgatggt gctaccagct 180

acgcccagaa gttcaagggt cgtgtgacca tgaccagaga caccagcacc agcaccgtgt 240

acatggagct gagctcttta aggagcgagg acaccgccgt gtactactgc gctcgtgact 300

actacggcag cagcccttta ggctattggg gacaaggtac tttagtgacc gtctcgagcg 360

cctcaaccaa aggaccctcc gtgtttcccc tcgccccctg ttcccgctcc acatccgagt 420

caaccgcggc gctgggctgc ctcgtgaagg actacttccc tgagcccgtc actngtgtcgt 480

ggaactccgg ggccctgacc tccggcgtgc acaccttccc tgccgtgctt caatcctccg 540

gactgtactc cctgtcctcg gtggtcaccg tgccgtcgag ctcgttggga accaagactt 600

acacttgcaa cgtggaccac aagccaagca acaccaaagt ggacaagaga gtcgaatcta 660

agtacggacc gccctgcccg ccttgccccg cccctgagtt tctcggcggt cctagcgtgt 720

tcctgttccc acccaagccc aaggacactc tgatgatctc ccggacccct gaagtgacct 780

gtgtggtcgt ggacgtgtcg caggaagatc cggaggtcca gttcaattgg tacgtggatg 840

gggtggaggt ccacaacgcc aagacgaagc cgagagaaga acagttcaac tcaacttacc 900

gggtggtgtc cgtgctgacc gtgctgcatc aggattggct caacggaaag gagtacaagt 960

gcaaagtgtc caacaagggc ctgcctagct caatcgaaaa gaccatttcc aaggccaagg 1020

gccagccgag ggaaccacag gtctatactc tgccaccgag ccaagaagag atgaccaaga 1080

accaagtgtc cctgacttgc ctggtcaagg ggttctaccc gtcggacatc gcagtggagt 1140

ggggagagcaa cggacagcct gaaaacaatt acaagaccac cccgcccgtg ctggatagcg 1200

acggttcctt cttcctttac tcgcgcctca ccgtcgacaa gagccggtgg caggagggca 1260

acgtgttctc ctgctccgtg atgcacgaag ctctgcataa ccactacact cagaagtcct 1320

tgtcgctgag cctcggaaag 1340

<210> 186

<211> 636

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 186

gacatccaga tgacccagag ccctagctct ttaagcgcct ctgtgggcga tcgtgtgacc 60

atcacttgta gcgccagccc cagcatcagc tacatgaact ggtaccagca gaagcccggc 120

aaggccccca agctgctgat ctacgccaca agctctttag ccagcggcgt gcctagcaga 180

tttagcggca gcggtagcgg cacagacttc actttaacca tcagctcttt acagccagaa 240

gacttcgcca cctactactg ccaccagagg agcagctacc ccaccttcgg ccaaggtacc 300

aagctcgaga tcaagagaac tgtggccgcg ccgtcagtgt ttatcttccc tccatcggat 360

gaacagctta agtccggcac ggcgtctgtg gtctgcctgc tcaataactt ttaccctagg 420

gaagctaaag tccaatggaa agtggataac gccctgcagt caggaaacag cccaggaatcg 480

gttaccgaac aggacagcaa ggacagcact tactccttgt cgtcgactct tactctgagc 540

aaggccgatt acgagaagca caaggtctac gcctgcgagg tcacccatca gggactctcg 600

tccccggtga ccaaatcctt caatagaggc gaatgc 636

<210> 187

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 187

caagttcagc tcgtgcagag cggtgctgaa gtgaagaagc ccggtgcctc tgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agctacaaca tgcactgggt gagacaagct 120

cccggtcaag gtttagagtg gatgggcgtg atcaaccccg gtgatggtgc taccagctac 180

aaccagaagt tccagggtcg tgtgaccatg accagagaca ccagcaccag caccgtgtac 240

atggagctga gctctttaag gagcgaggac accgccgtgt actactgcgc tcgtgactac 300

tacggcagca gccctttagg ctattgggga caaggtactt tagtgaccgt ctcgagcgcc 360

tcaaccaaag gaccctccgt gtttcccctc gccccctgtt cccgctccac atccgagtca 420

accgcggcgc tgggctgcct cgtgaaggac tacttccctg agcccgtcac tgtgtcgtgg 480

aactccgggg ccctgacctc cggcgtgcac accttccctg ccgtgcttca atcctccgga 540

ctgtactccc tgtcctcggt ggtcaccgtg ccgtcgagct cgttgggaac caagacttac 600

acttgcaacg tggaccacaa gccaagcaac accaaagtgg acaagagagt cgaatctaag 660

tacggaccgc cctgcccgcc ttgccccgcc cctgagtttc tcggcggtcc tagcgtgttc 720

ctgttcccac ccaagcccaa ggacactctg atgatctccc ggacccctga agtgacctgt 780

gtggtcgtgg acgtgtcgca ggaagatccg gaggtccagt tcaattggta cgtggatggg 840

gtggaggtcc acaacgccaa gacgaagccg agagaagaac agttcaactc aacttaccgg 900

gtggtgtccg tgctgaccgt gctgcatcag gattggctca acggaaagga gtacaagtgc 960

aaagtgtcca acaagggcct gcctagctca atcgaaaaga ccatttccaa ggccaagggc 1020

cagccgaggg aaccacaggt ctatactctg ccaccgagcc aagaagagat gaccaagaac 1080

caagtgtccc tgacttgcct ggtcaagggg ttctacccgt cggacatcgc agtggagtgg 1140

gagagcaacg gacagcctga aaacaattac aagaccaccc cgcccgtgct ggatagcgac 1200

ggttccttct tcctttactc gcgcctcacc gtcgacaaga gccggtggca ggagggcaac 1260

gtgttctcct gctccgtgat gcacgaagct ctgcataacc actacactca gaagtccttg 1320

tcgctgagcc tcggaaag 1338

<210> 188

<211> 636

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 188

gacatccaga tgacccagag ccctagctct ttaagcgcct ctgtgggcga tcgtgtgacc 60

atcacttgtc gtgccagcag cagcatcagc tatttaaact ggtaccagca gaagcccggc 120

aaggccccca agctgctgat ctacgccgcc tcttctttag cctctggcgt gccttctcgt 180

ttcagcggaa gcggcagcgg caccgacttc actttaacca tcagctcttt acagccagaa 240

gacttcgcca cctactactg ccaccagagg agcagcagcc ccaccttcgg acaaggtacc 300

aagctcgaga tcaagagaac tgtggccgcg ccgtcagtgt ttatcttccc tccatcggat 360

gaacagctta agtccggcac ggcgtctgtg gtctgcctgc tcaataactt ttaccctagg 420

gaagctaaag tccaatggaa agtggataac gccctgcagt caggaaacag cccaggaatcg 480

gttaccgaac aggacagcaa ggacagcact tactccttgt cgtcgactct tactctgagc 540

aaggccgatt acgagaagca caaggtctac gcctgcgagg tcacccatca gggactctcg 600

tccccggtga ccaaatcctt caatagaggc gaatgc 636

<210> 189

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 189

caagttcagc tggtgcagag cggagccgag gtgaaaaagc ccggtgcctc tgtgaaggtg 60

agctgcaagg ccagcggcta cacctttacc agctacaaca tgcactgggt gaggcaagct 120

cccggtcaag gtctggagtg gatcggcgtg atctaccccg gcgacggcag cacctcttac 180

aaccagaagt tccaaggtcg tgtgaccatg actcgtgaca ccagcaccag caccgtgtac 240

atggagctga gctctttaag gagcgaggat accgccgtgt actactgcgc tcgtgactac 300

tacggcagca gccctctggg ctattggggc caaggtactt tagtgaccgt ctcgagcgcc 360

tcaaccaaag gaccctccgt gtttcccctc gccccctgtt cccgctccac atccgagtca 420

accgcggcgc tgggctgcct cgtgaaggac tacttccctg agcccgtcac tgtgtcgtgg 480

aactccgggg ccctgacctc cggcgtgcac accttccctg ccgtgcttca atcctccgga 540

ctgtactccc tgtcctcggt ggtcaccgtg ccgtcgagct cgttgggaac caagacttac 600

acttgcaacg tggaccacaa gccaagcaac accaaagtgg acaagagagt cgaatctaag 660

tacggaccgc cctgcccgcc ttgccccgcc cctgagtttc tcggcggtcc tagcgtgttc 720

ctgttcccac ccaagcccaa ggacactctg atgatctccc ggacccctga agtgacctgt 780

gtggtcgtgg acgtgtcgca ggaagatccg gaggtccagt tcaattggta cgtggatggg 840

gtggaggtcc acaacgccaa gacgaagccg agagaagaac agttcaactc aacttaccgg 900

gtggtgtccg tgctgaccgt gctgcatcag gattggctca acggaaagga gtacaagtgc 960

aaagtgtcca acaagggcct gcctagctca atcgaaaaga ccatttccaa ggccaagggc 1020

cagccgaggg aaccacaggt ctatactctg ccaccgagcc aagaagagat gaccaagaac 1080

caagtgtccc tgacttgcct ggtcaagggg ttctacccgt cggacatcgc agtggagtgg 1140

gagagcaacg gacagcctga aaacaattac aagaccaccc cgcccgtgct ggatagcgac 1200

ggttccttct tcctttactc gcgcctcacc gtcgacaaga gccggtggca ggagggcaac 1260

gtgttctcct gctccgtgat gcacgaagct ctgcataacc actacactca gaagtccttg 1320

tcgctgagcc tcggaaag 1338

<210> 190

<211> 636

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 190

gacatccaga tgacccagag ccctagctct ttaagcgcct ctgtgggcga tcgtgtgacc 60

atcacttgta gcgccagcag cagcatcagc tacatgaact ggtaccagca gaagcccggc 120

aaggccccca agctgctgat ctacgccacc agcaagctgc ctagcggcgt gccctccaga 180

ttttctggca gcggctctgg caccgacttt actttaacca tcagctcttt acagccagaa 240

gacttcgcca cctactactg ccaccagagg agcagcaccc ctaccttcgg ccaaggtacc 300

aagctcgaga tcaagagaac tgtggccgcg ccgtcagtgt ttatcttccc tccatcggat 360

gaacagctta agtccggcac ggcgtctgtg gtctgcctgc tcaataactt ttaccctagg 420

gaagctaaag tccaatggaa agtggataac gccctgcagt caggaaacag cccaggaatcg 480

gttaccgaac aggacagcaa ggacagcact tactccttgt cgtcgactct tactctgagc 540

aaggccgatt acgagaagca caaggtctac gcctgcgagg tcacccatca gggactctcg 600

tccccggtga ccaaatcctt caatagaggc gaatgc 636

<210> 191

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 191

caagttcagc tcgtgcagag cggtgctgaa gtgaagaagc ccggtgcctc tgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc agctacaaca tgcactgggt gagacaagct 120

cccggtcaag gtttagagtg gatgggcgtg atcaaccccg gtgatggtgc taccagctac 180

gcccagaagt tcaagggtcg tgtgaccatg accagagaca ccagcaccag caccgtgtac 240

atggagctga gctctttaag gagcgaggac accgccgtgt actactgcgc tcgtgactac 300

tacggcagca gccctttagg ctattgggga caaggtactt tagtgaccgt ctcgagcgcc 360

tcaaccaaag gaccctccgt gtttcccctc gccccctgtt cccgctccac atccgagtca 420

accgcggcgc tgggctgcct cgtgaaggac tacttccctg agcccgtcac tgtgtcgtgg 480

aactccgggg ccctgacctc cggcgtgcac accttccctg ccgtgcttca atcctccgga 540

ctgtactccc tgtcctcggt ggtcaccgtg ccgtcgagct cgttgggaac caagacttac 600

acttgcaacg tggaccacaa gccaagcaac accaaagtgg acaagagagt cgaatctaag 660

tacggaccgc cctgcccgcc ttgccccgcc cctgagtttc tcggcggtcc tagcgtgttc 720

ctgttcccac ccaagcccaa ggacactctg atgatctccc ggacccctga agtgacctgt 780

gtggtcgtgg acgtgtcgca ggaagatccg gaggtccagt tcaattggta cgtggatggg 840

gtggaggtcc acaacgccaa gacgaagccg agagaagaac agttcaactc aacttaccgg 900

gtggtgtccg tgctgaccgt gctgcatcag gattggctca acggaaagga gtacaagtgc 960

aaagtgtcca acaagggcct gcctagctca atcgaaaaga ccatttccaa ggccaagggc 1020

cagccgaggg aaccacaggt ctatactctg ccaccgagcc aagaagagat gaccaagaac 1080

caagtgtccc tgacttgcct ggtcaagggg ttctacccgt cggacatcgc agtggagtgg 1140

gagagcaacg gacagcctga aaacaattac aagaccaccc cgcccgtgct ggatagcgac 1200

ggttccttct tcctttactc gcgcctcacc gtcgacaaga gccggtggca ggagggcaac 1260

gtgttctcct gctccgtgat gcacgaagct ctgcataacc actacactca gaagtccttg 1320

tcgctgagcc tcggaaag 1338

<210> 192

<211> 636

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 192

gacatccaga tgacccagag ccctagctct ttaagcgcca gcgtgggaga tcgtgtgacc 60

atcacttgtc gtgccagccc cagcatcagc tacatgaact ggtaccagca gaagcccggc 120

aaggccccca agctgctgat ctacgccacc agctctttag cctctggcgt gcctagcaga 180

ttcagcggca gcggaagcgg caccgacttc actttaacca tcagctcttt acagccagaa 240

gacttcgcca cctactactg ccaccagagg agcagcagcc ctaccttcgg ccaaggtacc 300

aagctcgaga tcaagagaac tgtggccgcg ccgtcagtgt ttatcttccc tccatcggat 360

gaacagctta agtccggcac ggcgtctgtg gtctgcctgc tcaataactt ttaccctagg 420

gaagctaaag tccaatggaa agtggataac gccctgcagt caggaaacag cccaggaatcg 480

gttaccgaac aggacagcaa ggacagcact tactccttgt cgtcgactct tactctgagc 540

aaggccgatt acgagaagca caaggtctac gcctgcgagg tcacccatca gggactctcg 600

tccccggtga ccaaatcctt caatagaggc gaatgc 636

<210> 193

<211> 636

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 193

gacatccaga tgacccagag ccctagctct ttaagcgcct ctgtgggcga tcgtgtgacc 60

atcacttgta gcgccagccc cagcatcagc tacatgaact ggtaccagca gaagcccggc 120

aaggccccca agctgctgat ctacgccaca agctctttac ccagcggcgt gcctagcaga 180

ttcagcggca gcggaagcgg caccgacttc actttaacca tcagctcttt acagccagaa 240

gacttcgcca cctactactg ccaccagaga agcagcagcc ccaccttcgg ccaaggtaca 300

aagctcgaga tcaagagaac tgtggccgcg ccgtcagtgt ttatcttccc tccatcggat 360

gaacagctta agtccggcac ggcgtctgtg gtctgcctgc tcaataactt ttaccctagg 420

gaagctaaag tccaatggaa agtggataac gccctgcagt caggaaacag cccaggaatcg 480

gttaccgaac aggacagcaa ggacagcact tactccttgt cgtcgactct tactctgagc 540

aaggccgatt acgagaagca caaggtctac gcctgcgagg tcacccatca gggactctcg 600

tccccggtga ccaaatcctt caatagaggc gaatgc 636

<210> 194

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 194

caagttcagc tggtgcagag cggtgctgag gtgaaaaaac ccggtgcttc cgtgaaggtg 60

agctgcaagg ccagcggcta cacctttacc agctacaaca tgcactgggt gagacaagcc 120

cccggtcaag gtttagagtg gatcggcgtg atctaccccg gcaacggcga cacctcttac 180

aaccagaagt tcaagggtcg tgtgaccatg actcgtgaca cctccaccag caccgtgtac 240

atggagctga gctctttaag gagcgaggac acagccgtgt actactgcgc tcgtgactac 300

tacggcagca gccctctggg ctattggggc caaggtactt tagtgaccgt ctcgagcgcc 360

tcaaccaaag gaccctccgt gtttcccctc gccccctgtt cccgctccac atccgagtca 420

accgcggcgc tgggctgcct cgtgaaggac tacttccctg agcccgtcac tgtgtcgtgg 480

aactccgggg ccctgacctc cggcgtgcac accttccctg ccgtgcttca atcctccgga 540

ctgtactccc tgtcctcggt ggtcaccgtg ccgtcgagct cgttgggaac caagacttac 600

acttgcaacg tggaccacaa gccaagcaac accaaagtgg acaagagagt cgaatctaag 660

tacggaccgc cctgcccgcc ttgccccgcc cctgagtttc tcggcggtcc tagcgtgttc 720

ctgttcccac ccaagcccaa ggacactctg atgatctccc ggacccctga agtgacctgt 780

gtggtcgtgg acgtgtcgca ggaagatccg gaggtccagt tcaattggta cgtggatggg 840

gtggaggtcc acaacgccaa gacgaagccg agagaagaac agttcaactc aacttaccgg 900

gtggtgtccg tgctgaccgt gctgcatcag gattggctca acggaaagga gtacaagtgc 960

aaagtgtcca acaagggcct gcctagctca atcgaaaaga ccatttccaa ggccaagggc 1020

cagccgaggg aaccacaggt ctatactctg ccaccgagcc aagaagagat gaccaagaac 1080

caagtgtccc tgacttgcct ggtcaagggg ttctacccgt cggacatcgc agtggagtgg 1140

gagagcaacg gacagcctga aaacaattac aagaccaccc cgcccgtgct ggatagcgac 1200

ggttccttct tcctttactc gcgcctcacc gtcgacaaga gccggtggca ggagggcaac 1260

gtgttctcct gctccgtgat gcacgaagct ctgcataacc actacactca gaagtccttg 1320

tcgctgagcc tcggaaag 1338

<210> 195

<211> 636

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 195

gacatccaga tgacccagag ccctagctct ttaagcgcct ctgtgggcga tcgtgtgacc 60

atcacttgta gcgccagcag cagcatcagc tacatgcact ggtaccagca gaagcccggc 120

aaggccccca agctgctgat ctacgacacc agcaagctgg ccagcggcgt gcctagcaga 180

ttcagcggca gcggaagcgg caccgacttc actttaacca tcagctcttt acagccagaa 240

gacttcgcca cctactactg ccaccagagg agcagctacc ccaccttcgg ccaaggtacc 300

aagctcgaga tcaagagaac tgtggccgcg ccgtcagtgt ttatcttccc tccatcggat 360

gaacagctta agtccggcac ggcgtctgtg gtctgcctgc tcaataactt ttaccctagg 420

gaagctaaag tccaatggaa agtggataac gccctgcagt caggaaacag cccaggaatcg 480

gttaccgaac aggacagcaa ggacagcact tactccttgt cgtcgactct tactctgagc 540

aaggccgatt acgagaagca caaggtctac gcctgcgagg tcacccatca gggactctcg 600

tccccggtga ccaaatcctt caatagaggc gaatgc 636

<210> 196

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 196

caagtgcagc tcgtgcagtc cggagccgaa gtcaagaagc ccggagcgtc agtgaaagtg 60

tcctgcaagg cctcgggcta cactttcaca agctacaaca tgcactgggt cagacaggca 120

cctgggcagg gtctggagtg gatgggagtg atctacccgg gcgacggcgc cacttcctac 180

gcccaaaagt tcaagggccg cgtgaccatg actagggaca cctcgacctc aaccgtgtac 240

atggaactga gctccctgcg gtccgaggat accgccgtgt actattgtgc tcgggactac 300

tacgggtcca gcccactggg atactgggga cagggtaccc ttgtcacggt gtcgtcagct 360

tccaccaagg gcccatccgt cttccccctg gcgccctgct ccaggagcac ctccgagagc 420

acagccgccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac gaagacctac 600

acctgcaatg tagatcacaa gcccagcaac accaaggtgg acaagagagt tgagtccaaa 660

tatggtcccc catgcccacc atgcccagca cctgagttcc tggggggacc atcagtcttc 720

ctgttccccc caaaacccaa ggacactctc atgatctccc ggacccctga ggtcacgtgc 780

gtggtggtgg acgtgagcca ggaagacccc gaggtccagt tcaactggta cgtggatggc 840

gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agttcaacag cacgtaccgt 900

gtggtcagcg tcctcaccgt cctgcaccag gactggctga acggcaagga gtacaagtgc 960

aaggtctcca acaaaggcct cccgtcctcc atcgagaaaa ccatctccaa agccaaaggg 1020

cagccccgag agccacaggt gtacaccctg cccccatccc aggaggat gaccaagaac 1080

caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 1140

gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1200

ggctccttct tcctctacag caggctaacc gtggacaaga gcaggtggca ggaggggaat 1260

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc 1320

tccctgtctc tgggtaaa 1338

<210> 197

<211> 636

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 197

gacatccaaa tgactcagtc cccgtcatcc ctgtcggcat ccgtgggaga cagagtcacc 60

attacgtgca gcgcgagccc gagcatttcc tatatgaact ggtaccagca gaagcccggg 120

aaagccccta agctgttgat ctacgccact tcctcactgg cttccggcgt gccatcccgg 180

ttctcgggtt ccggctcggg aaccgatttt acccttacta tctcgtccct gcaacccgag 240

gacttcgcca cctactactg tcaccagcgc tctagctacc ctacattcgg acagggcacc 300

aagctcgaaa tcaaacgaac tgtggctgca ccatctgtct tcatcttccc gccatctgat 360

gagcagttga aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga 420

gaggccaaag tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt 480

gtcacagagc aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgagc 540

aaagcagact acgagaaaca caaagtctac gcctgcgaag tcacccatca gggcctgagc 600

tcgcccgtca caaagagctt caacagggga gagtgt 636

<210> 198

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 198

caagtgcagc tcgtgcagtc cggagccgaa gtcaagaagc ccggagcgtc agtgaaagtg 60

tcctgcaagg cctcgggcta cactttcaca agctacaaca tgcactgggt cagacaggca 120

cctgggcagg gtctggagtg gattggagtg atctacccgg gcgacggctc cacttcctac 180

aaccaaaagt tccagggccg cgtgaccatg actagggaca cctcgacctc aaccgtgtac 240

atggaactga gctccctgcg gtccgaggat accgccgtgt actattgtgc tcgggactac 300

tacgggtcca gcccactggg atactgggga cagggtaccc ttgtcacggt gtcgtcagct 360

tccaccaagg gcccatccgt cttccccctg gcgccctgct ccaggagcac ctccgagagc 420

acagccgccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac gaagacctac 600

acctgcaatg tagatcacaa gcccagcaac accaaggtgg acaagagagt tgagtccaaa 660

tatggtcccc catgcccacc atgcccagca cctgagttcc tggggggacc atcagtcttc 720

ctgttccccc caaaacccaa ggacactctc atgatctccc ggacccctga ggtcacgtgc 780

gtggtggtgg acgtgagcca ggaagacccc gaggtccagt tcaactggta cgtggatggc 840

gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agttcaacag cacgtaccgt 900

gtggtcagcg tcctcaccgt cctgcaccag gactggctga acggcaagga gtacaagtgc 960

aaggtctcca acaaaggcct cccgtcctcc atcgagaaaa ccatctccaa agccaaaggg 1020

cagccccgag agccacaggt gtacaccctg cccccatccc aggaggat gaccaagaac 1080

caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 1140

gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1200

ggctccttct tcctctacag caggctaacc gtggacaaga gcaggtggca ggaggggaat 1260

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc 1320

tccctgtctc tgggtaaa 1338

<210> 199

<211> 635

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 199

acatccaaat gactcagtcc ccgtcatccc tgtcggcatc cgtgggagac agagtcacca 60

ttacgtgcag cgcgagctca agcatttcct atatgaactg gtaccagcag aagcccggga 120

aagcccctaa gctgttgatc tacgccactt ccaagctgcc gtccggcgtg ccatcccggt 180

tctcgggttc cggctcggga accgatttta cccttactat ctcgtccctg caacccgagg 240

acttcgccac ctactactgt caccagcgct ctagcacccc tacattcgga cagggcacca 300

agctcgaaat caaacgaact gtggctgcac catctgtctt catcttcccg ccatctgatg 360

agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc tatcccagag 420

aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc caggagagtg 480

tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg acgctgagca 540

aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag ggcctgagct 600

cgcccgtcac aaagagcttc aacagggggag agtgt 635

<210> 200

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223>HC

<400> 200

caagtgcagc tcgtgcagtc cggagccgaa gtcaagaagc ccggagcgtc agtgaaagtg 60

tcctgcaagg cctcgggcta cactttcaca agctacaaca tgcactgggt cagacaggca 120

cctgggcagg gtctggagtg gatgggagtg atcaacccgg gcgacggcgc cacttcctac 180

gcccaaaagt tcaagggccg cgtgaccatg actagggaca cctcgacctc aaccgtgtac 240

atggaactga gctccctgcg gtccgaggat accgccgtgt actattgtgc tcgggactac 300

tacgggtcca gcccactggg atactgggga cagggtaccc ttgtcacggt gtcgtcagct 360

tccaccaagg gcccatccgt cttccccctg gcgccctgct ccaggagcac ctccgagagc 420

acagccgccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac gaagacctac 600

acctgcaatg tagatcacaa gcccagcaac accaaggtgg acaagagagt tgagtccaaa 660

tatggtcccc catgcccacc atgcccagca cctgagttcc tggggggacc atcagtcttc 720

ctgttccccc caaaacccaa ggacactctc atgatctccc ggacccctga ggtcacgtgc 780

gtggtggtgg acgtgagcca ggaagacccc gaggtccagt tcaactggta cgtggatggc 840

gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agttcaacag cacgtaccgt 900

gtggtcagcg tcctcaccgt cctgcaccag gactggctga acggcaagga gtacaagtgc 960

aaggtctcca acaaaggcct cccgtcctcc atcgagaaaa ccatctccaa agccaaaggg 1020

cagccccgag agccacaggt gtacaccctg cccccatccc aggaggat gaccaagaac 1080

caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 1140

gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1200

ggctccttct tcctctacag caggctaacc gtggacaaga gcaggtggca ggaggggaat 1260

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc 1320

tccctgtctc tgggtaaa 1338

<210> 201

<211> 636

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 201

gacatccaaa tgactcagtc cccgtcatcc ctgtcggcat ccgtgggaga cagagtcacc 60

attacgtgcc gcgcgagccc gagcatttcc tatatgaact ggtaccagca gaagcccggg 120

aaagccccta agctgttgat ctacgccact tcctcactgg cttccggcgt gccatcccgg 180

ttctcgggtt ccggctcggg aaccgatttt acccttacta tctcgtccct gcaacccgag 240

gacttcgcca cctactactg tcaccagcgc tctagcagcc ctacattcgg acagggcacc 300

aagctcgaaa tcaaacgaac tgtggctgca ccatctgtct tcatcttccc gccatctgat 360

gagcagttga aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga 420

gaggccaaag tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt 480

gtcacagagc aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgagc 540

aaagcagact acgagaaaca caaagtctac gcctgcgaag tcacccatca gggcctgagc 600

tcgcccgtca caaagagctt caacagggga gagtgt 636

<210> 202

<211> 636

<212> DNA

<213> Artificial sequence

<220>

<223>LC

<400> 202

gacatccaaa tgactcagtc cccgtcatcc ctgtcggcat ccgtgggaga cagagtcacc 60

attacgtgca gcgcgagccc gagcatttcc tatatgaact ggtaccagca gaagcccggg 120

aaagccccta agctgttgat ctacgccact tcctcactgc cgtccggcgt gccatcccgg 180

ttctcgggtt ccggctcggg aaccgatttt acccttacta tctcgtccct gcaacccgag 240

gacttcgcca cctactactg tcaccagcgc tctagcagcc ctacattcgg acagggcacc 300

aagctcgaaa tcaaacgaac tgtggctgca ccatctgtct tcatcttccc gccatctgat 360

gagcagttga aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga 420

gaggccaaag tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt 480

gtcacagagc aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgagc 540

aaagcagact acgagaaaca caaagtctac gcctgcgaag tcacccatca gggcctgagc 600

tcgcccgtca caaagagctt caacagggga gagtgt 636

<---

Claims (119)

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to bile salt-stimulated lipase (BSSL) and contains: three complementarity determining regions (CDRs) of the heavy chain variable region (HCVR) (HCDR); and three light chain variable region (LCVR) CDRs (LCDRs), while the first HCDR consists of the amino acid sequence according to SEQ ID NO: 7, the second HCDR consists of the amino acid sequence according to SEQ ID NO: 8, the third HCDR consists of the amino acid sequence according to SEQ ID NO: 9, the first LCDR consists of the amino acid sequence according to SEQ ID NO: 10, the second LCDR consists of the amino acid sequence ATS, and the third LCDR consists of the amino acid sequence according to SEQ ID NO: 11; or the first HCDR consists of the amino acid sequence according to SEQ ID NO: 7, the second HCDR consists of the amino acid sequence according to SEQ ID NO: 18, the third HCDR consists of the amino acid sequence according to SEQ ID NO: 9, the first LCDR consists of the amino acid sequence according to SEQ ID NO: 10, the second LCDR consists of the amino acid sequence ATS, and the third LCDR consists of the amino acid sequence according to SEQ ID NO: 21; or the first HCDR consists of the amino acid sequence according to SEQ ID NO: 7, the second HCDR consists of the amino acid sequence according to SEQ ID NO: 8, the third HCDR consists of the amino acid sequence according to SEQ ID NO: 9, the first LCDR consists of the amino acid sequence according to SEQ ID NO: 20, the second LCDR consists of the amino acid sequence AAS, and the third LCDR consists of the amino acid sequence according to SEQ ID NO: 11; or the first HCDR consists of the amino acid sequence according to SEQ ID NO: 7, the second HCDR consists of the amino acid sequence according to SEQ ID NO: 19, the third HCDR consists of the amino acid sequence according to SEQ ID NO: 9, the first LCDR consists of the amino acid sequence according to SEQ ID NO: 20, the second LCDR consists of the amino acid sequence ATS, and the third LCDR consists of the amino acid sequence according to SEQ ID NO: 22. 2. An isolated antibody or antigen-binding fragment thereof according to claim 1, in which the first HCDR consists of the amino acid sequence according to SEQ ID NO: 7; the second HCDR consists of the amino acid sequence according to SEQ ID NO: 8; the third HCDR consists of the amino acid sequence according to SEQ ID NO: 9; the first LCDR consists of the amino acid sequence according to SEQ ID NO: 10; the second LCDR consists of the amino acid sequence ATS; and the third LCDR consists of the amino acid sequence according to SEQ ID NO: 11. 3. An isolated antibody or antigen-binding fragment thereof according to claim 2, wherein the isolated antibody or antigen-binding fragment thereof comprises: an extended second HCDR, which consists of the amino acid sequence according to SEQ ID NO: 12; an extended first LCDR, which consists of the amino acid sequence according to SEQ ID NO: 14 and an extended second LCDR, which consists of the amino acid sequence according to SEQ ID NO: 15. 4. An isolated antibody or antigen-binding fragment thereof according to claim 2 or 3, in which HCVR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 36 and/or LCVR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 37. 5. An isolated antibody or antigen-binding fragment thereof according to claim 2, wherein the isolated antibody or antigen-binding fragment thereof comprises: an extended second HCDR, which consists of the amino acid sequence according to SEQ ID NO: 12; an extended first LCDR, which consists of the amino acid sequence according to SEQ ID NO: 16 and an extended second LCDR, which consists of the amino acid sequence according to SEQ ID NO: 17. 6. An isolated antibody or antigen-binding fragment thereof according to claim 2 or 5, in which HCVR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 36 and/or LCVR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 38. 7. An isolated antibody or antigen-binding fragment thereof according to claim 1, in which the first HCDR consists of the amino acid sequence according to SEQ ID NO: 7; the second HCDR consists of the amino acid sequence according to SEQ ID NO: 18; the third HCDR consists of the amino acid sequence according to SEQ ID NO: 9; the first LCDR consists of the amino acid sequence according to SEQ ID NO: 10; the second LCDR consists of the amino acid sequence ATS; and the third LCDR consists of the amino acid sequence according to SEQ ID NO: 21. 8. An isolated antibody or antigen-binding fragment thereof according to claim 7, wherein the isolated antibody or antigen-binding fragment thereof comprises: an extended second HCDR, which consists of the amino acid sequence according to SEQ ID NO: 23; an extended first LCDR, which consists of the amino acid sequence according to SEQ ID NO: 16 and an extended second LCDR, which consists of the amino acid sequence according to SEQ ID NO: 15. 9. An isolated antibody or antigen-binding fragment thereof according to claim 7 or 8, in which HCVR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 30 and/or LCVR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 31. 10. An isolated antibody or antigen-binding fragment thereof according to claim 1, in which the first HCDR consists of the amino acid sequence according to SEQ ID NO: 7; the second HCDR consists of the amino acid sequence according to SEQ ID NO: 8; the third HCDR consists of the amino acid sequence according to SEQ ID NO: 9; the first LCDR consists of the amino acid sequence according to SEQ ID NO: 20; the second LCDR consists of the amino acid sequence AAS; and the third LCDR consists of the amino acid sequence according to SEQ ID NO: 11. 11. The isolated antibody or antigen-binding fragment thereof according to claim 10, wherein the isolated antibody or antigen-binding fragment thereof comprises: an extended second HCDR, which consists of the amino acid sequence according to SEQ ID NO: 24; an extended first LCDR, which consists of the amino acid sequence according to SEQ ID NO: 27 and an extended second LCDR, which consists of the amino acid sequence according to SEQ ID NO: 29. 12. An isolated antibody or antigen-binding fragment thereof according to claim 10 or 11, in which HCVR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 32 and/or LCVR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 33. 13. An isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the first HCDR consists of the amino acid sequence according to SEQ ID NO: 7; the second HCDR consists of the amino acid sequence according to SEQ ID NO: 19; the third HCDR consists of the amino acid sequence according to SEQ ID NO: 9; the first LCDR consists of the amino acid sequence according to SEQ ID NO: 20; the second LCDR consists of the amino acid sequence ATS; and the third LCDR consists of the amino acid sequence according to SEQ ID NO: 22. 14. The isolated antibody or antigen-binding fragment thereof according to claim 13, wherein the isolated antibody or antigen-binding fragment thereof comprises: an extended second HCDR, which consists of the amino acid sequence according to SEQ ID NO: 25; an extended first LCDR, which consists of the amino acid sequence according to SEQ ID NO: 26 and an extended second LCDR, which consists of the amino acid sequence according to SEQ ID NO: 28. 15. An isolated antibody or antigen-binding fragment thereof according to claim 13 or 14, in which HCVR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 34 and/or LCVR comprises, preferably consists of, the amino acid sequence according to SEQ ID NO: 35. 16. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the HCVR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36. 17. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the LCVR comprises, preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 38. 18. An isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a humanized antibody, and a chimeric antibody or antigen-binding fragment thereof. 19. An isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antigen-binding fragment is selected from the group consisting of a variable single-chain fragment (scFv), a Fab fragment, an F(ab') ₂ fragment, an F(ab') ₃ fragment, a Fab' fragment, an Fd fragment, an Fv fragment, a dAb fragment, an isolated complementarity determining region (CDR) and a nanobody, preferably scFv. 20. An isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the isolated antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof. 21. An isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the isolated antibody or antigen-binding fragment thereof has an isotype class selected from the group consisting of IgG, IgA, IgM, IgD and IgE, preferably IgG and more preferably IgG1 or IgG4. 22. An isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the isolated antibody or antigen-binding fragment thereof comprises at least one Fc silencing mutation that inhibits interaction with Fc receptors. 23. The isolated antibody or antigen-binding fragment thereof according to claim 22, wherein the isolated antibody or antigen-binding fragment thereof belongs to the IgG isotype class, and at least one Fc silencing mutation is selected from the group consisting of L234A, L235A, and P329G. 24. An isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the isolated antibody or antigen-binding fragment thereof comprises at least one stabilizing mutation that prevents or reduces Fab-arm turnover in vivo. 25. The isolated antibody or antigen-binding fragment thereof according to claim 24, wherein the isolated antibody or antigen-binding fragment thereof belongs to the IgG4 isotype subclass, and at least one stabilizing mutation is S228P. 26. An isolated antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the isolated antibody or antigen-binding fragment thereof specifically binds to human BSSL (hBSSL). 27. A pharmaceutical composition for reducing the pro-inflammatory effects of BSSL, comprising an isolated antibody and/or an antigen-binding fragment thereof according to any of the preceding claims and a pharmaceutically acceptable carrier or excipient. 28. Use of an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-26 or a pharmaceutical composition according to claim 27 in the treatment and/or prevention of an inflammatory disease 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 steatosis. 29. Use of an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-26 or a pharmaceutical composition according to claim 27 in the treatment and/or prevention of an inflammatory disease selected from the group consisting of a chronic inflammatory disease, a systemic inflammatory disease, an autoimmune disease, an autoinflammatory disease, and an inflammatory disease mediated by natural killer (NK) cells. 30. The use according to claim 29, wherein the inflammatory disease is a chronic inflammatory disease. 31. The use according to claim 29, wherein the inflammatory disease is a systemic inflammatory disease. 32. The use according to claim 29, wherein the inflammatory disease is an autoimmune disease. 33. The use according to claim 29, wherein the inflammatory disease is an autoinflammatory disease. 34. The use according to claim 29, wherein the inflammatory disease is an inflammatory disease mediated by natural killer (NK) cells. 35. The use according to claim 28, wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA) and psoriatic arthritis. 36. A polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-26. 37. An expression vector containing the polynucleotide according to claim 36. 38. A host cell for producing an antibody or antigen-binding fragment thereof according to any one of claims 1-26, wherein the host cell comprises an expression vector according to claim 37. 39. A method for producing an antibody or its antigen-binding fragment, comprising: culturing a cell according to claim 38, containing the expression vector according to claim 37, under conditions where the antibody or antigen-binding fragment thereof is expressed by the cell; and optionally, isolating the antibody or antigen-binding fragment thereof from the cell or from the culture medium in which the cell is cultured. 40. A method for detecting the presence or absence of bile salt stimulated lipase (BSSL) and/or quantifying the level of BSSL in a sample, comprising: contacting the sample with the isolated antibody or antigen-binding fragment thereof according to any one of paragraphs 1-26; and detecting the presence or absence of BSSL in a sample and/or quantifying the level of BSSL in a sample based on the amount of isolated antibody or antigen-binding fragment thereof bound to BSSL. 41. A method for diagnosing a disorder associated with bile salt-stimulated lipase (BSSL), comprising: a) contacting a sample obtained from a subject with an isolated antibody or antigen-binding fragment thereof according to any one of paragraphs 1-26; b) detecting the presence or absence of BSSL and/or quantifying the level of BSSL in a sample based on the amount of isolated antibody or antigen-binding fragment thereof bound to BSSL; and c) a conclusion based on the results of step b) as to whether the subject has a BSSL-related impairment or not.