EA040834B1 - HUMANIZED ANTIBODY TO IL-23p19 OR ITS ANTIGEN-BINDING FRAGMENT, ITS APPLICATION AND PHARMACEUTICAL COMPOSITION CONTAINING THE SPECIFIED ANTIBODY - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates generally to antibodies to IL-23p19 intended for diagnostic and therapeutic use. More specifically, humanized anti-IL-23p19 antibodies and their use in the treatment of various diseases or disorders are described. Pharmaceutical compositions containing these compounds are also described.

Prerequisites for the creation of the invention

Higher eukaryotic organisms have evolved a complex response to pathogens that first manifests itself as an innate immune response, after which an adaptive immune response is triggered. In combination with each other, these two mechanisms not only destroy the pathogens that infect the body, but also create a long-term immunological response against subsequent exposures. A deficiency in these responses can lead to increased susceptibility to infections and/or changes in the adaptive immune response, leading to chronic inflammation and autoimmunity. IL-12, a heterodimeric cytokine composed of p40 and p35 protein subunits, has been considered for many years as a cytokine that is a hallmark of the innate immune response that has a very large impact on adaptive immunity. However, research data on the biological role of this cytokine have led to mixed results. For example, while p40-deficient mice were resistant to collagen-induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE), p35-deficient mice were susceptible to both diseases and even experienced an exacerbation of the disease. These mysteries began to be resolved with the discovery in the late 1990s of a new member of the IL-12 cytokine family, which plays a special role in the immune response, namely, IL-23.

IL-23 consists of a subunit (p40) common to IL-12 and a unique p19 subunit. Despite the presence of a common p40 subunit, the roles of IL-23 and IL-12 are quite different. IL-12 plays an important role in Th1 responses by enhancing differentiation, proliferation and activation of Th1 cells. In contrast, IL-23 promotes the development and maintenance of a recently discovered population of CD4 T helper cells, designated Th17 cells because of their ability to produce IL-17 and related cytokines. There is strong evidence that IL-23 is involved in chronic autoimmune inflammation, and modulation of IL-23 activity may represent a promising therapeutic approach for the treatment of autoimmune diseases.

Thus, there is a need for IL-23 antagonist molecules with valuable pharmacological properties that can be used as therapeutic agents for the treatment of diseases, in particular immunological and autoimmune diseases in humans.

Thus, one of the objectives underlying the present invention was to develop anti-III-23 antagonist molecules, in particular anti-III-23 antagonist molecules, which have a high binding affinity for IL-23.

The next object of the present invention was to develop anti-IL-23 antagonist molecules that have high specificity for IL-23.

The next object of the present invention was to develop anti-III-23 antagonists that have a high blocking activity against the association of IL-23 with its receptor.

The next problem underlying the present invention was to develop anti-III-23 antagonists that have effective cellular activity.

The next object of the present invention was to develop anti-III-23 antagonists that have a preferred bioavailability.

The next object of the present invention was to develop anti-III-23 antagonists that have advantageous biophysical properties.

Further objects underlying the present invention are combinations of any of the above objectives.

Brief summary of the invention

The present invention addresses the above problems and provides antibodies that bind to the p19 subunit of IL-23 protein with high affinity, inhibit IL-23-stimulated production of IL-17 in mouse splenocytes. This antibody does not bind to IL-12 and does not antagonize IL-12, which is a closely related family member of IL-23.

One of the objects of the present invention is a humanized antibody to IL-23p19 or its antigennegative fragment, where the antibody or its antigennegative fragment contains:

a) a light chain variable region containing the amino acid 5 sequence of SEQ ID NO: 19 (CDR1-L); the amino acid sequence of SEQ ID NO: 20 (CDR2-L) and the amino acid sequence of SEQ ID NO: 21 (CDR3-L); And

b) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 66 or 67 (CDR1-H); amino acid 10 sequence of SEQ ID NO: 64 (CDR2-H) and

- 1 040834 amino acid sequence of SEQ ID NO: 65 (CDR3-H), wherein the antibody or antigen-binding fragment thereof has a K _D value for IL-23 of less than 40 pM.

In a preferred embodiment, the humanized anti-IL-23p19 antibody, or antigen-binding fragment thereof, comprises:

a) a light chain variable region containing the amino acid sequence of SEQ ID NO: 19 (CDR1-L); the amino acid sequence of SEQ ID NO: 20 (CDR2-L) and the amino acid sequence of SEQ ID NO: 21-20 (CDR3-L); And

b) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 66 (CDR1-H); the amino acid sequence of SEQ ID NO: 64 (CDR2-H); and the amino acid sequence of SEQ ID NO: 65 (CDR3-H).

In another preferred embodiment, the humanized anti-IL-23p19 antibody, or antigen-binding fragment thereof, comprises:

a) a light chain variable region containing the amino acid sequence of SEQ ID NO: 19 (CDR1-L); amino acid sequence 30 of SEQ ID NO: 20 (CDR2-L) and amino acid sequence of SEQ ID NO: 21 (CDR3-L); And

b) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 67 (CDR1-H); the amino acid sequence of SEQ ID NO: 64 (CDR2-H); and the amino acid sequence of SEQ ID NO: 65 (CDR3-H).

Said humanized anti-IL-23p19 antibody may be a monoclonal antibody.

Another object of this invention is the use of said antibody for the treatment of an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, or cancer.

Namely, for the treatment of psoriasis, inflammatory bowel disease, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis or ankylosing spondylitis.

In addition, the proposed antibody or its antigen-binding fragment can be used to treat asthma or chronic obstructive pulmonary disease (COPD).

Another object of this invention is a pharmaceutical composition containing the proposed antibody or antigennegative fragment and a pharmaceutically acceptable carrier.

Brief description of the drawings

The drawings show:

in fig. 1 - comparative analysis of the primary structure of the mouse and humanized variable regions. In FIG. 1a - 6B8 anti-IL-23p19 antibody with engineered Vk regions;

in fig. 1b - 6B8 anti-IL-23p19 antibody with engineered VH regions. The numbering of amino acids follows the standard Cabot numbering system. Regular font denotes sections of the human sequence; italicized/underlined portions of mouse sequence; shaded areas of the synthetic sequence; Bold/Italic/Underlined are CDRs;

in fig. 2 shows the results of a competitive binding assay of human IL-23 to IL-23R/Fc.

Detailed description of the invention

The IL-23 p19 subunit (also referred to as IL-23p19 and the p19 subunit) is a 189 amino acid polypeptide containing a 21 amino acid (aa) leader sequence (Oppmann et al., Immunity 13, 2000, p. 715, SEQ ID NO: 181). The biological activity of the molecule can only be detected after it has been paired with the IL-12p40 subunit to form IL-23. IL-23 is expressed mainly by activated dendritic cells (DC) and phagocytic cells. The IL-23 receptor has been shown to consist of the IL-12Re1 subunit of the IL-12 receptor paired with a unique subunit designated IL-23R (Parham et al., J. Immunol. 168, 2002, p. 5699). Receptor expression mainly occurs on memory T cells and NK cells. Thus, the expression of this cytokine:receptor pair is likely to be restricted to specific immune cell populations. Although at first it was assumed that IL-12 and IL-23 may have common functions, there is evidence that the picture is different. While IL-12 plays a major role in the production of Th1 cells, IL-23 has been shown to play a critical role in the production and maintenance of a recently discovered subpopulation of Th cells, designated Th17 (Kikly et al., Curr. Opin. Immunol 18, 2006, p.670, Kastelein et al., Ann. Rev. Immunol. 25, 2007, p. 221). These cells produce IL-17A, IL-17F, IL-22 and other pro-inflammatory cytokines such as IL-6 and TNF-α. As will be described below, studies in animal models of the role of these Th17 cells have demonstrated their importance as a driving force in chronic inflammation and autoimmunity.

The humanized antibodies of the invention were identified based on the sequence

- 2 040834 some of the most preferred (leader) mouse antibodies.

The leader mouse antibodies were derived from mouse hybridomas. Mice were immunized using various techniques. For example, antibodies specific for human IL-23p19 proteins or fragments thereof can be raised against an immunogenic antigen such as an isolated IL-23p19 protein, an isolated IL-23 protein, an isolated IL-23 fusion protein, and/or a fragment of any of the above. proteins (including synthetic peptides). For example, an IL-23 fusion protein is used to immunize mice, containing a mouse subunit of IL-23p40 and a human subunit of IL-23p19. Any suitable technique known in the art can be used to obtain immunogenic antigens and produce monoclonal antibodies.

The mouse antibody leader was chosen based on its high affinity for human IL-23. Selected mouse antibodies were humanized to obtain humanized antibodies. These humanized antibodies bind to human IL-23 with high affinity. Thus, an anti-IL-23p19 antibody has a KD value of less than 40 pM. The antibody binds to IL-23p19 with high affinity in the absence of human serum or in the presence of 50% human serum.

The humanized antibody of the present invention also binds to Cynomolgus monkey IL-23 with high affinity.

It binds to IL-23 but does not bind to IL-12. and does not interfere with the biological activity of IL-12, which is a member of the family closely related to IL-23.

The antibody of the present invention inhibits IL-23-stimulated production of IL-17 by mouse splenocytes and inhibits IL-23-induced STAT3 phosphorylation in DB cells.

The humanized antibody of the present invention antagonizes the action of IL-23 by binding to the p19 subunit of IL-23, for example, as measured by the inhibition of cytokines such as IL-17 and IL-22, whose production is stimulated by IL-23, and which is determined to reduce the levels of these cytokines.

The humanized antibody of the present invention has a preferred pharmacokinetic (PK) profile, such as in vivo half-life in cynomolgus monkeys, and has preferred biophysical properties such as, for example, quality, stability, or solubility.

The anti-IL-23p19 antibody may be a monoclonal antibody. An anti-IL-23p19 antibody is a humanized monoclonal antibody, such as a full length humanized monoclonal antibody.

The antibody, or antigen-binding fragment thereof, specifically recognizes an IL-23p19 antigenic epitope or an IL-23p19 epitope. In the context of the present description, these terms refer to a molecule (for example, a peptide) or a fragment of a molecule that/which has the ability to immunologically react with an antibody to IL-23p19, and, for example, includes an IL-23p19 antigenic determinant recognized by any of the antibodies having the following combination of light chain/heavy chain sequences: SEQ ID NOs: 84/121, 86/123, 88/125, 90/127, 91/128, 93/130, 95/132, 97/134, 99/136, 101 /138, 103/140, 105/142, 107/144, 109/146, 111/148, 113/150, 115/152, 117/154, 119/156, 160/166, 160/168, 158/166 or 158/168. Antigenic epitopes of IL-23p19 can be incorporated into proteins, protein fragments, peptides, and the like. Epitopes are in most cases proteins, short oligopeptides, oligopeptide mimetics (eg, organic compounds that mimic the binding ability of the IL-23p19 antigen), or combinations thereof. It is believed that the minimum size of a peptide or polypeptide epitope for an antibody should be about 4-5 amino acids. Peptide or polypeptide epitopes contain, for example, at least seven amino acids, or, for example, at least nine amino acids, or, for example, from about 15 to about 20 amino acids. Because an antibody can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids in an epitope do not need to be contiguous, in some cases they may not even be on the same peptide chain. Epitopes can be determined using various techniques known in the art, such as X-ray crystallography, deuterium/hydrogen exchange mass spectrometry (HXMS), site-directed mutagenesis, alanine-scanning mutagenesis, and peptide screening methods.

The general structure of an antibody or immunoglobulin is well known to those skilled in the art. These molecules are heterotetrameric glycoproteins, typically with a molecular weight of about 150,000 Da, consisting of two identical light (L) chains and two identical heavy (H) chains, and are generally referred to as full length antibodies. Each light chain is covalently linked to the heavy chain by a single disulfide bond to form a heterodimer, and a heterotetrameric molecule is formed by a covalent disulfide bond between two identical heterodimer heavy chains. Although light and heavy chains are linked by a single disulfide bond, the number of disulfide bonds between two heavy chains varies depending on

- 3 040834 bridges from the immunoglobulin isotype. Each heavy and light chain also has evenly distributed intrachain disulfide bridges. Each heavy chain has an amino-terminal variable domain (V _H ) followed by three or four constant domains ( _CH 1, CH2, CH3 and _CH 4) and a hinge between C _H 1 and CH2. Each light chain has two domains, an amino-terminal variable domain (V _L ) and a carboxy-terminal constant domain (C _L ). The VL domain is non-covalently linked to the VH domain, and the CL domain is generally covalently linked to the C _H 1 domain via a disulfide bond. It is believed that certain amino acid residues form the interface between the variable regions of the light and heavy chains (Chothia et al., J. Mol. Biol. 186, 1985, p. 651-663). In the context of the present description, variable domains are also referred to as variable regions.

Certain regions in the variable domains differ significantly among different antibodies, ie. they are hypervariable. These hypervariable regions contain residues that are directly involved in binding and determine the specificity of each particular antibody in relation to its specific antigenic determinant. Hypervariability in the variable domains of both the light chain and the heavy chain is concentrated in three segments, which are referred to as complementarity-determining regions (hypervariable regions) (CDRs) or hypervariable loops (HVLs). CDRs are determined by sequence comparison according to the method described by Kabat et al. in: Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991, a HVL (also referred to as CDR) is structurally defined based on the three-dimensional structure of the variable domain according to the method described in Chothia and Lesk, J. Mol. Biol. 196, 1987, p. 901-917. These two methods lead to some differences in CDR identification. According to Cabot's nomenclature, CDR-L1 is located at approximately residues 24-34, CDR-L2 at approximately residues 50-56, and CDR-L3 at approximately residues 89-97 in the light chain variable domain; CDR-H1 is located at approximately residues 31-35, CDR-H2 at approximately residues 50-65, and CDR-H3 at approximately residues 95-102 in the heavy chain variable domain. The exact numbers of residues that form a particular CDR will vary depending on the sequence and size of the CDR. Based on the amino acid sequence of the variable region of an antibody, those skilled in the art can determine which residues are contained in a particular CDR using conventional methods. Thus, the CDR1, CDR2, CDR3 of the heavy and light chains determine the unique and functional properties specific to a given antibody.

The three CDRs in each of the heavy and light chains are separated by framework regions (FRs) that contain sequences that tend to be less variable. Starting from the amino terminus towards the carboxy terminus of the heavy and light chain variable domains, FRs and CDRs are ordered as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The largely β-sheet conformation of FR allows the CDRs in each of the strands to be in close proximity to each other as well as to CDRs from the other strand. The resulting conformation is inherent in the antigen-binding center (see Kabat et al., NIH Publ. No. 91-3242, vol. I, 1991, p. 647-669), although it is not a necessary condition that all CDR residues are directly involved in antigen binding .

FR residues and Ig constant domains are not directly involved in antigen binding, but are involved in antigen-associated and/or antigen-mediated effector function. Some FR residues are likely to have a pronounced effect on antigen binding in at least three ways: by binding non-covalently directly to an epitope, by interacting with one or more CDR residues, and by influencing the interface between heavy and light chains. The constant domains are not directly involved in antigen binding, but mediate various Ig effector functions such as antibody involvement in antibody-mediated cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-mediated cellular phagocytosis (ADCP).

Vertebrate immunoglobulin light chains are assigned to one of two distinct classes, kappa (k) and lambda (λ), based on the amino acid sequence of the constant domain. By comparison, mammalian immunoglobulin heavy chains are classified into one of five major classes based on the sequence of constant domains: IgA, IgD, IgE, IgG, and IgM. IgG and IgA are further subdivided into subclasses (isotypes), for example, IgG1, IgG ₂ , IgG ₃ , IgG ₄ , IgA1 and IgA ₂ . Heavy chain constant domains that correspond to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of native immunoglobulin classes are well known.

The terms antibody, antibody to IL-23p19, humanized antibody to IL-23p19, humanized antibody to the IL-23p19 epitope, and humanized antibody variant to the IL-23p19 epitope refer, in particular, to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies); and antibody fragments, such as variable domains and other antibody regions, that have the desired biological activity, such as the ability to bind to IL-23p19. The term monoclonal antibody (MAb) refers to an antibody that is highly specific,

- 4 040834 a term is an individual antigenic determinant, an epitope. Thus, the adjective monoclonal indicates a distinguishing feature of antibodies that target an identical epitope and is not associated with a requirement to produce antibodies by any particular method. It should be obvious that monoclonal antibodies can be obtained using any technique or methodology known in this field; including, for example, the hybridoma method (Kohler et al., Nature 256, 1975, p. 495), or recombinant DNA methods known in the art (see, for example, US No. 4816567), or methods for isolating recombinantly obtained monoclonal Antibody DNA using antibody phage libraries using the techniques described in Clackson et al., Nature 352, 1991, p. 624-628 and Marks et al., J. Mol. Biol. 222, 1991, p. 581-597.

The term monomer refers to the homogeneous form of an antibody. For example, in the case of a full length antibody, monomer means a monomeric antibody that has two identical heavy chains and two identical light chains.

Chimeric antibodies consist of the heavy and light chain variable regions of an antibody from one species (e.g., non-human mammal, such as a mouse) and the heavy and light chain constant regions of an antibody from another species (e.g., a human) and can be generated by joining the sequences DNA encoding variable regions of an antibody from a first species (eg, mouse), with DNA sequences of constant regions of an antibody from a second species (eg, human), and transforming the host with an expression vector containing the linked sequences, allowing the host to produce a chimeric antibody. Alternatively, the chimeric antibody may also be an antibody in which one or more heavy and/or light chain regions or domains are identical, homologous, or a variant of the corresponding sequence in a monoclonal antibody from a different immunoglobulin class or isotype or from a consensus or germline sequence. lines. Chimeric antibodies may include fragments of these antibodies, provided that the antibody fragment has the desired biological activity of the parent antibody, such as the ability to bind to the same epitope (see, for example, US No. 4816567; and Morrison et al., Proc. Natl. Acad Sci USA 81, 1984, p.68516855).

The terms antibody fragment, antibody fragment to IL-23p19, antibody fragment to IL-23p19 epitope, fragment of humanized antibody to P.-23p19, fragment of humanized antibody to IL-23p19 epitope, fragment of variant humanized antibody to IL-23p19 epitope refer to part of the full-length antibodies to I.-23p19, which retains the variable region or its functional ability, for example, the specificity of binding to the epitope of I.-23p19. Examples of antibody fragments include, but are not limited to, Fab-, Fab'-, F(ab') _2- , Fd-, Fv-, scFv- and scFv-Fc fragment, dimeric antibody (diabodie), linear antibody, single chain antibody, minibody, dimeric antibody formed from antibody fragments, and multispecific antibodies formed from antibody fragments.

Full length antibodies can be treated with enzymes such as papain or pepsin to produce the desired antibody fragments. Papain digestion is used to produce two identical antigen-binding antibody fragments, referred to as Fab fragments, each with one antigen-binding site, the remainder being referred to as an Fc fragment. The Fab fragment also contains a light chain constant domain and a heavy chain C _n 1 domain. After treatment with pepsin, an F(ab')2 fragment is obtained which bears two antigen binding sites and still retains the ability to cross-link with the antigen.

Fab' fragments differ from Fab fragments by the presence of additional residues, including one or more cysteine residues from the antibody hinge region at the C-terminus of the C _n 1 domain. F(ab') ₂ antibody fragments are pairs of Fab' fragments linked by cysteine residues in the hinge region. Other chemical combinations of antibody fragments are known.

The Fv fragment contains a complete antigen-recognition and antigen-binding center, consisting of a dimer including the variable domain of one heavy and one light chain, which are in close non-covalent association. In this configuration, the three CDRs of each variable domain interact with a specific antigen-binding site on the surface of the VH-VL dimer. In general, six CDRs determine the specificity of an antibody for antigen binding.

A single chain Fv or scFv fragment of an antibody is a single chain Fv variant containing the V _H and VL domains of an antibody, the domains being present in a single polypeptide chain. The single chain Fv has the ability to recognize and bind to an antigen. The scFv polypeptide may optionally also contain a polypeptide linker located between the V _H - and VL domains, to facilitate the formation of the three-dimensional structure required for binding the scFv to the antigen (see, for example, Pluckthun, in: The Pharmacology of monoclonal Antibodies, vol. 113 , edited by Rosenburg and Moore, Springer-Verlag, New York, 1991, pp. 269-315).

The term dimeric antibody (diabodi) refers to small antibody fragments with two antigen-binding sites, the fragments containing a heavy chain variable domain (V _H ) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or

- 5 040834

_Vl - _Vh ). Dimeric antibodies are described in more detail, for example, in Holliger et al., Proc. Natl. Acad. sci. USA 90, 1993, p. 6444-6448.

Other recognizable antibody fragments include fragments that contain a pair of Fd segments arranged in tandem (V _h -C _h 1 -V _h -C _h 1 ) forming a pair of antigen-binding sites. Said linear antibodies may be bispecific or monospecific as described, for example, in Zapata et al., Protein Eng. 8(10), 1995, p. 1057-1062.

A humanized antibody or humanized antibody fragment is a specific type of chimeric antibody that includes a variant amino acid sequence of an immunoglobulin or fragment thereof that has the ability to bind to a predetermined antigen and that contains one or more FRs that have(s) an amino acid sequence, substantially corresponding to the sequence of a human immunoglobulin, and one or more CDRs which(s) have(s) an amino acid sequence which substantially corresponds to the amino acid sequence of a non-human immunoglobulin. Said non-human amino acid sequence, often referred to as an import sequence, is typically derived from the import domain of an antibody, in particular the variable domain. In general, a humanized antibody comprises at least the CDR or HVL of a non-human antibody inserted between the FR of a human heavy or light chain variable domain. The present invention describes specific humanized antibodies to IL-23p19 that contain CDRs derived from mouse monoclonal antibodies, or humanized CDRs that are shown in Tables 3 and 4, inserted between FRs in human germline heavy and light chain variable domain sequences. It should be apparent that certain mouse FR residues can be imported to provide humanized antibody function, and therefore certain residues of the human germline heavy and light chain variable domain sequences are modified to be the same as in the corresponding mouse sequence.

A humanized anti-IL-23p19 antibody may comprise substantially all of at least one, and typically two, variable domains (such as, for example, those found in Fab-, Fab'-, F(ab') ₂ -, Fabc- and Fv- fragments) in which all or substantially all of the CDRs correspond to those of non-human immunoglobulins and all of the CDRs are murine or humanized sequences detailed in Tables 1-4 below and all or substantially all of the FRs are FRs that have a consensus sequence or a germline sequence of a human immunoglobulin. In another aspect of the invention, the humanized anti-IL-23p19 antibody also includes at least a portion of the Fc region of an immunoglobulin, typically a human immunoglobulin. Typically, an antibody will contain both a light chain and at least a heavy chain variable domain. The antibody may also optionally include one or more of the following regions: C _h1 hinge region, C _h2 , C _h3 and/or C _h4 heavy chain.

The humanized IL-23p19 antibody can be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG ₃ , IgG ₄ , IgA1, and IgA2. For example, the constant domain may be a complement-fixing constant domain if the humanized antibody is required to have cytotoxic activity, and the isotype is typically IgG1. If the specified cytotoxic activity is not required, then the constant domain may be of another isotype, for example, IgG2. Alternatively, a humanized anti-IL-23p19 antibody may contain sequences from more than one class or isotype of immunoglobulins, and selection of specific constant domains to optimize desired effector functions is within the skill of ordinary skill in the art. Specifically, the antibodies may be antibodies of the IgG1 isotype, and more specifically, antibodies of the IgG1 isotype in which effector functions are disabled.

The FR and CDR or HVL regions of a humanized anti-IL-23p19 antibody do not have to exactly match the parental sequences. For example, one or more residues in an import CDR or HVL or in a consensus or germline FR sequence can be altered (eg, by mutagenesis) by substitution, insertion, or deletion such that the resulting amino acid residue is not identical to the original residue at the corresponding position of any parent sequence, but the antibody still retains a function, such as the ability to bind to IL-23p19. Said change, as a rule, should not be extensive and should be conservative changes. Typically, at least 75% of the residues, more often at least 90%, and most often more than 95% or more than 98%, or more than 99%, of the humanized antibody must match the residues of the parent consensus or germline FR sequence and import CDR sequences.

Immunoglobulin residues that affect the interface between the heavy and light chain variable regions (V _l -V _h interface) are residues that affect the proximity or orientation of the two chains relative to each other. Specific residues that

- 6 040834 can participate in interactions between chains, are residues V _L 34, 36, 38, 44, 46, 87, 89, 91, 96 and 98 and residues VH 35, 37, 39, 45, 47, 91, 93, 95, 100 and 103 (according to the numbering system proposed by Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987)). US Pat. No. 6,407,213 also discusses that residues such as V _L residues 43 and 85 and VH residues 43 and 60 may be involved in this interaction. Although these residues are only for human IgG, they can be considered for other species. Important antibody residues that are expected to be involved in interstrand interactions are selected for replacement in the consensus sequence.

The terms consensus sequence and consensus antibody refer to the amino acid sequence that contains the most frequently occurring amino acid residue at each position in all immunoglobulins of any class, isotype, or subunit structure, such as the variable domain of a human immunoglobulin. The consensus sequence may be based on immunoglobulins of specific species or multiple species. By consensus/consensus sequence, structure, or antibody is meant the human consensus sequence specified in specific embodiments of the invention, and refers to the amino acid sequence that contains the most frequently occurring residues at each position in all human immunoglobulins of a particular class, isotype, or subunit structure. Thus, the consensus sequence contains an amino acid sequence that has at each position an amino acid that is present in one or more known immunoglobulins, but which may not be an exact copy of the complete amino acid sequence of any individual immunoglobulin. The consensus sequence variable region is not derived from any naturally occurring antibody or immunoglobulin (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991) and its variants. Heavy and light chain FR consensus sequences and variants thereof are valuable sequences for generating humanized anti-IL-23p19 antibodies (see, for example, US Nos. 6,037,454 and 6,054,297).

Human germline sequences are naturally present in human antibody populations. The combination of these germline genes creates a variety of antibodies. The antibody light chain germline sequences are derived from the conserved v and j genes of the human kappa or lambda germline. Similarly, heavy chain sequences are derived from the v, d, and j germline genes (LeFranc M.-R. and LeFranc G., The Immunoglobulin Facts Book, Academic Press, 2001).

As used herein, each of variant, anti-IL-23p19 antibody variant, humanized anti-IL-23p19 antibody variant, or humanized anti-IL-23p19 antibody variant refers to a humanized anti-IL-23p19 antibody that has at least the murine variable region CDR sequence light chain, selected from any of the sequences presented in table. 1, or the sequence of the mouse heavy chain CDR derived from the mouse monoclonal antibody presented in table. 2. Variants include sequences having one or more amino acid substitutions in the variable regions of one or both of the light chains or heavy chains, provided that the amino acid substitution does not significantly affect the binding of the antibody to IL-23p19. Examples of humanized antibodies are the antibodies designated Antibody A, Antibody B, Antibody C and Antibody D and their various heavy and light chains as shown in SEQ ID NOs: 174 and 180 and SEQ ID NOs: 176 and 178, respectively.

An isolated antibody is one that has been identified and separated and/or purified from a component of its natural environment. Contaminant components of the natural environment of an antibody are substances that may interfere with the diagnostic or therapeutic use of the antibody and may be enzymes, hormones, or other proteinaceous or non-proteinaceous solutes. The isolated antibody should be an antibody purified to greater than 95% by weight.

An isolated antibody is an antibody in situ within the recombinant cells in which it is produced, if none of the components of the antibody's natural environment is present. However, as a rule, the selected antibody is obtained using at least one stage of purification, during which the recombinant cellular material is removed.

The term antibody characterization refers to factors that are involved in an antibody's recognition of an antigen or antibody performance in vivo. Changes in the amino acid sequence of an antibody can affect properties of the antibody, such as folding, and can affect physical factors, such as the initial rate of binding of the antibody to the antigen (k _a ), the dissociation constant of the antibody from the antigen (kd), the affinity constant of the antibody to the antigen ( Kd), antibody conformation, protein stability, and antibody half-life.

In the context of the present description, the term epitope labeling refers to an antibody to IL-23p19 fused with an epitope label. An epitope tag is a polypeptide consisting of

- 7 040834 the exact amount of amino acids to create an epitope for the production of antibodies, while it is created so that it does not affect the desired activity of humanized antibodies to IL-23p19. An epitope tag is typically largely unique, with the result that an antibody that is generated against an epitope tag does not overreact with other epitopes. Acceptable tag polypeptides typically contain at least 6 amino acid residues and typically contain about 8-50 amino acid residues, or about 930 residues.

Examples of epitope tags and antibodies that bind to an epitope are the flu HA polypeptide tag (HA of the influenza virus) and its corresponding 12CA5 antibody (Field et al., Mol. Cell. Biol. 8, 1988? 2159-2165); c-myc tag and its corresponding antibodies 8F9, 3C7, 6E10, G4, B7 and 9E10 (Evan et al., Mol. Cell. Biol. 5(12), 1985, p. 3610-3616); and a label comprising herpes simplex virus glycoprotein D (gD) and its corresponding antibody (Paborsky et al. Protein Engineering, 3(6), 1990, p. 547-553). An epitope tag is an epitope that binds to a rescue receptor. As used herein, a rescue receptor-binding epitope refers to an epitope in the Fc region of an IgG molecule (such as IgG1, IgG ₂ , IgG ₃ , or IgG ₄ ) responsible for prolonging the in vivo serum half-life of an IgG molecule.

Antibodies can be conjugated to a cytotoxic agent. It can be any substance that inhibits or interferes with cell function and/or causes cell destruction. The term includes radioactive isotopes (such as I ¹³¹ , I ¹²⁵ , Y ⁹⁰ and Re ¹⁸⁶ ), chemotherapeutic agents and toxins such as enzymatically active toxins derived from bacteria, fungi, plants or animals, and their fragments. Said cytotoxic agents can be coupled to humanized antibodies using standard procedures and used, for example, to treat a patient for whom antibody-based therapy is indicated.

A chemotherapeutic agent is a chemical compound that can be used to treat cancer. Numerous examples of chemotherapeutic agents are known that can be conjugated to therapeutic antibodies. Examples of these chemotherapeutic agents are alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamin; acetogenins (primarily bullatacin and bullatacinone); camptothecin (including the synthetic analog of topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin, auristatins (including analogues such as monomethyl-auristatin E and monomethyl-auristatin F); duocarmycin (including synthetic analogues of KW2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen analogues of mustard gas (mustard gas) such as chlorambucil, chlophasin, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlormethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trofosfamide, uramustine (a nitrogen and mustard derivative of uracil); nitrosureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as enediine antibiotics (e.g. calicheamincin, especially calicheamicin gamma 1I and calicheamincin phiI1 (see, for example, Agnew, Chem. Intl. Ed. Engl., 33, p. 183-186); dynemycin, including dynemycin A ; bisphosphonates such as clodronate; esperamycin; as well as neocarzinostatin chromophore and related chromophore enediin antibiotic chromophores), aclacinomysins, actinomycin, automycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinofelin, chromomycins, dactinomycin, deunorubicin -5-oxo-L-norleucine, doxorubicin (Adriamycin™) (including morpholino-doxorubicin, cyanmorpholino-doxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin , olivomycins, peplomycin, potfiromycin, puromycin, chelomycin, rhodarubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamitrine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuredine, doxyfluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenergics such as aminoglutethimide, mitotane, trilostane; a folic acid substitute such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; democolcin; diazichon; elformitin; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamed; pyrarubicin; loxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazone ki

- 8 040834 slots; triazichon; 2,2',2-trichlorotriethylamine; trichothecenes (primarily T-2 toxin, verracurin A, roridin A and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Anthony, France); chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (Navelbine™); novantron; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; SRT-11; topoisomerase inhibitor RFS-2000; difluoromethylornithine (DMPO); retinoids such as retinoic acid; capecitabine, and their pharmaceutically acceptable salts, acids or derivatives of any of the above compounds. Also included within the scope of this definition are antihormonal agents that regulate or inhibit the effect of a hormone on tumors, such as antiestrogen and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene. , keoxifene, LY117018, onapristone and toremifene (Fareston™); aromatase inhibitors, which inhibit the aromatase enzyme, regulate adrenal estrogen production, such as 4(5)-imidazoles, aminoglutethimide, megestrol acetate (Megace™), exemestane, formestane, fadrozole, vorozol (Rivisor™), letrozole (Femara™) ) and anastrozole (Arimidex™); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin, as well as pharmaceutically acceptable salts, acids and derivatives of any of the above compounds. Any one or more of these agents can be conjugated with humanized antibodies to provide a valuable therapeutic agent that can be used to treat a variety of disorders.

Antibodies can also be conjugated to prodrugs. A prodrug is a precursor or derivative of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and has the ability to be activated or converted to a more active parent form by enzymes (see, for example, Wilman in: Prodrugs in Cancer Chemotherapy, Biochemicals Society Transactions, 14, 1986, pp. 375-382, 615th Belfast Symposium (1986), and Stella et al in Prodrugs: A Chemical Approach to Targeted Drug Delivery, Directed Drug Delivery, edited by Borchardt et al., Humana Press, 1985, pp. 247-267). Suitable prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, prodrugs containing an optionally substituted phenoxyacetamide, or prodrugs containing an optionally substituted phenylacetamide , 5-fluorocytosine- and other 5-fluorouridine-containing prodrugs that can be converted to a more active cytotoxic free dosage form. Examples of cytotoxic drugs that can be derivatized as prodrugs include, but are not limited to, the chemotherapeutic agents described above.

For diagnostic purposes, as well as for monitoring treatment, antibodies can also be labeled, either labeled alone or labeled in combination with an additional second agent (prodrug, chemotherapeutic agent, etc.). The label, in contrast to the second additional agent, is a substance that is a detectable compound or a detectable composition, and can be conjugated directly or indirectly with a humanized antibody. The label itself can be a detectable substance (for example, radioisotope labels or fluorescent labels) or, as in the case of an enzymatic label, it can catalyze a chemical change in the compound or composition, which is/is a substrate, which can be detected. Labeled humanized anti-IL-23p19 antibody can be prepared and used for a variety of purposes, including in vitro and in vivo diagnostics.

Antibodies can be formulated to be part of a liposome formulation for efficient in vivo administration. A liposome is a small vesicle composed of various types of lipids, phospholipids and/or surfactants. Liposomes can be used to administer to a mammal a compound or composition, for example, a humanized anti-IL-23p19 antibody provided herein, optionally in combination or in combination with one or more pharmaceutical active ingredients and/or labels. The components of liposomes are usually arranged in a bilayer structure similar to the lipid structure of biological membranes.

An isolated nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one nucleic acid molecule of the contaminant with which it is normally associated in the natural source of the antibody nucleic acid. The isolated nucleic acid molecule is different from the nucleic acid molecule found in naturally occurring cells.

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One or more control sequences can be used to effect recombinant expression, ie. polynucleotide sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotic cells include, for example, promoter, operator, and ribosome binding site sequences. In eukaryotic cells, control sequences include, but are not limited to, promoters, polyadenylation signals, and enhancers. These control sequences can be used to express and produce a humanized anti-IL-23p19 antibody in prokaryotic and eukaryotic host cells.

A nucleic acid is operably linked if it is operably linked to another nucleotide sequence. For example, a presequence or secretory leader nucleic acid is operably linked to a nucleic acid encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is located such that it can facilitate translation. In general, operably linked means that the linked DNA sequences are contiguous, and in the case of a secretory leader, are contiguous and in reading frame. However, enhancers do not need to be contiguous. Linking is accomplished by insertion by ligation into appropriate restriction sites. If such sites do not exist, then synthetic oligonucleotide adapters or linkers can be used.

In the context of the present description, the terms cell, cell line, and cell culture are used interchangeably, and all such terms include progeny. Thus, the terms transformants and transformed cells include the primary cell of interest and cultures derived from it, regardless of the number of passages.

The term treated mammal refers to any animal that belongs to the class of mammals, including humans, domestic and farm animals, and zoo animals intended for sports or pets, such as dogs, horses, cats, cows, etc. .P. Preferably the mammal is a human.

In the context of the present description, the concept of a violation means any condition that can be alleviated by treatment with a humanized antibody to IL-23p19 presented in the present description. It includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question. Examples of disorders that can be treated according to the invention are (but not limited to) inflammatory, angiogenic, autoimmune and immunological disorders, respiratory disorders, cancer, hematological malignancies, benign and malignant tumors, leukemias and lymphoid malignancies.

The terms cancer and cancerous refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth. Examples of cancer are, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

As used herein, an IL-23-associated disorder or IL-23-associated disease refers to a condition in which IL-23 activity is involved in a disease and IL-23 is typically expressed abnormally. IL-23 associated disorders include diseases and disorders of the immune system such as autoimmune disorders and inflammatory disorders. These conditions include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, multiple sclerosis, psoriasis, psoriatic arthritis, inflammatory bowel disease (eg, ulcerative colitis and Crohn's disease), pneumonia, asthma and idiopathic thrombocytopenic purpura (ITP) and ankylosing spondylitis.

The term intravenous infusion refers to the introduction of an agent into the vein of a diseased animal or human over a period of time in excess of about 15 minutes, typically within about 30-90 minutes.

The term intravenous bolus or intravenous pulse refers to the administration of a drug to an animal or human such that the drug enters the body in about 15 minutes or less, typically 5 minutes or less.

Subcutaneous administration refers to the introduction of an agent under the skin of a diseased animal or human, preferably into a pocket between the skin and underlying tissue, by relatively slow, sustained administration from a drug-containing reservoir. By pinching or pulling the skin up and away from the underlying tissue, a pocket can be created.

The term subcutaneous infusion refers to the introduction of a drug under the skin of a diseased animal or human, preferably into a pocket between the skin and underlying tissue, by relatively slow, sustained administration from a drug-containing reservoir into

- 10 040834 for a period of time constituting (but not limited to) 30 minutes or less, or 90 minutes or less. The infusion may optionally be carried out by subcutaneous implantation of a drug pump implanted under the skin of a sick animal or human, the pump delivering the drug in a predetermined amount over a predetermined time period of 30 minutes, 90 minutes, or a period of time corresponding to duration of the treatment regimen.

The term subcutaneous bolus refers to the administration of a drug under the skin of a sick animal or human, wherein the duration of drug administration in the form of a bolus is less than about 15 minutes; less than 5 min, and still within less than 60 p. Insertion is made into a pocket between the skin and underlying tissue, where the pocket can be created by pinching or pulling the skin up and away from the underlying tissue.

In the context of the present description, the concept of a therapeutically effective amount refers to the amount of active substance, when used, the weakening or alleviation of one or more symptoms of the disorder to which treatment is directed. A therapeutically effective amount refers to the concentration in the patient's serum at which the active substance is found to be effective in slowing the progression of the disease. Efficacy can be assessed in conventional ways depending on the condition being treated.

In the context of the present description, the concepts of treatment and therapy, etc. refers to therapeutic, as well as preventive or suppressive measures for a disease or disorder that result in any clinically important or beneficial effect, including (but not limited to) alleviation or amelioration of one or more symptoms, regression, slowing or cessation of the development of the disease or violations. Thus, for example, treatment encompasses the administration of an agent before or after the onset of a symptom of a disease or disorder, which results in the prevention or elimination of one or more signs of the disease or disorder. In another example, the term refers to the administration of an agent after a clinical manifestation of a disease to control the symptoms of the disease. In addition, in the context of the present description, the term treatment or therapy refers to the administration of an agent after the onset and after the development of clinical symptoms, where the administration affects the clinical parameters of the disease or disorder, such as the degree of tissue damage or the number and spread of metastases, regardless of whether whether or not the treatment results in alleviation of the disease. In addition, if the use of compositions containing antibodies, either alone or in combination with another therapeutic agent, relieves or alleviates at least one of the symptoms of the disorder being treated in comparison with the indicated symptom without the use of a composition containing a humanized anti-IL-23p19 antibody, then the result should be considered as an effective treatment for the desired disorder, whether or not all symptoms of the disorder are alleviated.

The term package insert refers to the instructions that are commonly included in the packaging of therapeutic products for sale, which contain information about the indications, methods of use, administration, contraindications and / or precautions for the use of these therapeutic products.

Antibodies.

Humanized anti-IL-23p19 antibodies and binding agents can inhibit the production of Th17-associated cytokines that are involved in chronic autoimmune and inflammatory diseases. Thus, humanized anti-IL-23p19 antibodies and binding agents can be used to treat a variety of diseases or disorders. Both the humanized anti-IL-23p19 antibody and the IL-23p19 binding agent comprise at least a region that specifically recognizes an IL-23p19 epitope (ie, an antigen-binding fragment).

During the initial evaluation, mouse antibodies were selected based on their IL-23p19 binding characteristics.

Thus, such antibodies are characterized by a K _D value for IL-23, in particular for human IL-23, of less than 100 pM, or a K _D value of less than 40 pM, or a K _D value of less than 20 pM , or a K _D value of less than 10 pM, or a K _D value of less than 1 pM.

Selected mouse antibodies have light chain variable regions and heavy chain variable regions, which are presented in table. 1 and .:

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Table 1

Mouse leader antibodies to IL-23p19 VK sequences

2Dlvk GACATTGTGCTGACCCAATCTCCAGGTTCTTTGGCTGTGTCTCTAGGGCAGAGG GCCACCATATCCTGCAGAACCAGTGAAAGTGTTTATAGTTATGGCCAAAATTTT ATACACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCG TGCATCCAACCTGGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTA GGACAGACTTCACCCTCACCATGAATCCTGTGGAGGCTGATGATGTTGCAACC TATTACTGTCAGCAAACTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAA GCTGGAAATAAGA (SEQ ID NO: 83) DIVLTQSPGSLAVSLGQRATISCRTSESVYSYGQNFIHWYQQKPGQPPKLLIY RASNLESGIPARFSGSGSRTDFTLTMNPVEADDVATYYCQQTNEDPYTFGGG TKLEIR (SEQ ID NO: 84) 6B8Vk GACATTGTGATGACCCAGTCTCACAAATTCTTGTCCACATCAGTGGGAGA CAGGGTCACCATCACTTGCAAGGCCAGTCGGGATGTGGCTATTGCTGTAG CCTGGTATCAACAGAAACCAGGGCAATCTCCTAAACTACTTCTTTTCTGG GCATCCACCCGACACACTGGGGTCCCTGATCGCTTCACAGGCAGTGGATC TCGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGG CAGATTATTTCTGTCACCAATATAGCAGCTATCCATTCACGTTCGGCTCGG GGACAAAGTTGGAAATAAAG (SEQ ID NO: 85) DIVMTQSHKFLSTSVGDRVTITCKASRDVAIAVAWYQQKPGQSPKLLLFWAS TRHTGVPDRFTGSGSRTDFTLTISNVQSEDLADYFCHQYSSYPFTFGSGTKLEI K (SEQ ID NO: 86) 9D12-Vk GACATTGCGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTGGGGCAG AGGGCCACCATATCCTGCAGAGCCAGTGAAACTATTAATTTTTATGGCAC TAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGTCACCCAAACTCC

TCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGT GGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGC TGATGATGTTGCAACCTATTACTGTCAGCAAACTAATGAGGATCCGTACA CGTTCGGAGGGGGGACTAAGTTGGAAATAAAA (SEQ ID NO: 87) DIALTQSPASLAVSLGQRATISCRASETINFYGTSFMHWYQQKPGQSPKLLIYRASN LESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTNEDPYTFGGGTKLEIK (SEQ ID NO: 88) 15Cllvk GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGAT CAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGG AAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGC TCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTC AGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAACAGAGTGG AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCG TACACGTTCGGAGGGGGGACCCAGCTGGAAATAAAA (SEQ ID NO: 89) DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLL IYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPYTFGG GTQLEIK (SEQ ID NO: 90) 15Flvk DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQRSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLTINSVEPEDVGVYYCQNGHSFPFTFGSGTKLEIK (SEQ ID NO: 91) 18D3vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGAGTCTCTCTTTCCTGCAGGGCCAGTCAGAGTATTAGCGACTACTTAT ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATTT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCACTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCGACAGTGTGGAACCTGATGATGTTG GAGTCTTTTTCTGTCAAAATGGTCACAGCTTTCCGTTCACGTTCGGAGGGG GGACCAAGCTGGAAATAAAA (SEQ ID NO: 92) DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKFASQ SISGIPSRFTGSGSGSDFTLSIDSVEPDDVGVFFCQNGHSFPFTFGGGTKLEIK (SEQ ID NO: 93) 18C4vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGAGTACTTAC ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATAT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTG GAGTGTATTACTGTCAAAATGGTCACAGCTTTCCATTCACGTTCGGCTCG GGGACAAAGTTGGAAATAAAA (SEQ ID NO: 94) DIVMTQSPATLSVTPGDRVSLSCRASQSISEYLHWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPFTFGSGTKLEIK (SEQ ID NO: 95) 18E5vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGACTACTTAT ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATTT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCACTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCGACAGTGTGGAACCTGATGATGTTG GAGTCTTTTTCTGTCAAAATGGTCACAGCTTTCCGTTCACGTTCGGAGGGG GGACCAAGCTGGAAATAAAA (SEQ ID NO: 96) DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLYWYQQKSHESPRLLIKFASQ SISGIPSRFTGSGSGSDFTLSIDSVEPDDVGVFFCQNGHSFPFTFGGGTKLEIK (SEQ ID NO: 97) 20E8vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGAGTATTTAC ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATAT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTG GAGTTTATTACTGTCAAAATGGTCACAGCTTTCCATTCACGTTCGGCTCGG

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GGACAAAGTTGGAAATAAAA (SEQ ID NO: 98) DIVMTQSPATLSVTPGDRVSLSCRASQSISEYLHWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPFTFGSGTKLEIK (SEQ ID NO: 99) 22E2vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGAGTCTCTCTCTCCTGCAGGGCCAGCCAGAGTATTAGCGTCTACTTAC ACTGGTATCAACAAAAATCACCTGAGTCTCCAAGGCTTCTCATCAAATAT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTG GAGTTTATTACTGTCAAAATGGTCACAGCTTTCCATTCACGTTCGGCTCGG GGACAAAGTTGGAAATAAAA (SEQ ID NO: 100) DIVMTQSPATLSVTPGDRVSLSCRASQSISVYLHWYQQKSPESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPFTFGSGTKLEIK (SEQ ID NO: 101) 24A54vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAAA TAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGACTACTTAC ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATAT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATC AGGGTCAAATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTG GAGTGTATTATTGTCAAAATGGTCACAGCTTTCCATTCACGTTCGGCTCGG GGACAAAGTTGGAAATAAAA (SEQ ID NO: 102) DIVMTQSPATLSVTPGNRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSNFTLSINSVEPEDVGVYYCQNGHSFPFTFGSGTKLEIK (SEQ ID NO: 103) 26F7Vk GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTTTCTCTGGGGCAG AGGGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGATTCTCTGACTA TTTTTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCC TCATCTACCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGT GGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGA GGAGGATGCTGCAACCTATTACTGTCAGAACAGTAGGGAGCTTCCGTACA CGTTCGGAGGGGGGACCAAGCTGGAGATAAAA (SEQ ID NO: 104) DIVLTQSPASLAVSLGQRATISCRASKSVRFSDYFYMHWYQQKPGQPPKLLI YLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQNSRELPYTFGG GTKLEIK (SEQ ID NO: 105) 27G8vk GACATTGTGTTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAG AGGGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCTA TAGTTATATACACTGGTACCAACAGAAACCGGGACAGCCACCCAAATTCC TCATCTATCTTGCATCCAACCTAGATTCTGGGGTCCCTGCCAGGTTCAGTG GCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAG GAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGCTTCCGTACAC GTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 106) DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYIHWYQQKPGQPPKFLIY LASNLDSGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPYTFGGG TKLEIK (SEQ ID NO: 107) 31H9vk GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGACTACTTAC ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATAT GCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATC AGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTG GAGTGTATTACTGTCAAAATGGTCACAGCTTTCCGTACACGTTCGGAGGG GGGACCAAGCTGGAAATAAAA (SEQ ID NO: 108) DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQ SISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPYTFGGGTKLEIK (SEQ ID NO: 109) 34G3Vk GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGAT CAAGCCTCCATCTTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGG AAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGC

- 13 040834

TCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCCGACAGGTTCA GTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGA GGCTGAGGATCTGGGAGTTTATTCTGCTCTCAAAGTACACATGTTCCGT ACACGTTCGGAGGGGGACCAAGCTGGAAATAAAT (SEQ ID NO: 110) DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLL IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGG GTKLEIN (SEQ ID NO: 111) 34D9Vk GACATTATGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGA CAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGGGTAATGCTGTGG TCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGG GCATCCACCCGGCACATTGGAGTCCCTGATCGCTTCACAGGCAGTGGATC TGGGACAGATTTCACTCTCACCATTACCAATGTGCAGTCTGAAGACTTGG CAGATTATTTCTGTCAGCAATATAGCAGCTATCTCACGTTCGGTGCTGGG ACCAAGCTGGAGCTGAAA (SEQ ID NO: 112) DIMMTQSHKFMSTSVGDRVSITCKASQDVGNAVVWYQQKPGQSPKLLIYW ASTRHIGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYSSYLTFGAGTKL ELK (SEQ ID NO: 113) 43F5vk GATGTTGTGATGACCCAATCTCCACTCTCCCTGCCTGTCAGTCTTGGAGAT CAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGG AAACACCTATCTACATTGGTACCTGCTGAAGCCAGGCCAGTCTCCAAAGC TCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTC AGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGG AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCG TACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 114) DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLLKPGQSPKLLI YKVSNRFSGVPDRFSGSGSGTDFTLKISRVAEDLGVYFCSQSTHVPYTFGG GTKLEIK (SEQ ID NO: 115) 73H10Vk GACATCCAGATGACTCAGTCTCCAGTTTTCCTGTCTGCATCTGTGGGAGA AACTGTCACCATCACATGTCGAGCAAGTGAGAATATTGACAGTTATTTAG CATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTTTGCT GCACGAAACTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATC AGGCACACAGTATTCTCTCAAGATCAACAGAATGCAGTCTGAAGATGTTG CGAGATACTACTGTCAACATTATTATAGTACTCCATTCACGTTCGGCTCGG GGACAAAGTTGGAAATAGAA (SEQ ID NO: 116) DIQMTQSPVFLSASVGETVTITCRASENIDSYLAWYQQKQGKSPQLLVFAARNLAD GVPSRFSGSGSGTQYSLKINRMQSEDVARYYCQHYYSTPFTFGSGTKLEIE (SEQ ID NO: 117) 74H3Vk GACATCCAGATGACTCAGTCGCCAGCTTCCCTGTCTGCATCTGTGGGAGAAACT GTCATCTTCACATGTCGAGCAAGTGAGAATATTGACAGTTATTTAGCATGGTAT CAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATGCTGCAACAAACTT AGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTATT CTCTCAAGATCAACAGCCTGCAGTCTGAAGATGTTGCGAGATATTACTGTCTAC ATTATTATAGTACTCCATTCACGTTCGGCTCGGGGACAGAGTTGGAAATAAAA (SEQ ID NO: 118) DIQMTQSPASLSASVGETVIFTCRASENIDSYLAWYQQKQGKSPQLLVYAATNLAD GVPSRFSGSGSGTQYSLKINSLQSEDVARYYCLHYYSTPFTFGSGTELEIK (SEQ ID NO: 119)

- 14 040834

table 2

Mouse anti-IL-23p19 leader antibodies - VH sequences

2Dlvh CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAG CCTGTCCATCACATGCACTGTCTCTGGGTTCTCATTAACCACCTATGCTATA AGCTGGGTTCGCCAGTCACCAGGAAAGGGTCTGGAGTGGCTTGGAGTCAT ATGGACTGGTGGAGGCCAAAATATAATTTAGCTTCCAAATCCAGACTGA GCATCAGCAAAGACAACTCCAAGTAAGTCA CTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGAAAGGACTATAA TTACGGGGGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTC CTCA (SEQ ID NO: 120) QVQLKESGPGLVAPSQSLSITCTVSGFSLTTYAISWVRQSPGKGLEWLGVIWT GGGTKYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARKDYNYGG AMDYWGQGTSVTVSS (SEQ ID NO: 121) 6B8VH CAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGCACTTCA GTGAAGACATCCTGCAAAATTTCTGGCAACACCTTCACTGACCAAACTATT CACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATAT TTATCCTAGAGATGATAGTCCTAAGTACAATGAGAACTTCAAGGGCAAGG CCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAAC AGTCTGACATCTGAGGACTCTGCAGTCTATTTCTGTGCAATCCCAGACAGG TCAGGCTACGCCTGGTTTATTTACTGGGGCCAAGGGACTCTGGTCACTGTC TCTTCA (SEQ ID NO: 122) QVQLQQSDAELVKPGTSVKTSCKISGNTFTDQTIHWMKQRPEQGLEWIGYIYP RDD SPKYNENFKGKATLT ADKS S ST AYMQLNSLTSED S AVYFC AIPDRSGY A WFIYWGQGTLVTVSS (SEQ ID NO: 123) 9D12VH CAGGTGCAGCTGAAGGAGTCAGGACCTGTCCTGGTGGCGCCCTCACAGAG CCTGTCCATCACATGCACTGTCTCTGGGTTCTCATTAAACAACTTTGCTATA AGTTGGGTTCGTCAGCCACCAGGAAAGGGTCTGGAGTGGCTTGGAGCAAT ATGGACTGGTGGAGGCACAAATTATAATTCAGCTCTCAAATCCAGACTGA GCATCAGCAAAGACAACTCCAAGAGTCAAGTTTTCTTAAAAATGAACAGT CTGCAAACTGATGACACAGCCAGGTATTATTGTGTCAGAAAGGACTATAG TTACGGGGGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTC CTCA (SEQ ID NO: 124) QVQLKESGPVLVAPSQSLSITCTVSGFSLNNFAISWVRQPPGKGLEWLGAIWTGGGT NYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCVRKDYSYGGAMDYWGQ GTSVTVSS (SEQ ID NO: 125) 15Cllvh GAGGTCCAGCTGCAACAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTC AGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTATAT GAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTTA TTATTCCTTACAACGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAG GCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAAC AGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCACGAGATGGTCAC CGCTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 126) EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVI IP YNGGT S YNQKFKGK ATLT VDKS S ST AYMELN SLT SED S AVY YC ARD GHR WYFDVWGTGTTVTVSS (SEQ ID NO: 127) 15Flvh EVQLQQSGPELVKPGASVKMSCKASGYTFTCCIMHWVKQKPGQGLEWIGYI NP YND GTKYNEKFKGK ATLT SDKS S ST AYMEL S SLT SED S A VYYC ARRWDE AYWGQGTLVTVSA (SEQ ID NO: 128) 18D3vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTCAAGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTCGCTATCTTAT TCACTGGGTGAAACAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATA TTAATCCTTACAATGATGGTACTAAATACAATGAGAAGTTCAAAGGCAAG GCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAG CAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTACCTCTAACTGGGA CCTCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 129) EVQLQQSGPELVKPGASVKMSCKASGYTFTRYLIHWVKQKPGQGLEWIGYIN P YND GTKYNEKFKGK ATLT SDKS S ST AYMEL S SL TSED S AVY YCT SN WDLD Y WGQGTTLTVSS (SEQ ID NO: 130) 18C4vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAAGTGGTAAAGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCCTCTGGATACACATTCACTAGTTCTGTTAT ACACTGGGTGAAGCAGAAGGCTGGGCAGGGCCTTGAGTGGATTGGATATA

- 15 040834

TCAATCCCTATAATGATGGTACTAAGTACAATGAGAAGTTCAAAGGCAAG GCCACACTGACTTCAGACAGATCCTCCAGCACAGCCTACATGGAGCTCAG CAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTACAAGACGGTTGGA CGAGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 131) EVQLQQSGPEVVKPGASVKMSCKASGYTFTSSVIHWVKQKAGQGLEWIGYIN PYNDGTKYNEKFKGKATLTSDRSSSTAYMELSSLTSEDSAVYYCTRRLDEAY WGQGTLVTVSA (SEQ ID NO: 132) 18E5vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTGC AGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTCGCTATCTTAT TCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATA TTAATCCTTACAATGATGGTACTAAATATAATGAGAAGTTCAAAGGCAAG GCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAG CAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTACCTCTAATTGGGA CCTCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 133) EVQLQQSGPELVKPGAAVKMSCKASGYTFTRYLIHWVKQKPGQGLEWIGYIN PYNDGTKYNEKFKGK ATLTSDKS S ST AYMEL S SLTSED S AVYYCTSNWDLD Y WGQGTTLTVSS (SEQ ID NO: 134) 20E8vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTAAAGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCCTCTGGATACACATTCACTAGTTCTGTTAT GCACTGGGTGAAGCAGAAGGCTGGGCAGGGCCTTGAGTGGATTGGATATA TCAATCCCTATAATGATGGTACTCAGTACAATGAGAAGTTCAAAGGCAAG GCCACACTGACTTCAGACAAATTTTCCAGCACAGCCTACATGGAGCTCAGC AGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTACAAGACGGTTGGAC GAGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 135) EVQLQQSGPELVKPGASVKMSCKASGYTFTSSVMHWVKQKAGQGLEWIGYI NP YND GTQ YNEKFKGK ATLT SDKF S ST AYMEL S SLT SED S A VYYCTRRLDE A YWGQGTLVTVSA (SEQ ID NO: 136) 22E2vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTCTATTAT TCACTGGGTGAAGCAGAGGCCTGGGCAGGGCCTTGAGTGGATTGGATATA TTAATCCTTACGATGATGTTACTAAGTACAATGAGAAGTTCAAAGGCAAG GCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAG CAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGACGGTGGGA CGAGTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 137) EVQLQQSGPELVKPGASVKMSCKASGYTFTSSIIHWVKQRPGQGLEWIGYINP YDDVTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRWDESY WGQGTLVTVSA (SEQ ID NO: 138) 24A5vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCTTCTGGATACACTTTCACTACCTCTATTAT GCACTGGGTGAAACAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATA TTAATCCTTACGATGATGTTACTAAGTACAATGAAAAGTTCAAAGGCAAG GCCACATTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAG CAGCCTGACCTCTGAGGACTCTGCAGTCTATTACTGTGTAAGACGGTGGGA CGAGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 139) EVQLQQSGPELVKPGASVKMSCKASGYTFTSIMHWVKQKPGQGLEWIGYIN PYDDVTKYNEKFKGK ATLTSDKS S ST AYMEL S SLTSED SAVYYCVRRWDEA YWGQGTLVTVSA (SEQ ID NO: 140) 26F7VH GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTC AGTGAAGATATCCTGTAAGGCTCTGGATACACGTTTACTGACTACAT GAACTGGGTGAGGCAGAGCCATGGAGAGAGCCTTGAGTGGATTGGAGATT TTAATCATAACAATGATGTTATTACTTACAACCCGAAGTTCAAGGACCAAGG TCACATGAGCTGACTGTAGAGAAGCCTATCCACCCA

- 16 040834

GCCTGTCATCTGAGGACTCTGCAGTCTATTACTGTGCAAGGGGGCTACGAG GCTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCT CA (SEQ ID NO: 141) EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVRQSHGESLEWIGDFN HNND VITYNPKFKGKVTLT VEKS STT AYMELRSL SSED S AVY YCARGLRGY Y AMDYWGQGTSVTVSS (SEQ ID NO: 142) 27G8vh CAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGAGCTTC AGTGAAGATATCCTGCAAGGTTTCTGGCTACACCTTCACTGACCATACTAT TCACTGGATGAAGCAGAGGCCTGAACAGGGCCTGGAATGGATTGGATATA TTTATCCTAGAGATGGTTATCCTAAGTTCAATGAGAAGTTCAAGGGCAAGG CCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAAC AGCCTGACATCTGAGGACTCTGCAGTCTATTTCTGTGCAAGACGGCCCCCT TACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCGCCGTCTCCTCA (SEQ ID NO: 143) QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYIY PRDGYPKFNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCARRPPYYA MDYWGQGTSVAVSS (SEQ ID NO: 144) 31H9vh GAGGTCCAACTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGGTATCTTAT GCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGTTATA TTAATCCTTACAATGATGGTACTAATTACAATGAGAAGTTCAAAGGCAAG GCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAG CAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTTCCCTTAACTGGGA CTATGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 145) EVQLQQSGPELVKPGASVKMSCKASGYTFTRYLMHWVKQKPGQGLEWIGYI NP YND GTNYNEKFKGK ATLT SDKS S ST AYMEL S SLT SED S A VYYC SLN WD Y A YWGQGTLVTVSA (SEQ ID NO: 146) 34G3VH GAGTTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGCGCTTC AGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGACTACAACAT GAACTGGGTGAAGCAGAGCAAAGGAAAGAGCCTTGAGTGGATTGGAGTA ATTATTCCTAACTATGGTTTTACTAGCTACAATCAGAACTTCAAGGGCAAG GCCACTTTGACTGTAGACCAGTCTTCCAGCACAGCCCACATGCAGCTCAAC AGTGTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGAGATGGGGG AATACTCCTCTGGTATCTCGATGTCTGGGGCACAGGGACCACGGTCACCGT CTCCTCA (SEQ ID NO: 147) EFQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSKGKSLEWIGVIIP NYGFTSYNQNFKGKATLTVDQSSSTAHMQLNSVTSEDSAVYYCVRDGGILL WYLDVWGTGTTVTVSS (SEQ ID NO: 148) 34D9VH7 GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTC AGTGAAGATACCCTGCAAGGCTTCTGGATACACATTCACTGACTACAACAT GGACTGGGTGAAGAAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATA TCAATCCTCACAATGGTGGTACTATCTACAACCAGAAGTTCAAGGGCAAG GCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCCACATGGAGCTCCG CAGCCTGACATCTGAGGACACTGCAGTCTATTACTGTGCAAGAAATTACTA CGGTAGTAGTTACGGCTGGTACTTCGATGTCTGGGGCACAGGGACCACGG TCACCGTCTCGTCA (SEQ ID NO: 149) EVQLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKKSHGKSLEWIGDIN PHNGGTIYNQKFKGKATLTVDKSSSTAHMELRSLTSEDTAVYYCARNYYGSS YGWYFDVWGTGTTVTVSS (SEQ ID NO: 150) 43F5vh GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTC AGTGAAGATTTCCTGCAGGGCTTCTGGTTACTCATTCACTGGCTACTACAT GAACTGGGTGAAGCAAAGTCCTGAAAAGAGCCTTGAGTGGATTGGAGAGA TTATTCCTACCACTGGTGGTACTTCCTACAACCAGAAGTTCAAGGCCAAGG CCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAAG AGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGAGAGCGG TGGGTTCTACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGT CTCCTCA (SEQ ID NO: 151) EVQLQQSGPELVKPGASVKISCRASGYSFTGYYMNWVKQSPEKSLEWIGEIIP TTGGTSYNQKFKAKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARESGGFY WYFDVWGTGTTVTVSS (SEQ ID NO: 152) 73H10VH GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGGTATGTTAT GCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATA TTAATCCTTACAATGATGTTACTAAGTACAATGAGAAGTTCAAAGGCAAG GCCACACTGACTTCAGACAGATCCTCCAGCACAGCCTACATGAAACTCAG CAGCCTGACCTCTGAGGACTCTGCGGTCTATTATTGTGCAAGAAACTGGGA CGTTCCTTACTGGGGCCAAGGGACTCTGATCACTGTCTCTGCA (SEQ ID NO: 153) EVQLQQSGPELVKPGASVKMSCKASGYTFTRYVMHWVKQKPGQGLEWIGYINPY NDVTKYNEKFKGKATLTSDRSSSTAYMKLSSLTSEDSAVYYCARNWDVPYWGQG TLITVSA (SEQ ID NO: 154) 74H3VH GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTG AAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGGTATCTTATGCACTGG GTGAAGCAGAAGCCTGGACAGGGCCTTGAGTGGATTGGATATATTAATCCTTAC AATGATGGTACTAAGTACAATGAGAGGTTCAAAGGCAAGGCCACACTGACTTC AGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGA CTCTGCGGTCTATTACTGTGCAAGAAACTGGGACGTACCTTACTGGGGCCAAGG GACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 155) EVQLQQSGPELVKPGASVKMSCKASGYTFTRYLMHWVKQKPGQGLEWIGYINPYN DGTKYNERFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARNWDVPYWGQGTL VTVSA (SEQ ID NO: 156)

Human framework sequences were selected for each mouse leader antibody based on framework homology, CDR structure, conserved canonical residues, conserved interface residues, and other parameters.

CDR of mouse heavy chains and light chains of various mouse antibodies are presented in table. 3 and 4, respectively. In table. 4 also shows three heavy chain CDRs derived from mouse

- 17 040834 6B8 antibodies during the humanization process.

Table 3

Light chain CDR sequences

L-CDR1 L-CDR2 L-CDR3 18С4 RASQSISEYLH (SEQ ID NO: 1) YASQSIS (SEQ ID NO: 2) QNGHSFPFT (SEQ ID NO: 3) 18E5 RASQSISDYLY (SEQ ID NO: 4) FASQSIS (SEQ ID NO: 5) QNGHSFPFT (SEQ ID NO: 3) 18D3 RASQSISDYLY (SEQ ID NO: 4) FASQSIS (SEQ ID NO: 5) QNGHSFPFT (SEQ ID NO: 3) 20E8 RASQSISEYLH (SEQ ID NO: 1) YASQSIS (SEQ ID NO: 2) QNGHSFPFT (SEQ ID NO: 3) 22E2 RASQSISVYLH (SEQ ID NO: 6) YASQSIS (SEQ ID NO: 2) QNGHSFPFT (SEQ ID NO: 3) 24A5 RASQSISDYLH YASQSIS QNGHSFPFT

L-CDR1 L-CDR2 L-CDR3 (SEQ ID NO: 7) (SEQ ID NO: 2) (SEQ ID NO: 3) 15C11 RSSQSLVHSNGNTYLH (SEQ ID NO: 8) KVSNRFS (SEQ ID NO: 9) SQSTHVPYT (SEQ ID NO: 10) 43F5 RSSQSLVHSNGNTYLH (SEQ ID NO: 8) KVSNRFS (SEQ ID NO: 9) SQSTHVPYT (SEQ ID NO: 10) 27G8 RASKVSTSGYSYIH (SEQ ID NO: 11) LASNLDS (SEQ ID NO: 12) QHSRELPYT (SEQ ID NO: 13) 31H9 RASQSISDYLH (SEQ ID NO: 7) YASQSIS (SEQ ID NO: 2) QNGHSFPYT (SEQ ID NO: 14) 2D1 RTSESVYSYGQNFIH (SEQ ID NO: 15) RASNLES (SEQ ID NO: 16) QQTNEDPYT (SEQ ID NO: 17) 9D12 RASETINFYGTSFMH (SEQ ID NO: 18) RASNLES (SEQ ID NO: 16) QQTNEDPYT (SEQ ID NO: 17) 6B8 KASRDVAIAVA (SEQ ID NO: 19) WASTRHT (SEQ ID NO: 20) HQYSSYPFT (SEQ ID NO: 21) 73H10 RASENIDSYLA (SEQ ID NO: 22) AARNLAD (SEQ ID NO: 23) QHYYSTPFT (SEQ ID NO: 24) 74H3 RASENIDSYLA (SEQ ID NO:22) AATNLAD (SEQ ID NO: 25) LHYYSTPFT (SEQ ID NO: 26) 35H8 RSSQSLVHSNGNTYLH (SEQ ID NO: 8) KVSNRFS (SEQ ID NO: 9) SQSTHVPYT (SEQ ID NO: 10) 26F7 RASKSVRFSDYFYMH (SEQ ID NO: 27) LASNLES (SEQ ID NO: 28) QNSRELPYT (SEQ ID NO: 29) 34G3 RSSQSLVHSNGNTYLH (SEQ ID NO:8) KVSNRFS (SEQ ID NO: 9) SQSTHVPYT (SEQ ID NO: 10) 34D9 KASQDVGNAVV (SEQ ID NO: 30) WASTRHI (SEQ ID NO: 31) QQYSSYLT (SEQ ID NO: 32)

Table 4

Heavy chain CDR sequences

H-CDR1 H-CDR2 H-CDR3 18C4 GYTFTSSVIH (SEQ ID NO: 33) YINPYNDGTKYNEKFKG (SEQ ID NO: 34) RLDEAY (SEQ ID NO: 35) 18E5 GYTFTRYLIH (SEQ ID NO: 36) YINPYNDGTKYNEKFKG (SEQ ID NO: 34) NWDLDY (SEQ ID NO: 37) 18D3 GYTFTRYLIH (SEQ ID NO: 36) YINPYNDGTKYNEKFKG (SEQ ID NO: 34) NWDLDY (SEQ ID NO: 37) 20E8 GYTFTSSVMH (SEQ ID NO: 38) YINPYNDGTQYNEKFKG (SEQ ID NO: 39) RLDEAY (SEQ ID NO: 35) 22E2 GYTFTSSIH (SEQ ID NO: 40) YINPYDDVTKYNEKFKG (SEQ ID NO: 41) RWDESY (SEQ ID NO: 42) 24A5 GYTFTSIMH (SEQ ID NO: 43) YINPYDDVTKYNEKFKG (SEQ ID NO: 41) RWDEAY (SEQ ID NO: 44) 15C11 GYTFTDYYMN (SEQ ID NO: 45) VIIPYNGGTSYNQKFKG (SEQ ID NO: 46) DGHRWYFDV (SEQ ID NO: 47) 43F5 GYSFTGYYMN (SEQ ID NO: 48) EIIPTTGGTSYNQKFKA (SEQ ID NO: 49) ESGGFYWYFDV (SEQ ID NO: 50) 27G8 GYTFTDHTTIH (SEQ ID NO: 51) YIYPRDGYPKFNEKFKG (SEQ ID NO: 52) RPPYYAMDY (SEQ ID NO: 53) 31H9 GYTFTRYLMH (SEQ ID NO: 54) YINPYNDGTNYNEKFKG (SEQ ID NO: 55) NWDYAY (SEQ ID NO: 56)

- 18 040834

2D1 GFSLTTYAIS (SEQ ID NO: 57) VIWTGGGTKYN S ALK S (SEQ ID NO: 58) KDYNYGGAMDY (SEQ ID NO: 59) 9D12 GFSLNNFAIS (SEQ ID NO: 60) AIWTGGGTNYN S ALK S (SEQ ID NO: 61) KDYSYGGAMDY (SEQ ID NO: 62) 6B8 GNTFTDQTIH (SEQ ID NO: 63) YIYPRDDSPKYNENFKG (SEQ ID NO: 64) PDRSGYAWFIY (SEQ ID NO: 65) Hu 6В8-2 GYTFTDQTIH (SEQ ID NO: 66) YIYPRDDSPKYNENFKG (SEQ ID NO: 64) PDRSGYAWFIY (SEQ ID NO: 65) None 6V8-5 GFTFTDQTIH (SEQ ID NO: 67) YIYPRDDSPKYNENFKG (SEQ ID NO: 64) PDRSGYAWFIY (SEQ ID NO: 65) Ni_6V836/65 GGTFTDQTIH (SEQ ID NO: 68) YIYPRDDSPKYNENFKG (SEQ ID NO: 64) PDRSGYAWFIY (SEQ ID NO: 65) 73H10 GYTFTRYVMH (SEQ ID NO: 69) YINPYNDVTKYNEKFKG (SEQ ID NO: 70) NWDVPY (SEQ ID NO: 71) 74НЗ GYTFTRYLMH (SEQ ID NO: 54) YINPYNDGTKYNERFKG (SEQ ID NO: 72) NWDVPY (SEQ ID NO: 71) 35H8 GYTFTDYYMN (SEQ ID NO: 45) VIIPYNGGISYNQKFKG (SEQ ID NO: 73) NDYDWYFDV (SEQ ID NO: 74) 26F7 GYTFTDYYMN (SEQ ID NO: 45) DFNHNNDVITYNPKFKG (SEQ ID NO: 75) GLRGYYAMDY (SEQ ID NO: 76) 34G3 GYSFTDYNMN (SEQ ID NO: 77) VIIPNYGFTSYNQNFKG (SEQ ID NO: 78) DGGILLWYLDV (SEQ ID NO: 79) 34D9 GYTFTDYNMD (SEQ ID NO: 80) DINPHNGGTIYNQKFKG (SEQ ID NO: 81) NYYGSSYGWYFDV (SEQ ID NO: 82)

The CDRs shown in Tables 3 and 4 above are defined according to the Hotia nomenclature (Al-Lazikani et al., JMB 273, 1997, p. 927-948).

Fab fragments found to have improved or equivalent binding compared to the chimeric parent Fab fragment were selected for conversion to IgG format. 6B8 was converted to IgG1KO format. IgG1KO (knock-out effector functions) has two mutations in the Fc region, namely Leu234Ala and Leu235Ala, which reduce effector function such as FcyR and complement binding. The IgG format has been described in the literature (see, for example, Hezareh et al., Journal of Virology 75, 2001, p. 12161-12168). Example 1 describes the humanization process in more detail. As a result of the implementation of this humanization, it was possible to create sequences of humanized antibodies. Representative examples of light chain and heavy chain variable regions derived from mouse 6B8 antibody are shown in Tables 5 and 6. Primary structure comparison of humanized light chain and heavy chain variable regions derived from mouse 6B8 antibody and light chain and heavy chain variable regions from mouse antibody 6B8 is shown in FIG. 1.

Selected combinations of humanized light chain and heavy chain variable regions derived from mouse antibody 6B8 allowed the creation of antibodies A, B, C and D:

Antibody A: 6B8-IgG1KO-2 with IgK-66 (heavy chain variable region 6B8CVH-02 and light chain variable region 6B8CVK-66);

Antibody B: 6B8-IgG1KO-5 ch IgK-66 (heavy chain variable region 6B8CVH-05 and light chain variable region 6B8CVK-66);

Antibody C: 6B8-IgG1KO-2 with IgK-65 (heavy chain variable region 6B8CVH-02 and light chain variable region 6B8CVK-65);

Antibody D: 6B8-IgG1KO-5 with IgK-65 (heavy chain variable region 6B8CVH-05 and light chain variable region 6B8CVK-65).

Antibodies A, B, C and D have the sequence of heavy and light chains, which are presented in table. 7.

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Table 5

Humanized sequences 6B8-VK

6B8CVK-65 Gacatccagatgacccagagcccaagcagcctgagcgccagcgtgggcgaccgcgtgaccatcacctgc aaggccagccgcgacgtggccatcgccgtggcctggtaccagcagaagccaggcaaggtgccaaagctg ctgctgttctgggccagcacccgccacaccggcgtgccagaccgcttcagcggcagcggcagcggcacc gacttcaccctgaccatcagcagcctgcagccagaggacctggccgactactactgccaccagtacagcag ctacccattcaccttcggccagggcaccaagctggagatcaag (SEQ ID NO: 157) DIQMTQSPSSLSASVGDRVTITCKASRDVAIAVAWYQQKPGKVPKLLL FWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDLADYYCHQYSSYPFT FGQGTKLEIK (SEQ ID NO: 158) 6B8CVK-66 Gacatccagatgacccagagcccaagcagcctgagcgccagcgtgggcgaccgcgtgaccatcacctgc aaggccagccgcgacgtggccatcgccgtggcctggtaccagcagaagccaggcaaggtgccaaagctg ctgatctactgggccagcacccgccacaccggcgtgccaagccgcttcagcggcagcggcagccgcacc gacttcaccctgaccatcagcagcctgcagccagaggacgtggccgactacttctgccaccagtacagcag ctacccattcaccttcggcagcggcaccaagctggagatcaag (SEQ ID NO: 159) DIQMTQSPSSLSASVGDRVTITCKASRDVAIAVAWYQQKPGKVPKLLI YWASTRHTGVPSRFSGSGSRTDFTLTISSLQPEDVADYFCHQYSSYPFT FGSGTKLEIK (SEQ ID NO: 160) 6B8CVK-67 Gacatccagatgacccagagcccaagcagcctgagcgccagcgtgggcgaccgcgtgaccatcacctgc aaggccagccgcgacgtggccatcgccgtggcctggtaccagcagaagccaggcaaggtgccaaagctg ctgctgtactgggccagcacccgccacaccggcgtgccaagccgcttcagcggcagcggcagccgcacc gacttcaccctgaccatcagcagcctgcagccagaggacgtggccacctactactgccaccagtacagcag ctacccattcaccttcggcagcggcaccaagctggagatcaag (SEQ ID NO: 161) DIQMTQSPSSLSASVGDRVTITCKASRDVAIAVAWYQQKPGKVPKLLL YWASTRHTGVPSRFSGSGSRTDFTLTISSLQPEDVATYYCHQYSSYPFT FGSGTKLEIK (SEQ ID NO: 162) 6B8CVK-78 Gacatccagatgacccagagcccaagcagcctgagcgccagcgtgggcgaccgcgtgaccatcacctgc aaggccagccgcgacgtggccatcgccgtggcctggtaccagcagaagccaggcaaggtgccaaagctg ctgctgttctgggccagcacccgccacaccggcgtgccagaccgcttcagcggcagcggcagccgcacc gacttcaccctgaccatcagcagcctgcagccagaggacctggccgactactactgccaccagtacagcag ctacccattcaccttcggcagcggcaccaagctggagatcaag (SEQ ID NO: 163) DIQMTQSPSSLSASVGDRVTITCKASRDVAIAVAWYQQKPGKVPKLLL FWASTRHTGVPDRFSGSGSRTDFTLTISSLQPEDLADYYCHQYSSYPFT FGSGTKLEIK (SEQ ID NO: 164)

Table 6

Humanized 6B8-VH sequences

6B8CVH-02 Caggtgcagctggtgcagagcggcgccgaggtgaagaagccaggcagcagcgtgaaggtgagctgcaa ggccagcggctacaccttcaccgaccagaccatccactggatgcgccaggccccaggccagggcctgga gtggatcggctacatctacccacgcgacgacagcccaaagtacaacgagaacttcaagggcaaggtcacc atcaccgccgacaagagcaccagcaccgcctacatggagctgagcagcctgcgcagcgaggacaccgcc gtgtactactgcgccatcccagaccgcagcggctacgcctggttcatctactggggccagggcaccctggt gaccgtgagcagc (SEQ ID NO: 165) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDQTIHWMRQAPGQGLE WIGYIYPRDDSPKYNENFKGKVTITADKSTSTAYMELSSLRSEDTAVY YCAIPDRSGYAWFIYWGQGTLVTVSS (SEQ ID NO: 166) 6B8CVH-05 Caggtgcagctggtgcagagcggcgccgaggtgaagaagccaggcagcagcgtgaaggtgagctgcaa ggccagcggcttcaccttcaccgaccagaccatccactgggtgcgccaggccccaggccagggcctgga gtggatgggctacatctacccacgcgacgacagcccaaagtacaacgagaacttcaagggcaaggtcacc ctgaccgccgacaagagcaccagcaccgcctacatggagctgagcagcctgcgcagcgaggacaccgcc gtgtactactgcgccatcccagaccgcagcggctacgcctggttcatctactggggccagggcaccctggt gaccgtgagcagc (SEQ ID NO: 167) QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDQTIHWVRQAPGQGLEW MGYIYPRDDSPKYNENFKGKVTLTADKSTSTAYMELSSLRSEDTAVYY CAIPDRSGYAWFIYWGQGTLVTVSS (SEQ ID NO: 168) 6B8CVH-36 Caggtgcagctggtgcagagcggcgccgaggtgaagaagccaggcagcagcgtgaagaccagctgcaa ggccagcggcggcaccttcaccgaccagaccatccactgggtgcgccagcgcccaggccagggcctgga gtggatgggctacatctacccacgcgacgacagcccaaagtacaacgagaacttcaagggccgcgtcacc atcaccgccgacaagagcaccagcaccgcctacatggagctgagcagcctgcgcagcgaggacaccgcc gtgtactactgcgccatcccagaccgcagcggctacgcctggttcatctactggggccagggcaccctggt gaccgtgagcagc (SEQ ID NO: 169) Q VQL VQ S G AE VKKPGS S VKT S CK AS GGTFTDQTIH W VRQRP GQGLE W MGYIYPRDDSPKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYY CAIPDRSGYAWFIYWGQGTLVTVSS (SEQ ID NO: 170) 6B8CVH-65 Caggtgcagctggtgcagagcggcgccgaggtgaagaagccaggcagcagcgtgaaggtgagctgcaa ggccagcggcggcaccttcaccgaccagaccatccactgggtgcgccaggccccaggccagggcctgga gtggatgggctacatctacccacgcgacgacagcccaaagtacaacgagaatttcaagggccgcgtcaccc tgaccgccgacaagagcaccagcaccgcctacatggagctgagcagcctgcgcagcgaggacaccgcc gtgtacttctgcgcccgcccagaccgcagcggctacgcctggttcatctactggggccagggcaccctggt gaccgtgagcagc (SEQ ID NO: 171) QVQLVQSGAEVKKPGSSVKVSCKASGGTFTDQTIHWVRQAPGQGLEW MGYIYPRDDSPKYNENFKGRVTLTADKSTSTAYMELSSLRSEDTAVYF CARPDRSGYAWFIYWGQGTLVTVSS (SEQ ID NO: 172)

- 20 040834

Table 7

DNA sequences and amino acid sequences of the heavy and light chains of Antibodies A, B, C and D

Antibody A IgK light chain #66 Gacatccagatgacccagagcccaagcagcctgagcgccagcgtgg gcgaccgcgtgaccatcacctgcaaggccagccgcgacgtggccatc gccgtggcctggtaccagcagaagccaggcaaggtgccaaagctgct gatctactggggccagcacccgccacaccggcgtgccaagccgcttca gcggcagcggcagccgcaccgacttcaccctgaccatcagcagcctg cagccagaggacgtggccgactacttctgccaccagtacagcagctac ccattcaccttcggcagcggcaccaagctggagatcaagcgtactgtg gctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatct KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG (SEQ ID NO: 178) Antibody C IgK light chain #65 Gacatccagatgacccagagcccaagcagcctgagcgccagcgtgg gcgaccgcgtgaccatcacctgcaaggccagccgcgacgtggccatc gccgtggcctggtaccagcagaagccaggcaaggtgccaaagctgct gctgttctggggccagcacccgccacaccggcgtgccagaccgcttca gcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctg cagccagaggacctggccgactactactgccaccagtacagcagcta cccattcaccttcggccagggcaccaagctggagatcaagcgtactgt ggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatc tggaactgcctctgttgtgtgcctgctgaataacttctatcccagagagg ccaaagtacagtggaaggtggataacgccctccaatcgggtaactccc aggagagtgtcacagagcaggacagcaaggacagcacctacagcct cagcagcaccctgacgctgagcaaagcagactacgagaaacacaaa gtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcaca aagagcttcaacaggggagagtgt (SEQ ID NO: 179) DIQMTQSPSSLSASVGDRVTITCKASRDVAIAV AW YQQKPGKVPKLLLFWASTRHTGVPDRF SG SGSGTDFTLTISSLQPEDLADYYCHQYSSYPFT FGOGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQD SKD ST YSLS S TLTLSK AD YEKHK VYAC EVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 180) IgGlKO, heavy chain, #2 (SEQ ID NO: 175) (SEQ ID NO: 176) Antibody D IgK light chain #65 (SEQ ID NO: 179) (SEQ ID NO: 180) IgGlKO, heavy chain, #5 (SEQ ID NO: 177) (SEQ ID NO: 178)

In table. 7, the variable regions of the light chains and heavy chains of Antibodies A, B, C, and D are underlined.

Humanized antibody to IL-23p19, disclosed in the description, has at least one of the following properties. It may have any combination of at least two or at least 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the following properties, or all of the following properties.

K _D for human IL-23 is <1 pM (no association rate shift in 50% human serum).

Blocks the binding of IL-23 to human IL-23R/Fc in vitro.

Does not bind to human IL-12.

Inhibits human IL-23-induced IL-17 production in mouse splenocytes, characterized by an IC ₅₀ <20 pM.

Inhibits human IL-23 induced STAT3 phosphorylation in human DB cells, characterized by an IC ₅₀ <40 pM.

The presence of ADCC/CDC activity is not predicted.

K _D <1pM against IL-23 of Cynomolgus monkeys.

No cross-reactivity with mouse or rat IL-23.

Inhibits human IL-23-induced production of IL-17 and IL-22 in mouse ear

- 21 040834 (>80% inhibition of both cytokines at a concentration of 1 mg/kg).

Stable up to 83°C (melting point 83°C measured by differential scanning calorimetry).

Solubility >100 mg/mL (as measured by UV spectroscopy and turbidity monitoring).

When administered subcutaneously at a dose of 1.0 mg/kg to three cynomolgus monkeys, a long-term (> 10 nM) exposure occurs for about 28 days with a bioavailability of about 70%.

Not predictive of ADCC/CDC activity as used herein means that a humanized anti-IL-23p19 antibody has reduced affinity for the Fc receptor and is therefore predicted to lack ADCC/CDC activity.

An anti-IL-23p19 antibody may have at least one of the following properties. It may have any combination of at least two, or at least 3, 4, 5, 6, 7, 8, 9, or 10 of the following properties, or all of the following properties.

K _D for human IL-23 is <1 pM (no association rate shift in 50% human serum).

Blocks the binding of IL-23 to human IL-23R/Fc in vitro.

Does not bind to human IL-12.

Inhibits human IL-23-induced IL-17 production in mouse splenocytes, characterized by an IC ₅₀ <20 pM.

Inhibits human IL-23 induced STAT3 phosphorylation in human DB cells, characterized by an IC ₅₀ <40 pM.

The presence of ADCC/CDC activity is not predicted. K _D <1 pM against Cynomolgus IL-23.

No cross-reactivity with mouse or rat IL-23.

Inhibits human IL-23-induced production of IL-17 and IL-22 in mouse ear (>80% inhibition of both cytokines at 1 mg/kg).

Stable up to 83°C (melting point 83°C measured by differential scanning calorimetry).

Solubility >100 mg/mL (as measured by UV spectroscopy and turbidity monitoring).

The humanized antibody has at least one of the following binding features (properties A). A humanized anti-IL-23p19 antibody may have any combination of at least two or at least three of the following properties, or all of the following properties.

KD for human IL-23 is < 1pM (no association rate shift in 50% human serum).

Does not bind to human IL-12.

KD<1 pM against Cynomolgus monkey IL-23.

No cross-reactivity with mouse or rat IL-23. In particular, the humanized antibody has a K _D for human IL-23 of < 1 pM (no association rate shift in 50% human serum) and does not bind to human IL-12.

The humanized antibody has at least one of the following functional properties (properties B). A humanized anti-IL-23p19 antibody has any combination of at least two or at least three of the following, or all of the following.

Blocks the binding of IL-23 to human IL-23R/Fc in vitro.

Inhibits human IL-23-induced IL-17 production in mouse splenocytes, characterized by an IC50 <20 pM.

Inhibits human IL-23 induced STAT3 phosphorylation in human DB cells, characterized by an IC ₅₀ <40 pM.

Inhibits human IL-23-induced production of IL-17 and IL-22 in mouse ear (>80% inhibition of both cytokines at 1 mg/kg).

The humanized antibody has at least one of the following functional properties (Properties B). A humanized anti-IL-23p19 antibody has any combination of at least two or at least three of the following properties, or or all of the following properties.

The presence of ADCC/CDC activity is not predicted.

Stable up to 83°C (melting point 83°C measured by differential scanning calorimetry).

- 22 040834

Solubility >100 mg/mL (as measured by UV spectroscopy and turbidity monitoring).

When administered subcutaneously at a dose of 1.0 mg/kg to three cynomolgus monkeys, a long-term (>10 nM) exposure occurs for about 28 days with a bioavailability of about 70%.

The humanized antibody has at least one of the following functional properties (Properties B). Humanized antibody to IL-23p19 has any combination of at least two of the following properties or has all of the following properties.

The presence of ADCC/CDC activity is not predicted.

Stable up to 83°C (melting point 83°C measured by differential scanning calorimetry).

Solubility >100 mg/mL (as measured by UV spectroscopy and turbidity monitoring).

A humanized antibody has at least one property A, at least one property B, and at least one property C. A humanized anti-IL-23p19 antibody has any combination of at least two or at least three properties A, B, and C.

The humanized antibody has blocking activity, while it reduces the binding of IL-23 to the IL-23 receptor by at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. The ability of an antibody to block IL-23 binding to the IL-23 receptor can be assessed using competitive binding assays known in the art. The blocking activity of an antibody can be measured by assessing the biological actions of IL-23, such as the production of IL-17 and IL-22, to determine whether IL-23 receptor-mediated signaling is inhibited.

Humanized antibody to IL-23p19 may have favorable biophysical properties. The humanized anti-IL-23p19 antibody may be present in the buffer at least 90% in monomeric form, or at least 92% in monomeric form, or at least 95% in monomeric form. The humanized anti-IL-23p19 antibody is buffered to at least 90% monomeric or at least 92% monomeric or at least 95% monomeric for 1 month or 4 months.

The humanized antibody may be Antibody A, Antibody B, Antibody C, or Antibody D. Thus, a humanized antibody may comprise the light chain sequence of SEQ ID NO: 174 and the heavy chain sequence of SEQ ID NO: 176 (Antibody A). The humanized antibody may comprise the light chain sequence of SEQ ID NO: 174 and the heavy chain sequence of SEQ ID NO: 178 (Antibody B). The humanized antibody may comprise the light chain sequence of SEQ ID NO: 180 and the heavy chain sequence of SEQ ID NO: 176 (Antibody C). The humanized antibody may comprise the light chain sequence of SEQ ID NO: 180 and the heavy chain sequence of SEQ ID NO: 178 (Antibody D).

The humanized antibody may consist of the light chain sequence of SEQ ID NO: 174 and the heavy chain sequence of SEQ ID NO: 176 (Antibody A). The humanized antibody may consist of the light chain sequence of SEQ ID NO: 174 and the heavy chain sequence of SEQ ID NO: 178 (Antibody B). The humanized antibody may consist of the light chain sequence of SEQ ID NO: 180 and the heavy chain sequence of SEQ ID NO: 176 (Antibody C). The humanized antibody may consist of the light chain sequence of SEQ ID NO: 180 and the heavy chain sequence of SEQ ID NO: 178 (Antibody D).

Humanized anti-IL-23p19 antibodies, including their antigen-binding fragments such as heavy and light chain variable regions, may contain an amino acid sequence derived from Antibody A (light chain sequence of SEQ ID NO: 174; heavy chain sequence: SEQ ID NO: 176 ), Antibody B ((light chain sequence SEQ ID NO: 174; heavy chain sequence: SEQ ID NO: 178), Antibody C (light chain sequence SEQ ID NO: 180; heavy chain sequence SEQ ID NO: 176) or Antibody D (light chain sequence SEQ ID NO: 180; heavy chain sequence SEQ ID NO: 178).

An anti-IL-23p19 antibody, or antigen-binding fragment thereof, can bind to human IL-23p19 at an epitope consisting of amino acid residues 108-126 and amino acid residues 137151 of SEQ ID NO: 181.

An anti-IL-23p19 antibody, or antigen-binding fragment thereof, can compete for binding to a human IL-23p19 subunit from an antibody, such as Antibody A, Antibody B, Antibody C, or Antibody D as described herein. The ability of an antibody or antigen-binding fragment thereof to compete for binding to IL-23p19 can be assessed using competitive binding assays known in the art.

Humanized antibodies to IL-23p19 optionally include specific amino acid substitutions in consensus framework regions or germline framework regions. specific

- 23 040834 This substitution of amino acid residues at these framework positions can improve various aspects of antibody performance, such as binding affinity and/or stability, compared to those of humanized antibodies obtained by direct exchange of CDRs or HVLs with human germline frameworks. lines.

The application describes other monoclonal antibodies having the light chain variable region amino acid sequence shown in SEQ ID NOs: 84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111 . , 142, 144, 146, 148, 150, 152, 154 or 156 (see Tables 1 and 2 above). CDR sequences of these mouse antibodies are presented in table. 3 and 4. Introduction of these CDRs into the FRs of the human heavy and light chain consensus variable regions can result in acceptable humanized antibodies.

In particular, monoclonal antibodies are described having the following combination of light chain variable regions and heavy chain variable regions: SEQ ID NO: 84/121, 86/123, 88/125, 90/127, 91/128, 93/130, 95/ 132, 97/134, 99/136, 101/138, 103/140, 105/142, 107/144, 109/146, 111/148, 113/150, 115/152, 117/154 or 119/156. These variable regions can be combined with human constant regions.

The application describes humanized antibodies whose light chain variable region sequences have the amino acid sequence shown in SEQ ID NO: 158, 160, 162 or 164 and other humanized antibodies whose heavy chain variable region sequences have the amino acid sequence shown in SEQ ID NO: 166, 168, 170 or 172 (see tables 5 and 6 above). The CDR sequences of these antibodies are shown in Table 1. 3 and 4, in particular, monoclonal antibodies having the following combinations of light chain variable regions and heavy chain variable regions: SEQ ID NO: 160/166, 160/168, 158/166 or 158/168. These variable regions can be combined with human constant regions.

An anti-IL-23p19 antibody, or antigen-binding fragment thereof, may comprise a humanized light chain variable domain comprising the CDR regions of SEQ ID NO: 160 and framework regions having an amino acid sequence that is at least 90% identical to the amino acid sequence of the framework regions of the variable domain. light chain that has the amino acid sequence of SEQ ID NO: 160, and a humanized heavy chain variable domain containing the CDR regions of SEQ ID NO: 166 and framework regions having an amino acid sequence that is at least 90% identical to the amino acid sequence of the framework regions of the heavy chain variable domain, which has the amino acid sequence of SEQ ID NO: 166.

An anti-IL-23p19 antibody, or antigen-binding fragment thereof, may comprise a humanized light chain variable domain comprising the CDR regions of SEQ ID NO: 160 and framework regions having an amino acid sequence that is at least 90% identical to the amino acid sequence of the framework regions of the variable domain. light chain that has the amino acid sequence of SEQ ID NO: 160, and a humanized heavy chain variable domain containing the CDR regions of SEQ ID NO: 168 and framework regions having an amino acid sequence that is at least 90% identical to the amino acid sequence of the framework portions of the heavy chain variable domain, which has the amino acid sequence of SEQ ID NO: 168.

An anti-IL-23p19 antibody, or antigen-binding fragment thereof, may comprise a humanized light chain variable domain comprising the CDR regions of SEQ ID NO: 158 and framework regions having an amino acid sequence that is at least 90% identical to the amino acid sequence of the framework regions of the variable a light chain domain that has the amino acid sequence of SEQ ID NO: 158, and a humanized heavy chain variable domain that contains the CDR regions of SEQ ID NO: 166 and framework regions having an amino acid sequence that is at least 90% identical to the amino acid sequence framework regions of the heavy chain variable domain, which has the amino acid sequence of SEQ ID NO: 166.

An anti-IL-23p19 antibody, or antigen-binding fragment thereof, may comprise a humanized light chain variable domain comprising the CDR regions of SEQ ID NO: 158 and framework regions having an amino acid sequence that is at least 90% identical to the amino acid sequence of the framework regions of the variable domain. a light chain that has the amino acid sequence of SEQ ID NO: 158, and a humanized heavy chain variable domain containing the CDR regions of SEQ ID NO: 168 and framework regions having an amino acid sequence that is at least 90% identical to the amino acid sequence of the framework portions of the heavy chain variable domain, which has the amino acid sequence of SEQ ID NO: 168.

Humanized antibodies to IL-23p19 may contain at least the variable domain of th

- 24 040834 of a gel or light chain containing the CDRs or HVLs of the mouse monoclonal antibodies or humanized antibodies shown in Tables 1-6 above and the FRs of the human germline heavy and light chain variable domains.

CDR of these sequences are presented in table. 3 and 4. Thus, an antibody or antigen-binding fragment thereof may comprise the light chain CDR1 (L-CDR1) sequence of SEQ ID NOs: 1, 4, 6, 7, 8, 11, 15, 18, 19, 22, 27, or 30; light chain CDR2 sequence (L-CDR2) SEQ ID NO: 2, 5, 9, 12, 16, 20, 23, 25, 28 or 31; light chain CDR3 sequence (L-CDR3) SEQ ID NO: 3, 10, 13, 14, 17, 21, 24, 26, 29 or 32; heavy chain CDR1 sequence CDR1 (H-CDR1) SEQ ID NO: 33, 36, 38, 40, 43, 45, 48, 51, 54, 57, 60, 63, 66, 67, 68, 69, 77, or 80; heavy chain CDR2 sequence (H-CDR2) SEQ ID NO: 34, 39, 41, 46, 49, 52, 55, 58, 61, 64, 70, 72, 73, 75, 78 or 81 and the heavy chain CDR3 sequence ( H-CDR3) SEQ ID NO: 35, 37, 42, 44, 47, 50, 53, 56, 59, 62, 65, 71, 74, 76, 79, or 82. Anti-IL-23p19 antibody or antigen-binding fragment thereof may contain a light chain variable region comprising the above L-CDR1, the above L-CDR2 and the above L-CDR3, and a heavy chain variable region comprising the above H-CDR1, the above H-CDR2 and the above H-CDR3.

An anti-IL-23p19 antibody, or antigen-binding fragment thereof, may also contain:

a) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 1, 2, 3, 33, 34 and 35, respectively; or

b) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 4, 5, 3, 36, 34 and 37, respectively; or

c) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 1, 2, 3, 38, 39 and 35, respectively; or

d) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 6, 2, 3, 40, 41 and 42, respectively; or

e) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 7, 2, 3, 43, 41 and 44, respectively; or

e) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 8, 9, 10, 45, 46 and 47, respectively; or

g) the sequence L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NO: 8, 9, 10, 48, 49 and 50, respectively; or

h) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 11, 12, 13, 51, 52 and 53, respectively; or

i) the sequence L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 7, 2, 14, 54, 55 and 56, respectively; or

j) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 15, 16, 17, 57, 58 and 59, respectively; or

k) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 18, 16, 17, 60, 61 and 62, respectively; or

l) the sequence L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NO: 19, 20, 21, 63, 66, 67 or 68, 64 and 65, respectively; or

m) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 22, 23, 24, 69, 70 and 71, respectively; or

o) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 22, 25, 26, 55, 72 and 71, respectively; or

n) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 8, 9, 10, 45, 73 and 74, respectively; or

p) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 27, 28, 29, 45, 75 and 76, respectively; or

c) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 8, 9, 10, 77, 78 and 79, respectively; or

m) the sequence of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 SEQ ID NOs: 30, 31, 32, 80, 81 and 82, respectively.

An anti-IL-23p19 antibody, or antigen-binding fragment thereof, may comprise a light chain variable region comprising the above combination of L-CDR1, L-CDR2 and L-CDR3, and a heavy chain variable region comprising the above combination of H-CDR1, H-CDR2 and H-CDR3.

It is sometimes implied that based on chimeric antibodies with the replacement (switching) of CDR regions (i.e., for example, with the replacement of one or two CDRs of one of the mouse antibodies or humanized antibodies derived from them, with similar CDRs from another mouse antibody or derived from it a humanized antibody) between these exemplary immunoglobulins, valuable antibodies can be obtained.

The humanized anti-IL-23p19 antibody may be an antibody fragment. Various

- 25 040834 antibody fragments are described in general above and developed methods for obtaining antibody fragments. Fragments can be obtained by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24, 1992, p. 107-117 and Brennan et al., Science 229, 1985, p. 81). Alternatively, fragments can be generated directly from recombinant host cells. For example, Fab'-SH fragments can be isolated directly from E. coli and chemically linked to form F(ab') ₂ fragments (see, for example, Carter et al., Bio/Technology, 10, 1992, p. 163 -167). According to another approach, F(ab') ₂ fragments can be isolated directly from the culture of the recombinant host cell. Other techniques for obtaining antibody fragments should be obvious to a person skilled in the art.

Thus, antibody fragments may contain the CDRs described herein, in particular one of the combinations of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, and H-CDR3 described herein. Others are antibody fragments containing the variable regions described herein, for example, one of the combinations of the light chain variable regions and the heavy chain variable regions described herein.

The F(ab') ₂ fragment of a humanized anti-IL-23p19 antibody may comprise any of the light chain sequences of SEQ ID NO: 174 or SEQ ID NO: 180 in combination with the heavy chain sequence of SEQ ID NO: 176 or SEQ ID NO: 178. Moreover, the intact antibody, in turn, may contain these F(ab') ₂ fragments.

An antibody or antibody fragment may include a constant region that mediates an effector function. The constant region may mediate antibody-mediated cell-dependent cytotoxicity (ADCC), antibody-mediated cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC) responses to a target cell expressing IL-23. The effector domain(s) may be, for example, the Fc region of an Ig molecule.

The effector domain of an antibody can be derived from any suitable vertebrate species and any isotype. Isotypes from different animal species differ in their ability to mediate effector functions. For example, in terms of their ability to mediate CDC and ADCC/ADCP, human immunoglobulins tend to be in the order IgM®IgG1®IgG ₃ >IgG ₂ >IgG ₄ and IgG1®IgG ₃ >IgG ₂ /IgM/IgG ₄ , respectively. Mouse immunoglobulins, in terms of their ability to mediate CDC and ADCC/ADCP, are generally arranged in the following order: mouse IgM®IgG ₃ >>IgG _2b >IgG _2a >>IgG1 and IgG _2b >IgG _2a >IgG1>>IgG ₃ , respectively. In another example, mouse IgG _2a mediates ADCC, and mouse IgG _2a and IgM both mediate CDC.

Antibody modifications.

Humanized anti-IL-23p19 antibodies and agents may include modifications to a humanized anti-IL-23p19 antibody or antigen-binding fragment thereof. For example, it may be desirable to modify the antibody with respect to its effector function, thereby increasing the effectiveness of the antibody in the treatment of cancer. One such modification is the introduction of cysteine residue(s) into the Fc region, which results in the formation of an interchain disulfide bond in this region. The homodimeric antibody thus obtained may have improved internalization capacity and/or increased complement-dependent cytotoxicity and antibody-mediated cell-dependent cytotoxicity (ADCC) (see, e.g., Caron et al., J. Exp. Med., 176, 1992, p. 1191-1195 and Shopes, J. Immunol., 148, 1992, pp. 2918-2922). Homodimeric antibodies with enhanced antitumor activity can also be obtained using the heterobifunctional cross-linkers described in Wolff et al., Cancer Research, 53, 1993, p. 2560-2565. Alternatively, an antibody can be engineered to have twice the number of Fc regions, whereby it may have an increased ability for complement-dependent lysis and ADCC (see Stevenson et al., Ant-Cancer Drug Design, 3, 1989, p. 219- 230).

Antibodies with increased ability to maintain ADCC were obtained by modifying the glycosylation pattern of their Fc region. This is possible because glycosylation of the antibody at the asparagine residue, N297, in the C _H 2 domain is involved in the interaction between IgG and Fcγ receptors that is required for ADCC. Host cell lines were designed to express antibodies with altered glycosylation, for example, with increased levels of bisector N-acetylglucosamine or reduced levels of fucose. Decreased fucose provides a more significant increase in ADCC activity than an increase in bisecting N-acetylglucosamine. In addition, the increased ADCC activity of low fucose antibodies is independent of the FcyRIIIa V/F polymorphism.

Modification of the amino acid sequence of the Fc region of antibodies is an alternative to glycosylation in terms of increasing ADCC activity. The binding site on human IgG1 for Fcy receptors was determined using extended mutation analysis. This made it possible to create humanized antibodies of the IgG1 isotype with mutations in the Fc region, which increased the binding affinity for FcyRIIIa and enhanced ADCC activity in vitro. In addition, Fc variants with multiple

- 26 040834 assigned different permutations of the binding ability, for example, with increased ability to bind to specific FcγR receptors, but with unchanged or reduced ability to bind to other FcγR receptors.

Immunoconjugates may comprise a humanized antibody or fragment thereof conjugated/conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatic bacterial, fungal, plant, or animal toxin or fragments thereof), or a radioactive isotope (i.e., a radioconjugate ).

Chemotherapeutic agents that can be used to obtain such immunoconjugates are described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria toxin A chain, non-binding active diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modescin A chain, alpha-sarcin, proteins Aleurites fordii, dianthine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), inhibitor from Momordica charantia, curcin, crotin, inhibitor from Sapaonaria officinalis, gelonin, mitogellin, restrictocin, fenomycin, enomycin, tricothecenes, etc. Various radionuclides can be used to generate radioconjugates of humanized anti-IL-23p19 antibodies. Their examples are ²¹² Bi, ¹³¹ 1, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Conjugates of a humanized anti-IL-23p19 antibody and a cytotoxic or chemotherapeutic agent can be prepared by known methods using a variety of bifunctional protein binding agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyladipimidate-HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazido compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis(p- diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene-2,6diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared according to the method described in Vitetta et al., Science, 238, 1987, p. 1098). C ¹⁴ labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of a radionucleotide to an antibody. Conjugates can also be made using a cleavable linker.

Humanized antibodies to IL-23p19, presented in the present description, can also be prepared in the form of immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. sci. USA 82, 1985, p. 3688; Hwang et al., Proc. Natl. Acad. sci. USA 77, 1980, p. 4030; and US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation life are described, for example, in US Pat. No. 5,013,556.

The most preferred liposomes can be obtained by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through filters with a certain pore size, obtaining liposomes of the required diameter. Fab' fragments of an antibody can be incorporated into liposomes according to the method described in Martin et al., J. Biol. Che., 257, 1982, p. 286-288 via a disulfide bond formation reaction. A chemotherapeutic agent (such as doxorubicin) can optionally be included in the liposomes (see, for example, Gabizon et al., J. National Cancer Inst., 81(19), 1989, p. 1484).

The antibodies provided herein can be used in ADEPT (Antibody Directed Enzyme Prodrug Therapy Procedures) by conjugating the antibody to a prodrug activating enzyme that converts the prodrug (e.g., a peptidyl chemotherapeutic agent) into an active anticancer drug (see e.g. , WO 81/01145, WO 88/07378 and US No. 4975278). An enzyme component of the immunoconjugate that can be used in ADEPT may be an enzyme that has the ability to effect a prodrug in such a way that it is converted to a more active cytotoxic form. Specific enzymes that can be used in ADEPT include, but are not limited to, alkaline phosphatase for converting phosphate-containing prodrugs to free drug substances; arylsulfatase for converting sulfate-containing prodrugs into free drug substances; cytosine deaminase to convert the non-toxic 5-fluorocytosine into the anti-cancer drug substance 5-fluorouracil; proteases such as Serratia proteinase, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L) for converting peptide-containing prodrugs into free drug substances; D-alanylcarboxypeptidases for converting prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase to convert glycosylated prodrugs to free drug substances; β-lactamase for converting prodrugs derivatized with β-lactams into free drug substances; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, for converting drug substances derivatized at the amine nitrogens of the phenoxyacetyl or phenylacetyl group

- 27 040834 pamy, respectively, into free medicinal substances. Alternatively, antibodies with enzymatic activity (abzymes) can be used to convert prodrugs into free active drug substances (see, for example, Massey, Nature, 328, 1987, p. 457-458). Antibody-abzyme conjugates can be prepared using methods known in the art for introducing an abzyme into a tumor cell population, for example, by covalently linking the enzyme to humanized anti-GE-23p19 antibody/heterobifunctional crosslinking reagents as described above. Alternatively, fusion proteins that contain at least an antigen-binding region of an antibody linked to at least a functionally active region of an enzyme provided herein can be constructed using recombinant DNA techniques (see, e.g., Neuberger et al., Nature, 312, 1984, pp. 604-608).

Occasionally, it may be desirable to use a fragment of a humanized anti-IL-23p19 antibody instead of an intact antibody, for example, to increase tissue penetration, for example, it may be necessary to modify the antibody fragment to increase serum half-life. For this purpose, it is possible, for example, to include an epitope that binds to a rescue receptor in an antibody fragment. According to one method, the corresponding region of the antibody fragment can be changed (for example, mutated), or the epitope can be inserted into a peptide tag, which is then fused to the antibody fragment at either end or in the middle, for example, using DNA or peptide synthesis (see, for example WO 96/32478).

Covalent modifications of the humanized anti-IL-23p19 antibody may also be used. Covalent modifications include modification of cysteinyl residues, histidyl residues, lysinyl and amino terminal residues, arginyl residues, tyrosyl residues, carboxyl side groups (aspartyl or glutamyl), glutaminyl and asparaginyl residues, or seryl or threonyl residues. Another type of covalent modification involves the chemical or enzymatic cross-linking of glycosides with an antibody. These modifications can be carried out by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if possible. For the introduction of other types of covalent modifications into the antibody molecule, the interaction of labeled amino acid residues of the antibody with an organic derivatizing agent that can interact with selected side chains or amino-terminal or carboxy-terminal residues can be used.

Removal of any carbohydrate moieties present on the antibody can be done chemically or enzymatically. Chemical deglycosylation is described in Hakimuddin et al., Arch. Biochem. Biophys. 259, 1987, p. 52 and in Edge et al., Anal. Biochem., 118, 1981, p. 131. Enzymatic cleavage of carbohydrate fragments on antibodies can be performed using various endo- and exoglycosidases according to the method described in Thotakura et al., Meth. Enzymol., 138, 1987, p. 350.

Another type of suitable covalent modification involves linking the antibody to one of numerous non-protein polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, using the methods described in one or more of US No. 4640835, US No. 4496689, US No. 4301144, US No. 4670417, US No. 4791192 and US No. 4179337.

Humanization and amino acid sequence variants.

Amino acid sequence variants of an anti-IL-23p19 antibody can be generated by introducing appropriate nucleotide changes into the DNA of an anti-IL-23p19 antibody or by peptide synthesis. These variants include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the anti-IL-23p19 antibodies exemplified herein. Any combination of deletions, insertions and substitutions is used to obtain the final construct, provided that the final construct has the required characteristics. Amino acid substitutions can also alter the post-translational processing of the humanized anti-IL-23p19 antibody or variant, such as changing the number or position of glycosylation sites.

A useful method for identifying specific residues or regions in an anti-IL-23p19 antibody that are preferred sites for mutagenesis is so-called alanine-scanning mutagenesis, as described in Cunningham and Wells, Science, 244, 1989, p. 1081-1085. It identifies a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) and replaces it with a neutral or negatively charged amino acid (usually alanine) to affect the interaction of amino acids with antigen IL-23p19. Those amino acid positions that have been shown to be functionally sensitive to substitutions are then improved by the introduction of additional or other variants at or for substitution sites. Thus, although the site for the introduction of amino acid sequence variation is predetermined, the nature of the mutation itself is not predetermined. For example, to analyze the efficiency of mutation at the site in question, an alanine scan or non-specific mutagenesis of the target codon or target region is performed and the resulting anti-IL-23p19 antibody variants are screened for the desired activity.

Insertions into the amino acid sequence include amino- and/or carboxy-terminal fusions

- 28 040834 ions of various lengths from one residue to polypeptides that contain one hundred or more residues, as well as insertions into the sequence of one or more amino acid residues. An example of a terminal insertion is an anti-IL-23p19 antibody fused to a tag epitope. Other insertion variants of the anti-IL-23p19 antibody molecule include fusion to the N- or C-terminus of the anti-IL-23p19 antibody of an enzyme or polypeptide that prolongs the serum half-life of the antibody.

Another type of variant is a variant comprising an amino acid substitution. In these embodiments, at least one amino acid residue in the anti-IL-23p19 antibody molecule is removed and another residue is inserted in its place. The sites of greatest interest for mutagenesis by substitution are the hypervariable regions, but changes to the FR can also be made. Conservative substitutions are presented in table. 8 titled preferred substitutions. If such substitutions result in a change in biological activity, then more significant substitutions, referred to as Examples of substitutions, or other substitutions described below with reference to the amino acid class, can be introduced, and then the products can be screened.

Table 8

original balance Substitution examples Preferred Substitutions Ala (A) Val; Leu; lie Val Arg(R) Lys; gin; Asn Lys Asn(N) gin; His; asp; Lys; Arg gin Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gin (Q) asn; Glu Asn Glu(E) asp; gin asp Gly (G) Ala Ala His(H) Arg; asn; gin; Lys Arg He(I) Leu; Val; met; Ala; Ph; norleucine Leu Leu(L) lie; norleucine; Val; met; Ala; Phe lie Lys (K) Arg; Ph; lie Arg Met(M) Leu; Ph; lie Leu Phe(F) Tyr; Leu; Val; lie; Ala Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser TRP(W) Tyr; Phe Tyr Tyr (Y) Ph; trp; Thr; Ser Phe Val(V) Leu; lie; met; Ph; Ala; norleucine Leu

It is generally accepted in protein chemistry that the biological properties of an antibody can be modified by selecting substitutions that vary widely in their effect on maintaining (a) the backbone structure of the polypeptide at the site of the substitution, such as a folded or helical conformation, (b) the charge or hydrophobicity of the molecule in the target site or (in) the size of the side chain. Naturally occurring residues are divided into groups based on the common properties of the side chains:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions involve replacing a representative of one of these classes with a representative of another class.

Any of the cysteine residues not involved in maintaining the proper conformation of the humanized anti-IL-23p19 antibody or variant can also be replaced, usually with serine, to increase the oxidative stability of the molecule and prevent abnormal cross-linking, or to create points for conjugation with a cytotoxic or cytostatic compound. Conversely, cysteine bond(s) can be introduced into the antibody to improve its stability (especially if the antibody is an antibody fragment, such as an Fv fragment).

One type of variant generated by substitution involves substitution of one or more hypervariable region residues of the parent antibody (eg, humanized or human antibody). As a rule, the resulting variant(s) selected for further improvement should have improved biological properties relative to the parent antibody from which it(they) is(are) derived. A suitable way to generate such substitution-based variants involves affinity maturation using phage display. In general, the method is as follows: several hypervariable region sites (eg, 6-7 sites) are mutated to give all possible amino acid substitutions at each site. The antibody variants thus obtained are displayed in monovalent form on the surface of filamentous phage particles as fusions with the gene III product of M13 phage, which is packaged in each particle. Options to

- 29 040834 which are exposed to phage and then screened for their biological activity (eg binding affinity). In order to identify hypervariable region sites that are promising for modification, alanine-scanning mutagenesis can be performed to identify hypervariable region residues that are most important for antigen binding. Alternatively or additionally, it may be useful to analyze the crystal structure of the antigen-antibody complex in order to identify points of contact between the antibody and human IL-23p19. Such contact residues and adjacent residues are candidates for replacement using the methods developed in the creation of the present invention. Once such variants have been generated, a panel of variants is subjected to the screening described herein, and antibodies with improved properties in one or more of the appropriate assays can be selected for further development.

Another way to obtain an amino acid variant of an antibody is to change the original glycosylation pattern of the antibody. By alteration is meant the deletion of one or more carbohydrate moieties present in the antibody and/or the addition of one or more glycosylation sites not present in the antibody.

Glycosylation of antibodies usually occurs via either N-linking or O-linking. N-linking involves the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, are recognizable sequences designed to enzymatically attach a carbohydrate moiety to the side chain of an asparagine residue. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation involves the attachment of one of the sugars, such as N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Thus, in order to glycosylate a given protein, eg an antibody, an amino acid sequence of the protein is created that contains one or more of the tripeptide sequences described above (for N-glycosylation sites). The change may also be the addition or replacement of one or more serine or threonine residues in the sequence of the parent antibody (for O-glycosylation sites).

Nucleic acid molecules that encode variants of the amino acid sequences of antibodies to IL-23p19, get using various methods known in this field. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants), or preparation by oligonucleotide mutagenesis (or site-directed mutagenesis), PCR mutagenesis, and cassette mutagenesis of a previously generated variant, or non-variant version of the antibody to IL-23p19.

Polynucleotides, Vectors, Host Cells, and Recombination Methods The isolated polynucleotides can encode any desired form of anti-IL-23p19 antibody, including, for example, full-length monoclonal antibodies, Fab-, Fab'-, F(ab') ₂ - and Fv fragments, dimeric antibodies, linear antibodies, single chain antibody molecules, and multispecific antibodies derived from antibody fragments.

The isolated polynucleotides may contain sequences encoding the light chain variable region of an antibody or antibody fragment that has any of the amino acid sequences of SEQ ID NOs: 84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, or 119. Examples of polynucleotide sequences encoding said amino acid sequences are SEQ ID NOs: 83, 85, 87, 89, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116 and 118. The isolated polynucleotides may contain sequences encoding the heavy chain variable region of an antibody or antibody fragment that has the amino acid sequence of SEQ ID NOs: 121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, or 156. Examples of polynucleotide sequences encoding said amino acid sequences are SEQ ID NOs: 120, 122, 124, 126, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, or 155. The isolated polynucleotides may comprise sequences encoding the light chain variable region of an antibody or antibody fragment that has any of the amino acid sequences of SEQ ID NO: 158, 160, 162, or 164. Examples of polynucleotide sequences encoding said amino acid sequences are SEQ ID NO: 157, 159, 161, or 163. The isolated polynucleotides may contain sequences encoding the heavy chain variable region of an antibody or antibody fragment that has the amino acid sequence of SEQ ID NO: 166, 168, 170, or 172. Examples of polynucleotide sequences encoding these amino acid sequences are SEQ ID NO: 165, 167, 169 or 171.

The isolated polynucleotides may contain sequences encoding an antibody light chain having any of the amino acid sequences of SEQ ID NO: 174 or SEQ ID NO: 180. Examples of polynucleotide sequences encoding said amino acid sequences

- 30 040834 values are SEQ ID NO: 173 or SEQ ID NO: 179. The isolated polynucleotides may contain sequences encoding the heavy chain of an antibody having the amino acid sequence of SEQ ID NO: 176 or SEQ ID NO: 178. Examples of polynucleotide sequences encoding these amino acid sequences are SEQ ID NO: 175 or SEQ ID NO: 177.

The isolated(s) polynucleotide(s) sequence(s) may encode an antibody or antibody fragment having a variable region of the light chain and heavy chain, which contain the amino acid sequences of SEQ ID NO: 174 and SEQ ID NO: 176, respectively; SEQ ID NO: 174 and SEQ ID NO: 178, respectively; SEQ ID NO: 180 and SEQ ID NO: 176, respectively; SEQ ID NO: 180 and SEQ ID NO: 178, respectively. Examples of polynucleotide sequences encoding the indicated amino acid sequences are SEQ ID NO: 173 and SEQ ID NO: 175, respectively, SEQ ID NO: 173 and SEQ ID NO: 177, respectively, SEQ ID NO: 179 and SEQ ID NO: 175, respectively, SEQ ID NO: 179 and SEQ ID NO: 177, respectively.

The polynucleotide(s) which contain(s) a sequence encoding a humanized anti-IL-23p19 antibody, or fragment or chain thereof, may be fused to one or more regulatory or control sequences known in the art and may be incorporated into acceptable expression vectors or host cell known in the art. Each of the polynucleotide molecules encoding the heavy or light chain variable domains can be independently fused to a polynucleotide sequence that encodes a constant domain, such as a human constant domain, allowing intact antibodies to be generated. Alternatively, the polynucleotides, or portions thereof, can be fused together to create a template to produce a single chain antibody.

For production by recombination techniques, the polynucleotide encoding the antibody is inserted into a replicable vector for cloning (DNA amplification) or for expression. Many suitable vectors for the expression of a recombinant antibody are known and available. The components of a vector typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription terminator sequence.

Humanized antibodies to IL-23p19 can also be prepared as fusion polypeptides in which the antibody is fused to a heterologous polypeptide, such as a signal sequence or another polypeptide, bearing a specific cleavage site at the amino terminus of the mature protein or polypeptide. The heterologous signal sequence chosen is typically one that is recognized and processed (ie, cleaved off by a signal peptidase) in the host cell. For prokaryotic host cells that cannot recognize and process the humanized anti-IL-23p19 antibody signal sequence, the signal sequence can be replaced with a prokaryotic signal sequence. The signal sequence may be, for example, alkaline phosphatase, penicillinase, lipoprotein or thermostable enterotoxin II leaders, and the like. For secretion from yeast, the native signal sequence can be replaced, for example, with a leader sequence derived from yeast invertase α-factor (including Saccharomyces and Kluyveromyces α-factor leader sequences), acid phosphatase, C. albicans glucoamylase, or the signal sequence described in WO 90 /13646. Mammalian signal sequences can be used for expression in mammalian cells, as well as viral secretory leader sequences, such as the herpes simplex virus gD signal. DNA encoding said precursor region is ligated in-frame to DNA that encodes a humanized anti-IL-23p19 antibody.

Expression and cloning vectors contain a nucleotide sequence that allows the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is a sequence that allows for viral replication independent of the host's chromosomal DNA and includes an origin of replication or autonomously replicating sequences. Such sequences are well known for various bacteria, yeasts and viruses. The origin of replication from plasmid pBR322 is suitable for most Gram-negative bacteria, the origin of plasmid 2μ is suitable for yeast, and various viral origins (SV40 (simian virus 40), polyoma virus, adenovirus, VSV (vesicular stomatitis virus) or BPV (large papilloma virus) cattle)) are suitable for cloning vectors in mammalian cells. Generally, mammalian expression vectors do not require an origin component (generally, only the SV40 origin can be used because it contains an early promoter).

Expression and cloning vectors may contain a gene that encodes a selectable marker to facilitate identification or expression. Typically, selectable marker genes

- 31 040834 proteins that confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, or alternatively replenish auxotrophic deficiency, or alternatively replenish critical nutrients that are not present in complex environments, for example the gene encoding D-alanine racemase for Bacilli.

In one exemplary selection scheme, a drug is used to stop the growth of a host cell. In those cells that are successfully transformed with the heterologous gene, the protein conferring drug resistance is produced and the cells therefore survive this selection scheme. Examples of drugs used for such dominant selection are neomycin, mycophenolic acid and hygromycin. Common selectable markers for mammalian cells are those that allow the identification of cells competent to take up a nucleic acid that encodes a humanized antibody to IL-23p19, such as DHFR (dihydrofolate reductase), thymidine kinase, metallothionein I and -II (for example, primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, and the like. Cells transformed with the DHFR gene used for selection are identified primarily by culturing all transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. A suitable host cell when wild-type DHFR is used is a Chinese Hamster Ovary (CHO) cell deficient in DHFR activity (eg, DG44).

Alternatively, host cells (primarily wild-type hosts that contain an endogenous DHFR gene) transformed or co-transformed with DNA sequences that encode an anti-IL-23p19 antibody, a wild-type DHFR protein, and another selectable marker such as aminoglycoside-3 '-phosphotransferase (APH) can be selected for cell growth in a medium containing a selection agent for a selection marker, such as an aminoglycoside antibiotic, eg kanamycin, neomycin, or G418 (see, eg, US Pat. No. 4,965,199).

When recombinant production is carried out using a yeast cell as the host cell, the TRP1 gene which is present in the yeast-derived plasmid YRp7 (Stinchcomb et al., Nature, 282, 1979, p. 39) can be used as a marker. The TRP1 gene is a selection marker for a mutant yeast strain lacking the ability to grow in a medium supplemented with tryptophan, such as strain ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85, 1977, p. 12). Thus, the presence of a trp1 lesion in the yeast host cell genome provides an efficient environment for detecting growth-based transformation in the absence of tryptophan. Similarly, Leu2p deficient yeast strains such as ATCC 20622 and 38626 are supplemented with known plasmids carrying the LEU2 gene.

In addition, vectors derived from the 1.6 μm pKD1 circular plasmid can be used to transform the yeast Kluyveromyces. On the other hand, an expression system for the large scale production of recombinant calf chymosin in K. lactis is known (Van den Berg, Bio/Technology 8, 1990, p. 135). Stable multicopy expression vectors for the secretion of mature recombinant human serum albumin using industrial strains of Kluyveromyces have also been described (Fleer et al., Bio/Technology 9, 1991, p. 968-975).

Expression and cloning vectors typically contain a promoter recognized by the host organism and operably linked to a nucleic acid molecule that encodes an anti-IL-23p19 antibody or polypeptide chain thereof. Promoters that are suitable for use in prokaryotic hosts are the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and hybrid promoters such as the tac promoter. Other bacterial promoters may also be used. Promoters intended for use in bacterial systems also contain a Shine-Dalgarno (S.D.) sequence operably linked to DNA that encodes a humanized anti-IL-23p19 antibody.

Many eukaryotic promoter sequences are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the transcriptional initiation site. Another sequence found at a distance of 70-80 bases from the transcription start site of many genes is the CNCAAT region, in which N can be any nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence, which may represent a signal to add a poly-A tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukryotic expression vectors.

Examples of promoter sequences that can be used in yeast host cells include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase , phosphoglucose isomerase and glucokinase.

Inducible promoters have the additional transcriptional advantage of

- 32 040834 which is controlled by growth conditions. These include the yeast promoter regions of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, metalthionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. In addition, suitable vectors and promoters that can be used in yeast expression are described in EP 73657. Yeast enhancers are also advantageously used in combination with yeast promoters.

Transcription of the humanized anti-IL-23p19 antibody from vectors in mammalian host cells is controlled, for example, by promoters derived from the genomes of viruses such as polyoma virus, avian pox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40), using heterologous mammalian promoters, for example, an actin promoter or an immunoglobulin promoter, or heat shock promoters, provided that such promoters are compatible with host cell systems.

The early and late promoters of the SV40 virus are expediently obtained in the form of an SV40 restriction fragment, which also contains the site of initiation of SV40 virus replication. The immediate early promoter of human cytomegalovirus is expediently obtained as a restriction HindIII E fragment. A DNA expression system in mammalian host cells using bovine papillomavirus as a vector is described in US No. 4,419,446. A modification of this system is described in US No. 4,601,978 (see also Reyes et al. 598-601 description of human p-interferon cDNA expression in mouse cells under the control of the thymidine kinase promoter from the herpes simplex virus). Alternatively, the Rous sarcoma virus long terminal repeat can be used as a promoter.

Another important element that can be used in a recombinant expression vector is an enhancer sequence that is used to increase the transcription of DNA encoding a humanized anti-IL-23p19 antibody in higher eukaryotic organisms. Many enhancer sequences of mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin genes) are known. Typically, however, an enhancer from a eukaryotic cell virus is used. Examples are the SV40 enhancer located far from the origin of replication (base pairs 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer located far from the origin of replication, and adenovirus enhancers (see also Yaniv Nature 297, 1982, pp. 17-18 description of enhancer elements used to activate eukaryotic promoters). The enhancer can be spliced into the vector at the 5' or 3' position relative to the anti-IL-23p19 antibody coding sequence, but is preferably placed at a site located 5' from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells of other multicellular organisms) must also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are usually derived from the 5' and sometimes from the 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated region of the mRNA that encodes the anti-IL-23p19 antibody. One suitable transcription termination component is the bovine growth hormone polyadenylation region (see WO 94/11026 and the expression vector presented therein). Humanized antibodies to IL-23p19 can be expressed using the CHEF system (see, for example, US No. 5888809, the description of which is incorporated herein by reference).

Suitable host cells for cloning or expression of DNA in the vectors described herein are prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotes include, but are not limited to, eukaryotic bacteria (eubacteria) such as gram-negative or gram-positive organisms, for example, fam. Enterobacteriaceae such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia, e.g. Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis (e.g. strain B. licheniformis 41P, which is described in DD 266710 published April 12, 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli strain that is used as a cloning host is E. coli 294 (ATCC 31446), although other E. coli B strains, E. coli X1776 (ATCC 31537) and E. coli can be used. W3110 (ATCC 27325). These examples are given by way of illustration and do not limit the scope of the invention.

In addition to prokaryotic organisms, eukaryotic microorganisms such as filamentous fungi or yeast can be used as hosts for cloning or expression of vectors that encode a humanized anti-III-23p19 antibody. The most commonly used lower eukaryotic host microorganism is Saccharomyces cerevisiae, ie. regular baker's yeast. However, it is possible to use numerous known and available representatives of other genera, species and strains, such as Schizosaccharomyces pombe; hosts from the family. Kluyveromyces such as

- 33 040834 example, as K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 36906), K. thermotolerans and K. marxianus; Yarrowia (EP 402226); Pichiapastoris (EP 183070); Candida Trichoderma reesia (EP 244234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76, 1979, p. 5259-5263); Schwanniomyces such as Schwanniomyces occidentalism and filamentous fungi such as Neurospora, Penicillium, Tolypocladium and hosts p. Aspergillus such as A. nidulans and A. niger.

Host cells suitable for expression of a glycosylated humanized anti-IL23p19 antibody are obtained from multicellular organisms. Examples of invertebrate cells are plant cells and insect cells, e.g., numerous baculovirus strains and variants thereof and corresponding insect host cells such as Spodoptera frugiperda (caterpillar), Aedes aegipti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori (silkworm). A variety of virus strains are widely known that can be used for transfection, such as Autographa californica NPV variant L-1 and Bombyx mori Bm-5 NPV strain, and such viruses can be used primarily for transfection of Spodoptera frugiperda cells.

Cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.

Expression of a humanized anti-IL-23p19 antibody can be carried out in vertebrate cells. Propagation of vertebrate cells in culture (tissue culture) has now become a standard procedure and the corresponding techniques are widely accepted. Examples of suitable host mammalian cell lines are monkey kidney cell line CVI transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cell line subcloned for suspension culture, Graham et al., J. Gen. Virol., 36, 1977, p. 59); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA, 77, 1980, p. 4216); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23, 1980, p. 243-251); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); bovine rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Ann als N.Y. Acad. Sci., 383, 1982, pp. 44-68); MRC 5 cells; FS4 cells and a human hepatoma cell line (Hep G2).

Host cells are transformed with the expression or cloning vectors described above to produce a humanized anti-IL-23p19 antibody and cultured in suitable nutrient media suitably modified to induce promoters, select transformants, or amplify genes encoding the desired sequences.

Host cells that are used to produce the humanized anti-IL-23p19 antibody provided herein can be cultured in a wide variety of media. Commercially available media can be used to culture host cells, such as Ham's F10 medium (Sigma-Aldrich Co., St. Louis, MO), Minimal Maintenance Medium ((MEM), Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.) and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma-Aldrich Co.). In addition, any of the media described in one or more of the following publications can be used as host cell culture media: Ham et al., Meth. Enz. 58, 1979, p. 44, Barnes et al., Anal. Biochem. 102, 1980, p. 255, US No. 4767704, US No. 4657866, US No. 4927762, US No. 4560655, US No. 5122469, WO 90/103430 and WO 87/00195. Any of these media can be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium, calcium, magnesium chloride and phosphate), buffers (such as HEPES), nucleotides ( such as adenosine and thymidine), antibiotics (such as gentamicin), micronutrients (ie, inorganic compounds that are typically present in final concentrations in the micromolar range), and glucose or an equivalent energy source. It will be apparent to those skilled in the art that any other necessary additives may also be included at appropriate concentrations. Culture conditions such as temperature, pH, and the like are those that have been used previously for host cells selected for expression, and should be obvious to one of ordinary skill in the art.

When using recombination techniques, the antibody can be produced inside the cell, in the periplasmic space, or directly released into the environment. If the antibody is produced within the cell, then in the first step, the cells can be destroyed to release the protein. Particulate debris, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. In Carter et al., Bio/Technology, 10, 1992, p. 163-167 describe a method for isolating antibodies secreted into the periplasmic space of E. coli. In general, the method is as follows: the cell mass in the form of a paste is thawed for approx.

- 34 040834 for 30 minutes in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF). Cellular debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatants of such expression systems are typically first concentrated using a commercially available protein concentration filter, such as an ultrafiltration device such as Amicon or Millipore Pellicon. At any of the previous steps, a protease inhibitor such as PMSF may be added to inhibit proteolysis, and antibiotics may be added to prevent the build up of unwanted impurities. A wide variety of techniques can be used to isolate an antibody from a host cell.

The antibody composition derived from the cells can be purified, for example, by hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification method. The possibility of using protein A as an affinity ligand depends on the type and isotype of the Fc region of any immunoglobulin present in the antibody. Protein A can be used to purify antibodies based on human heavy gamma 1, gamma 2 or gamma 4 chains (Lindmark et al., J. Immunol. Meth., 62, 1983, p. 1-13). Protein G is recommended for use in all mouse isotypes and for the human gamma 3 chain (Guss et al., EMBO J., 5, 1986, p. 1567-1575). Agarose is most commonly used as the matrix to which the affinity ligand binds, but other matrices can also be used. Mechanically resistant matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow higher flow rates and shorter processing times to be achieved compared to the performance achieved with agarose. If the antibody contains a _CH 3 domain, a Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) can be used for purification. Depending on the antibody to be isolated, other protein purification methods can also be used, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, SEPHAROSE™ heparin chromatography, chromatography on an anion or cation exchange resin (e.g. column with polyaspartic acid), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate.

After any of the previous purification step(s), the mixture containing the antibody of interest and impurities can be subjected to hydrophobic interaction chromatography at low pH using an elution buffer that has a pH of about 2 .5 to 4.5, chromatography is preferably carried out at low salt concentrations (eg using approximately 0-0.25M salts).

Nucleic acids that hybridize under the non-stringent, moderate, or stringent conditions described herein to all or part (e.g., the region that encodes a variable region) of a nucleotide sequence are isolated polynucleotide sequence(s) coding(s) antibody or antibody fragment, Hybridizing region of the hybridizing nucleic acid, as a rule, consists of at least 15 (for example, 20, 25, 30 or 50) nucleotides. The hybridizing region of the hybridizing nucleic acid is at least 80%, e.g., at least 90%, at least 95%, or at least 98% identical to the sequence of the region or all of the nucleic acid that encodes anti-[L-23p19 a polypeptide (eg, a heavy chain or light chain variable region), or its complement. Hybridizable nucleic acids of the type described herein can be used, for example, as a cloning probe, a primer, such as a PCR primer, or a diagnostic probe.

The isolated polynucleotides may include sequences that encode an antibody or antibody fragment having/having any of the amino acid sequences of SEQ ID NOs: 84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, or 119 and which are at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the polynucleotide sequences of SEQ ID NO: 83, 85, 87, 89, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, or 118.

The isolated polynucleotides may include sequences that encode an antibody or antibody fragment having/having any of the amino acid sequences of SEQ ID NO: 158, 160, 162, or 164, and which are at least 80%, at least 90%, at least at least 95%, at least 98%, or at least 99% identical to the polynucleotide sequences of SEQ ID NO:157, 159, 161, or 163.

The isolated polynucleotides may include sequences that encode an antibody or antibody fragment having/having any of the amino acid sequences of SEQ ID NOs: 121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, or 156 and which are at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the polynucleotide sequences of SEQ ID NO: 120, 122, 124, 126, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 or 155.

Isolated polynucleotides may include sequences that encode an antibody

- 35 040834 or an antibody fragment having/having any of the amino acid sequences of SEQ ID NO: 166, 168, 170 or 172, and which are at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the polynucleotide sequences of SEQ ID NO: 165, 167, 169, or 171.

The isolated polynucleotides may include sequences that encode an antibody or antibody fragment having/having a light chain variable region amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% % or at least 99% identical to any of the amino acid sequences of SEQ ID NOs: 84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115 , 117, or 119. The isolated polynucleotides may include sequences that encode an antibody or antibody fragment having/having a light chain variable region amino acid sequence that is at least 80%, at least 90%, at least 95% at least 98% or at least 99% identical to any of the amino acid sequences of SEQ ID NO: 158, 160, 162, or 164. The isolated polynucleotides may include sequences that encode an antibody or antibody fragment, having/having a heavy chain variable region amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any of the amino acid sequences SEQ ID NOs: 121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, or 156. The isolated polynucleotides may include sequences that encode an antibody or antibody fragment having/having a heavy chain variable region amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any of the amino acid sequences of SEQ ID NO: 166, 168, 170 or 172. In the context of the present description, the concept of identical or percent identity regarding two or more nucleotide or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotides or amino acid residues when compared and aligned for maximum match. To determine percent identity, sequences are aligned for optimal comparison (eg, gaps can be introduced into the sequence of the first amino acid or nucleotide sequence for optimal primary structure comparison with a second amino acid or nucleotide sequence). Amino acid residues or nucleotides are then compared at the corresponding amino acid positions or nucleotide positions. When a position in the first sequence has the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions in the sequences (ie, % identity = number of identical positions/total number of positions (eg, overlapping positions) x 100). The two sequences to be compared may be of the same length after introduction, if gaps in the sequence are necessary (eg, excluding additional sequence extending beyond the sequences to be compared). For example, when variable region sequences are compared, leader and/or constant domain sequences are not considered. When comparing the sequences of two sequences, the corresponding CDR refers to the CDR located in the same position in both sequences (eg, CDR-H1 of each sequence). Determining the percent identity or percent similarity between two sequences can be done using a mathematical algorithm. A preferred example of a mathematical algorithm that can be used to compare two sequences is (but not limited to) the algorithm of Karlin and Altschul, Proc. Natl. Acad. sci. USA 87, 1990, pp. 2264-2268 modified according to Karlin and Altschul, Proc. Natl. Acad. sci. USA 90, 1993, pp. 5873-5877. This algorithm is included in the NBLAST and XBLAST programs described in Altschul et al., J. Mol. Biol. 215, 1990, p. 403-410. Nucleotide analysis with BLAST can be performed using the NBLAST program, in which score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid encoding the protein of interest. BLAST analysis of proteins can be performed using the XBLAST program, in which score = 50, word length = 3, to obtain amino acid sequences homologous to the protein of interest. To perform comparative analyzes of the primary structure, including gaps, you can use the Gapped BLAST program described in Altschul et al., Nucleic Acids, 25, 1997, p. 3389-3402. Alternatively, PSI-Blast can be used to perform an iterative search to determine distant relationships between molecules (Id.). When using the BLAST, Gapped BLAST and PSI-Blast programs, the default parameters of the respective programs (eg XBLAST and NBLAST) can be used. Another preferred example (but not limited to) of a mathematical algorithm that can be used to compare sequences is

- 36 040834 algorithm described in Myers and Miller, CABIOS, 1989. This algorithm is included in the program ALIGN (version 2.0), which is part of the software package for comparative analysis of the primary structure of GCG sequences. When the ALIGN program is used to compare amino acid sequences, a PAM120 weighted residue table, gap length penalty of 12, and gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, Comput. . Appl. biosci. 10, 1994, p. 3-5; and FASTA, described by Pearson and Lipman, Proc. Natl. Acad. sci. USA 85, 1988, p. 2444-2448. In the FASTA program, the ktup parameter denotes a control parameter that sets the sensitivity and speed of the search. If ktup = 2, then similar regions in the two sequences to be compared are identified by looking at pairs of aligned residues; if ktup = 1, then the individual aligned amino acids are evaluated. For protein sequences, ktup can be set to 2 or 1, and for DNA sequences, values ranging from 1 to 6. The default value of ktup, if not specified, is 2 for proteins and 6 for DNA. Alternatively, comparative primary structure analysis of protein sequences can be performed using the CLUSTAL W algorithm described in Higgins et al., Methods Enzymol. 266, 1996, p. 383-402.

non-therapeutic use.

The antibodies provided herein can be used as affinity-based purification agents. In this process, the antibodies are immobilized on a solid phase, such as a protein A resin, by methods well known in the art. The immobilized antibody is brought into contact with a sample containing the IL-23p19 protein (or fragment thereof) to be purified, and then the support is washed with an acceptable solvent, which should remove substantially all material in the sample except for the IL-23p19 protein bound to the immobilized antibody. Finally, the support is washed with another acceptable solvent, which should separate the IL-23p19 protein from the antibody.

Anti-IL-23p19 antibodies, such as humanized anti-IL-23p19 antibodies, can also be used in diagnostic assays to detect and/or quantify IL-23 protein, for example, by determining the expression of IL-23 in specific cells, tissues, or serum. Anti-IL-23p19 antibodies can be used for diagnostic purposes, eg to monitor the development or progression of a disease, as part of a clinical testing procedure, eg to determine the efficacy of a given treatment and/or prophylaxis regimen. Detection can be facilitated by binding the IL-23p19 antibody. Examples of detectable substances are various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting materials, which are detected using positron emission tomography, and non-radioactive paramagnetic metal ions (see, for example, US No. 4741900, in which describes metal ions that can be conjugated with antibodies for use in diagnostics.

Anti-IL-23p19 antibodies can be used in methods for diagnosing an IL-23-associated disorder (eg, a disorder characterized by abnormal expression of IL-23) or to determine whether an individual is at increased risk of developing an IL-23-associated disorder. Such methods include contacting a biological sample from an individual with an anti-IL-23p19 antibody and determining the binding of the antibody to IL-23p19. By biological sample is meant any biological sample obtained from an individual, cell line, tissue culture or other source of cells potentially expressing IL-23. Methods for obtaining biopsies of mammalian tissue and total body water are well known in the art.

The method may also be to compare the level of IL-23 in a sample from the patient's body and in a control sample (for example, from the body of a patient who does not have an IL-23-associated disorder) to determine whether the patient has an IL-23-associated disorder or the risk of developing an IL-23-associated disorder.

It is expedient, for example, for diagnostic purposes, to label an antibody with a detectable fragment. Numerous detectable labels are known and available, such as radioisotopes, fluorescent labels, enzyme substrate labels, and the like. The label can be indirectly conjugated to the antibody using various known techniques. For example, an antibody can be conjugated to biotin, and any of the above three broad categories of labels can be conjugated to avidin, or vice versa. Biotin binds selectively to avidin and therefore the label can be conjugated to the antibody in an indirect manner. Alternatively, to achieve indirect label-to-antibody conjugation, the antibody can be conjugated to a small hapten (such as digoxin) and one of the various types of labels mentioned above conjugated to an anti-hapten antibody (eg, anti-digoxin). In this way, indirect conjugation of the label with the antibody can be carried out.

Examples of radioisotope labels are ³⁵ S, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I. An antibody can be labeled with a radioisotope using the techniques described, for example, in Current Protocols in Immunology, vols. 1 and 2, 1991,

- 37 040834 ed. Coligen et al., Wiley-Interscience, New York, N.Y., 1991. Radioactivity can be measured, for example, using a scintillation counter.

Examples of fluorescent labels are known labels based on rare earth chelates (europium chelates), or labels based on fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lyssamine, phycoerythrin and Texas red. Fluorescent labels can be conjugated to the antibody using known techniques, such as those described in Current Protocols in Immunology, supra. Fluorescence can be quantified using a fluorometer.

Various well-characterized enzyme-substrate tags are known in the art (see, for example, the review cited in US No. 4,275,149). The enzyme typically catalyzes a chemical change in the chromogenic substrate, which can be assessed using a variety of techniques. For example, the change may be a change in the color of the substrate, which can be assessed spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Methods for quantifying the change in fluorescence are described above. As a result of a chemical reaction, an electronically excited state arises in the chemiluminescent substrate, as a result of which it can emit light, which can be assessed, for example, using a chemiluminometer, or it is an energy donor for a fluorescent acceptor.

Examples of enzymatic labels are luciferases such as firefly luciferase and bacterial luciferase (US No. 4737456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase, such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase , lysozyme, saccharide oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic compound oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described, for example, in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in: Methods in Enzym., ed. J. Langone and H. Van Vunakis, Academic Press, N.Y., 73, 1981, p. 147-166.

Examples of enzyme-substrate combinations are, for example: horseradish peroxidase (HRPO) and hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor such as orthophenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB); alkaline phosphatase (AP) and para-nitrophenylphosphonate as a chromogenic substrate; and β-O-galactosidase (β-D-Gal) and a chromogenic substrate such as, for example, para-nitrophenyl-β-D-galactosidase or a fluorogenic substrate such as 4-methylumbelliferyl-β-O-galactosidase.

Numerous other enzyme-substrate combinations are known to those skilled in the art. A general overview is presented, for example, in US No. 4275149 and US No. 4318980.

The humanized anti-IL-23p19 antibody can be used unlabeled and is detected by a labeled antibody linked to a humanized anti-IL-23p19 antibody.

The antibodies provided herein can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation-based assays (see, for example, Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., 1987, pp. 147-158).

An anti-IL-23p19 antibody, or antigen-binding fragment thereof, can be used to inhibit the binding of IL-23 to the IL-23 receptor. These methods involve introducing an anti-IL-23p19 antibody or antigen-binding fragment thereof into a cell (eg, a mammalian cell) or the cell's environment, thereby inhibiting IL-23 receptor-mediated signaling. These methods can be carried out in vitro or in vivo. By cell-environment is meant the tissue, medium, or extracellular matrix that surrounds the cell. An anti-IL-23p19 antibody or antigen-binding fragment thereof is introduced into the cell environment in such a way that the antibody or fragment thereof has the ability to bind to IL-23 molecules outside the cellular environment or in the cellular environment, thereby preventing IL-23 from binding to its receptor.

diagnostic kits.

You can use a humanized antibody to IL-23p19, which is part of the diagnostic kit, which is a package that contains a combination of reagents taken in predetermined quantities, in combination with instructions for performing a diagnostic analysis. If the antibody is labeled with an enzyme, the kit may include substrates and cofactors necessary for the enzyme, such as a precursor substrate that forms a detectable chromophore or fluorophore. In addition, other additives such as stabilizers and buffers (eg blocking buffer or lysis buffer) and the like may be included. The relative amounts of the various reagents can vary widely, providing concentrations of reagents in solution that greatly optimize the sensitivity of the assay. The reagents may be dry powders, typically lyophilized, including excipients which, when dissolved, should form a reagent solution having an appropriate concentration.

Therapeutic application.

- 38 040834

The humanized anti-IL-23p19 antibody provided herein can be used to treat various disorders associated with the expression of IL-23p19 as described herein. Treatments for an IL-23-associated disorder comprise administering a humanized anti-IL-23p19 antibody in a therapeutically effective amount to the individual in need thereof.

The humanized antibody or agent against IL-23p19 is administered by any suitable route, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal routes of administration and, if topical immunosuppressive treatment is required, by administration to the affected area (including perfusion or other means of bringing the graft into contact with the antibody before transplantation). The humanized antibody and agent for IL-23p19 can be administered, for example, by infusion or bolus. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In addition, the humanized anti-IL-23p19 antibody can be administered by pulsatile infusion, primarily using lower doses of the antibody. Dosing can be by injection, most preferably intravenous or subcutaneous injection, depending on whether the treatment is short-term or prolonged.

To prevent or treat a disease, the appropriate dose of an antibody will depend on a number of factors such as the type of disease being treated, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, prior therapy, the patient's medical history and response to the antibody, and from the prescription of the attending physician. The antibody can be administered to a patient in a single dose or in a series of treatments.

Depending on the type and severity of the disease, the patient may initially be administered from about 1 μg/kg to 20 mg/kg (e.g., 0.1-15 mg/kg) of antibody as the initial intended dose, by, for example, through one or more administrations. or by continuous infusion. Typically, the daily dose will be from about 1 μg/kg to 100 mg/kg or more, depending on the above factors. For repeated administrations over several days or longer, depending on the condition, treatment is continued until the desired suppression of the present symptoms of the disease. However, other dosing regimens may be used. The success of this therapy is easily assessed by conventional methods and assays. An example of a dosing regimen is provided in WO 94/04188.

In the context of the present description, the concept of suppression has the same meaning as the concepts of relief and relief, and refers to the weakening of one or more characteristics of the disease.

The antibody composition can be formulated, dosed and administered in accordance with good clinical practice. Factors of importance in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, site of administration of the agent, method of administration, schedule of administration, and other factors known to the medical practitioner. In this regard, the therapeutically effective amount of the antibody to be administered is adjusted to be the minimum amount necessary to prevent, alleviate, or treat a disorder associated with IL-23 expression.

The antibody is optional, but in certain instances, included in the formulation in combination with one or more agents currently used to prevent or treat the disorder in question. The effective amount of these other agents depends on the amount of humanized anti-IL-23p19 antibody present in the formulation, the type of disorder or treatment, and other factors mentioned above. They are generally used at the same doses and by the same route of administration in which they have been previously used, or at a dose of from about 1% to about 99% of their accepted doses.

IL-23 associated disorders.

Anti-IL-23p19 antibodies or agents can be used to treat or prevent an immunological disorder characterized by abnormal expression of IL-23, such as inappropriate activation of immune cells (eg, lymphocytes or dendritic cells). Said abnormal expression of IL-23 may be associated, for example, with elevated levels of IL-23 protein. Anti-IL-23p19 antibodies, or antigen-binding fragments thereof, may also find use in the treatment or prevention of respiratory disorders, metabolic disorders such as diabetes mellitus, and certain types of cancer. Treatment or prevention of an immunological disorder, respiratory disorder, metabolic disorder, or cancer using the methods described herein is carried out by administering to an individual in need of such treatment or prevention an effective amount of an anti-IL-23p19 antibody or agent, wherein the antibody reduces IL-23 activity associated with the disease state.

Immunological diseases that are characterized by inappropriate activation of immune cells and that can be treated or prevented using the methods provided herein can be classified, for example, according to the type(s) of hypersensitivity reaction(s) that

- 39 040834 paradise(s) underlies the violation. These reactions are generally classified into 4 types: anaphylactic reactions, cytotoxic (cytolytic) reactions, immune complex reactions, or cell-mediated immunity (CMI) reactions (also referred to as delayed hypersensitivity (DTH) reactions (see e.g. Fundamental Immunology, William E. Paul, Raven Press, N.Y., 3rd ed., 1993. Immunological diseases include inflammatory diseases and autoimmune diseases.

Specific examples of these immunological diseases are rheumatoid arthritis, autoimmune demyelinating diseases (eg, multiple sclerosis, allergic encephalomyelitis), endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (eg, Crohn's disease or ulcerative colitis), anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, inflammatory myositis, multiple endocrine insufficiency, Schmidt's syndrome, autoimmune uveitis, Addison's disease, inflammation of the adrenal gland, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypo parathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's disease, esophagitis (esophageal dysmotility), sclerodactyly and telangiectasia), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas disease, sarcoidosis, rheumatic attack, asthma, habitual miscarriage , antiphospholipid syndrome, exogenous allergic alveolitis, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, birder's allergy, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic al veolitis, fibrous alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, transient arteritis, schistosomiasis, gingantocellular arteritis, ascariasis, aspergillosis, Sumpter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Kaplan's syndrome , Kawasaki disease, Dengue fever, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, persistent elevated erythema, psoriasis, psoriatic arthritis, fetal erythroblastosis, eosinophilic fasciitis, Schulmann's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuchs cyclitis, IgA nephropathy, Henoch-Schönlein purpura, graft-versus-host disease, graft rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenström macroglobulemia, Evans syndrome, acute respiratory distress syndrome, pneumonia, osteoporosis, delayed hype reaction sensitivity and autoimmune gonadal dysfunction.

The immunological disorder may be a T cell mediated immunological disorder and thus the anti-IL-23p19 antibodies or agents provided herein may also be used to treat or prevent T cell mediated immunological disorders.

Anti-IL-23p19 antibodies or agents can be used to treat or prevent a respiratory disorder in which IL-23 is abnormally expressed. Treatment or prevention of a respiratory disorder using the methods described herein is accomplished by administering to an individual in need of such treatment or prevention an effective amount of an anti-IL-23p19 antibody or agent, wherein the antibody reduces disease-associated IL-23 activity. state. These include (but are not limited to) complaints related to respiratory function, obstructive pulmonary diseases of various origins, emphysema of the lungs of various origins, obliterating pulmonary diseases, interstitial pulmonary diseases, interstitial lung disease, cystic fibrosis, bronchitis of various origins, bronchiectasis, ARDS (respiratory adult distress syndrome) and all forms of pulmonary edema; obstructive pulmonary diseases selected from COPD (chronic obstructive pulmonary disease), asthma, bronchial asthma, pediatric asthma, severe asthma, acute asthma attacks and chronic bronchitis; emphysema of the lungs, the origin of which is associated with COPD (chronic obstructive pulmonary disease) or deficiency of an a1-proteinase inhibitor; obliterating pulmonary diseases selected from allergic alveolitis, obliterating pulmonary diseases that are triggered by exposure to harmful toxic substances such as asbestosis or silicosis, and obliteration associated with lung tumors such as carcinomatous lymphagiosis, bronchoalveolar carcinoma and lymphomas; pneumonia caused by infections, such as those associated with viruses, bacteria, fungi, protozoa, helminths or other pathogens, pneumonitis,

- 40 040834 called by various factors, such as, for example, aspiration and left ventricular heart failure, radiation-induced pneumonitis or fibrosis, collagenosis, such as, for example, lupus erythematosus, systemic sclerosis or sarcoidosis, granulomatosis, such as, for example, Beck's disease, idiopathic interstitial pneumonia or idiopathic pneumosclerosis (IPF); cystic fibrosis, bronchitis caused by a bacterial or viral infection, allergic bronchitis and toxic bronchitis; bronchiectasis; pulmonary edema, for example, toxic pulmonary edema after aspiration or inhalation of toxic substances and foreign substances; rhinitis, arthritis and related arthropathies, psoriasis, myeloid leukemia, multiple sclerosis, Alzheimer's disease, glomerulonephritis and chronic atopic dermatitis.

Antibodies or agents against IL-23p19 can also be used to treat various types of cancer in which IL-23 is abnormally expressed.

Cancer types that express IL-23 that can be treated using the methods provided herein include, for example, leukemia, eg, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia (eg, myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia or erythroleukemia), chronic leukemia, chronic myelocytic (granulocytic) leukemia or chronic lymphocytic leukemia; true polycythemia; lymphoma (eg, Hodgkin's disease or non-Hodgkin's lymphoma); multiple myeloma, Waldenström's macroglobulinemia; heavy chain disease; solid (solid) tumors, such as sarcomas and carcinomas (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, obstructive carcinoma, leiomyosarcoma colon, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, fetal carcinoma, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, malanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, or esophageal carcinoma).

Pharmaceutical compositions and their administration.

A composition containing an IL-23p19 binding agent (eg, an anti-IL-23p19 antibody) may be administered to an individual who suffers from or is at risk of developing an immunological disorder, respiratory disorder, or cancer. An IL-23p19 binding agent (eg, an anti-IL-23p19 antibody) can be used in the manufacture of a medicament for the prevention or treatment of cancer, a respiratory disorder, or an immunological disorder. The term "individual" as used herein means any diseased mammal to which an IL-23p19 binding agent can be administered, including, for example, humans and non-human mammals such as primates, rodents, and dogs. Using the methods presented in the present description, you can treat, in particular people. Antibodies or agents can be administered alone or in combination with other compositions for the prevention or treatment of an immunological disorder, a respiratory disorder, or cancer. Such compositions that can be used in combination with antibodies or agents include methotrexate (MTX) and immunomodulators, such as antibodies or small molecules.

Examples of antibodies for use in these pharmaceutical compositions are antibodies that are a humanized antibody or antibody fragment that has any of the light chain variable region amino acid sequences of SEQ ID NOs: 84, 86, 88, 90, 91, 93, 95, 97 , 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 or 119. Examples of antibodies for use in these pharmaceutical compositions are also antibodies that are a humanized antibody or an antibody fragment that has any of the amino acids heavy chain variable region sequences SEQ ID NOs: 121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, or 156.

Further examples of antibodies for use in these pharmaceutical compositions are antibodies that are a humanized antibody or antibody fragment that has any of the amino acid sequences of the light chain variable region of SEQ ID NO: 158, 160, 162, or 164. Preferred antibodies for use in these pharmaceutical compositions are also antibodies that are a humanized antibody or antibody fragment that has any of the amino acid sequences of the heavy chain variable region of SEQ ID NO: 166, 168, 170, or 172.

Additional examples of antibodies for use in these pharmaceutical compositions are antibodies that are a humanized antibody or antibody fragment that has any of the amino acid sequences of the light chain variable region and

- 41 040834 heavy chain variable region SEQ ID NOS: 160 and 166, SEQ ID NOS: 160 and 168, SEQ ID NOS: 158 and 166, or SEQ ID NOS: 158 and 168.

Further examples of antibodies for use in said pharmaceutical compositions are those which are humanized antibodies which have any of the light chain variable region amino acid sequences of SEQ ID NO: 174 or 180. Preferred antibodies for use in said pharmaceutical compositions are also antibodies which are a humanized antibody that has any of the heavy chain variable region amino acid sequences of SEQ ID NO: 176 or 178.

Additional examples of antibodies for use in these pharmaceutical compositions are antibodies that are Antibody A, Antibody B, Antibody C or Antibody D.

Various administration systems are known and can be used to administer the IL-23p19 binding agent. Methods of introduction include (but are not limited to) intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The IL-23p19 binding agent may be administered, for example, by infusion, bolus or injection, and may be administered together with other biologically active agents such as chemotherapeutic agents. The introduction can be systemic or local. Preferably, administration is by subcutaneous injection. Formulations for such injections can be prepared, for example, in the form of pre-filled syringes, and they can be used at weekly intervals.

The IL-23p19 binding agent is administered by injection, by catheter, by suppository, or by an implant, where the implant is composed of a porous, non-porous, or gel-like material, including a membrane such as a Silastic membrane or fiber. As a rule, when the composition is administered, substances are used that do not absorb the antibody or agent to IL-23p19.

The anti-IL-23p19 antibody or agent can be administered using a controlled release system, or a pump can also be used (see, for example, Langer, Science, 249, 1990, p. 1527-1533; Sefton, CRC Crit. Ref. Biomed. Eng. 14, 1989, p 201; Buchwald et al., Surgery, 88, 1980, p. 507; Saudek et al., N. Engl. J. Med., 321, 1989, p 574). Polymeric materials can also be used (see, for example, Medical Applications of Controlled Release, ed. Langer and Wise, CRC Press, Boca Raton, Florida, 1974); Controlled Drug Bioavailability, Drug Product Design and Performance, ed. Smolen and Ball, Wiley, New York, 1984); Ranger and Peppas, Macromol. sci. Rev. macromol. Chem. 23, 1983, p. 61; see also Levy et al., Science 228, 1985, p. 190; During et al., Ann. Neurol. 25, 1989, p. 351; Howard et al., J. Neurosurg. 81, 1989, p. 105). Langer, supra, discusses other controlled release systems.

An IL-23p19 binding agent (eg, an anti-IL-23p19 antibody) may be administered as pharmaceutical compositions containing a therapeutically effective amount of the binding agent and one or more pharmaceutically compatible ingredients.

The pharmaceutical composition is prepared according to conventional procedures to obtain a pharmaceutical composition suitable for intravenous or subcutaneous administration to humans. Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. If necessary, the pharmaceutical agent may also contain a solubilizing agent and a local anesthetic such as lipocaine to relieve pain at the injection site. Typically, the ingredients are either supplied individually or mixed to form a unit dose of the drug, for example, as a dry lyophilized powder or a water-free concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active ingredient. Where the pharmaceutical agent is to be administered by infusion, it may be filled into infusion bottles containing pharmaceutical grade sterile water or saline. Where the pharmaceutical agent is administered by injection, an ampoule of sterile water for injection or saline may be used so that the ingredients can be mixed prior to administration.

In addition, the pharmaceutical composition can be included in a pharmaceutical kit that contains (a) a container with an IL-23p19 binding agent (e.g., anti-IL-23p19 antibody) in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. A pharmaceutically acceptable diluent may be used to reconstitute or dilute the lyophilized anti-IL-23p19 antibody or agent. Optionally, the specified container(s) may be accompanied by a notice in a form approved by an official agency authorizing the manufacture, use, or sale of pharmaceuticals or biological products, where the notice reflects the agency's approval for manufacture, use, or sale for the purpose of administration to humans.

The amount of an IL-23p19 binding agent (eg, anti-IL-23p19 antibody) that is effective in treating or preventing an immunological disorder or cancer can be determined by conventional clinical testing methods. Additionally, in vitro assays may optionally be used, the results of which may help determine optimal dosage ranges. Accurate

- 42 040834 the dose for use in the formulation should also depend on the route of administration and the stage of development of the immunological disorder or cancer, and it should be taken on the basis of the advice of the attending physician and the circumstances specific to each patient. Effective doses can be determined by extrapolation from dose-response curves obtained from in vitro experiments or animal model test systems.

Typically, the dose of anti-IL-23p19 antibody or GE-23p19 binding agent administered to a patient with an immunological disorder or cancer characterized by IL-23p19 expression is about 0.1 to about 100 mg/kg of the individual's body weight. . The dose administered to an individual is about 0.1 to about 50 mg/kg, about 1 to about 30 mg/kg, about 1 to about 20 mg/kg, about 1 to about 15 mg/kg, or about 1 to about 10 mg/kg of an individual's body weight.

Examples of doses are (but are not limited to) doses ranging from 1 ng/kg to 100 mg/kg. Sometimes the dosage is about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15 or about 16 mg/kg. The dose can be administered, for example, every day, once a week (every week), twice a week, three times a week, four times a week, five times a week, six times a week, once every two weeks or monthly, every two months or every three months. Specifically, the dosage may be about 0.5 mg/kg/week, about 1 mg/kg/week, about 2 mg/kg/week, about 3 mg/kg/week, about 4 mg/kg/week, about 5 mg/week. kg/week, approximately 6 mg/kg/week, approximately 7 mg/kg/week, approximately 8 mg/kg/week, approximately 9 mg/kg/week, approximately 10 mg/kg/week, approximately 11 mg/kg/week week, about 12 mg/kg/week, about 13 mg/kg/week, about 14 mg/kg/week, about 15 mg/kg/week, or about 16 mg/kg/week. Dose ranges may be from about 1 to about 15 mg/kg/week.

Pharmaceutical compositions containing an IL-23p19 binding agent may further include a therapeutic agent conjugated or unconjugated to the binding agent. The anti-IL-23p19 antibody or IL-23p19 binding agent may be administered in conjunction with one or more therapeutic agents used to treat or prevent immunological disorders or cancer.

Such combination therapy may have an additive or synergistic effect in relation to the signs of the disease (for example, the severity of the symptom, the number of symptoms, or the frequency of relapses).

In combination regimens, the anti-IL-23p19 antibody or GE-23p19 binding agent is administered simultaneously with the therapeutic agent, or the therapeutic agent is administered before or after administration of the anti-IL-23p19 antibody or IL-23p19 binding agent for at least a period from 1 hour to several months, for example, for at least 1 hour, 5 hours, 12 hours, one day, week, month, or three months before or after administration of an antibody to IL-23p19 or IL-23p19-binding agent.

Products.

The product includes a container and a label. Suitable containers are, for example, bottles, vials, syringes and laboratory tubes. Containers can be made from various materials such as glass or plastic. The container contains a composition that is effective for treating a particular condition and may have a sterile access port. For example, the container may be an intravenous solution bag or vial having a closure device permeable to a hypodermic injection needle. The active substance in the composition is a humanized antibody to IL-23p19. The label on or associated with the container indicates that the composition is used to treat the selected condition. The article of manufacture may further include a second container containing a pharmaceutically acceptable buffer such as phosphate buffered saline, Ringer's solution and dextrose solution. It may additionally include other substances whose presence is desirable from a commercial and consumer point of view, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The invention is described below with the help of the presented examples, which are not intended to limit the scope of the invention.

Examples

Example 1. Obtaining humanized antibodies to IL-23p19.

The mouse 6B8 leader antibody was converted into a chimeric antibody consisting of the mouse 6B8 variable domain and the human IgG1KO constant domain. Mouse antibody 6B8 is presented above in table. 1 and 2. IgG1KO (knock out) carries two substitution mutations (transversions) (Leu234Ala and Leu235Ala) that eliminate ADCC and CDC activity by reducing effector functions such as binding to FcyR and complement. The variable domains of mouse and chimeric antibodies are identical. Chimeric antibodies were created to confirm the function

- 43 040834 antibodies and to ensure that the correct sequence is obtained. The antibody variable region was then humanized through a design and screening process. A library was prepared in which human and mouse residues were varied such that either a human or a mouse residue could be in any particular position. The specified library was created for those amino acid residues that were different in the human germline and mouse antibodies. Only those clones were selected that retained the function of the parent mouse antibody. Representative humanized variable regions of the 6B8 antibody are shown in Table 1. 5 and 6.

Thus, Antibody A, Antibody B, Antibody C, and Antibody D were humanized antibodies derived from mouse 6B8 antibody (cloned in a human IgG1-KO (KO knock-out)/kappa framework). Antibodies A, B, C and D are presented in table. 7.

Example 2 Binding of antibodies to recombinant IL-23 protein.

A) Data on the kinetics and binding affinity of mouse antibodies to IL-23p19 with recombinant human IL-23 are presented below (table. 9). Binding kinetics and affinity were evaluated using Fortebio Octet technology (Fortebio, Menlo Park, CA) using hybridoma derived material after a single column purification. Because Octet is not a liquid-based technology, this method does not accurately determine dissociation rates. In some cases, it was possible to evaluate only the affinity

Table 9

Antibody to _a (1/Ms) k _d (l/c) K _D (nM) 18С4 3.84E+05 2.14Е-06 5.57 18E 5 3.29E+05 2.61E-06 7.93 18D3 ZD9E+05 2.16E-06 6.78 20 E8 4.21E+05 2.69Е-04 638 22 E2 3.46E+05 3.53E-04 1024 24A5 2.02E+05 4.57Е-06 22.6 15C11 4D1E+05 1.07Е-05 26 43F5 1.72E+05 5.96E-06 34.6 27G8 1.57E+05 4.26E-06 27.2 31H9 2.99E+05 3.45Е-06 I,5 2D1 < 1E-6 < 1 9D12 < 1E-6 < 1 6B8 < 1E-6 < 1 73H10 5.29E+04 5.24Е-06 99.2 74НЗ 3.06E+04 2.09Е-06 68.3 35H8 26F7 4.76E+05 1.34Е-05 28.1 34G3 9D8E+05 3.10E-05 32.8 34D9 3.44E+03 1.87Е-06 544

B) The affinity of humanized antibodies derived from mouse 6B8 antibody was assessed. Binding kinetic data measured with the ProteON XPR36 device (Biorad, Hercules, Calif.) and a global fit based on a 1:1 binding model demonstrated that interactions between recombinant IL-23 that either contained a 21 amino acid linker , covalently bound to subunits p19 and p40, or did not contain the indicated linker, were characterized by high affinity in the range of 1-100 pM (Table 10). The 6H12 antibody (described in WO 2007/027714), the QF20 antibody (described in WO 2007/024846) and the C1273 antibody (described in WO 2007/005955) were also tested.

Table 10

Antibody Human IL-23 with linker Human IL-23 without linker to _a (1/Ms) k _d (1/s) K _D (pM) to _a (1/Ms) k _d (1/s) K _D (pM) Mouse 6V8 5.57E+05 1.38E-05 24.5 Antibody A 6.27E+05 < 1E-6 < 1 5.51E+05 < 1E-6 < 1

- 44 040834

Antibody B 3.56E+05 < 1E-6 < 1 5.17E+05 < 1E-6 < 1 Antibody C 3.74E+05 1.19E-05 31.8 4.54E+05 Ts65E-05 36.3 Antibody D 3.82E+05 4.07Е-05 107 3.66E+05 4.93E-05 135 S-1273 3.60E+05 5.75Е-06 15.8 6Н12 4.99E+05 Ts07E-04 214 QF20 2.03E+05 5.89Е-06 2.91

C) The binding affinity and kinetics of anti-IL-23p19 antibodies to cynomolgus monkey IL-23 was measured using the ProteON XPR36 device and a global fit based on a 1:1 binding model (Table 11). The 6H12 antibody (described in WO 2007/027714), the QF20 antibody (described in WO 2007/024846) and the C1273 antibody (described in WO 2007/005955) were also tested.

Table 11

Antibody K _D (pM) to _a (1/Ms) k _d (1/s) Antibody A < 1 2.95E+06 < 1E-6 Antibody B < 1 2.99E+06 < 1E-6 Antibody C 2.9 3.23E+06 9.36Е-06 Antibody D 15.9 2.07E+06 3.29E-05 S-1273 >5000 n/a n/a 6Н12 157 9.91E+05 1.56Е-04 QF20 1.2 3.90E+06 4.78E-06

D) Selectivity at the molecular level for human IL-12.

Anti-IL-23p19 antibodies were also injected onto the surface of human IL-12 at a concentration of 100nM. The binding signal of these antibodies, measured using Fortebio Octet technology, was zero, indicating that these antibodies selectively bind to human IL-23. The binding of anti-IL-23p19 antibodies to IL-23 was also analyzed in the presence of 50% human serum and the found lack of significant effect of serum on the rate of association demonstrates high specificity.

Example 3 Competitive binding assay of human IL-23 binding to human IL-23R/Fc.

Human IL-23R-Fc was immobilized on the biosensor surface and 10 nM human IL-23 was injected. The sensorgrams indicated specific binding between IL-23 and the IL-23 receptor (FIG. 2, top panel). The antibodies were then co-injected with 10nM human IL-23 to determine if binding of the antibody to IL-23 could inhibit the interaction between IL-23 and the IL-23 receptor. In this example, it was found that if the antibody binds to human IL-23 and has the ability to inhibit the interaction, then there is reduced binding or no binding (Fig. 2, bottom graph). In this example, Antibody A, which was used at an equivalent molar concentration, was co-injected with 10 nM recombinant human IL-23.

Example 4 Cell Functional Assays, Inhibition of IL-17 Production in IL-23 Stimulated Mouse Splenocytes.

One of the functional cellular assays of antibodies to IL-23p19 measured the ability to inhibit IL-23 stimulated production of IL-17 in mononuclear cells isolated from the spleen of mice. Human recombinant IL-23 protein has the ability to stimulate the release of IL-17 from mouse splenocytes. In addition, a naturally occurring source of human IL-23 found in the supernatant of activated human monocytic THP-1 cells can be used to stimulate the production of IL-17 in mouse mononuclear cells.

Human recombinant IL-23 or naturally occurring IL-23 from activated THP-1 cells were preincubated with titrated anti-IL-23p19 antibodies. The IL-23/antibody combinations were then added to freshly isolated mouse splenocytes. A sample containing only recombinant IL-23 was used as a positive control. After culturing for 2 days, cell supernatants were collected and analyzed for IL-17 by ELISA (R&D Systems, Minneapolis, MN). The following are representative IC ₅₀ values for anti-IL-23p19 antibodies. The antibodies tested were hybridoma-derived mouse antibodies (lines 1-19, see Tables 1 and 2), chimeric antibodies (lines 20-23) and AD antibodies. The 6H12 antibody (described in WO 2007/027714), the QF20 antibody (described in WO 2007/024846) and the C1273 antibody (described in WO 2007/005955) were also tested.

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Table 12

Antibody IC50 values (pM), recombinant human IL-23 IC50 values (pM), naturally occurring human IL-23 18С4 471 100, 413 18E5 did not define 9, 9, 13 18D3 234 did not define 20E8 did not define 438, 561 22E2 61, 130 117, 35 24А5 22, 37 85, 31 15С11 126 232 43F5 250, 8000 8000 27G8 235 5000 31H9 960 2000 2D1 did not define 2336, 1911, 1597 9D12 59 281, 138 6B8 13 8, 2 73H10 1411 did not define 74НЗ 1352 did not define 36H8 did not define did not define 26F7 27 2, 8 34G3 336 27, 25 34D9 510 456 chimeric 18E5 31 8, 36, 10, 9 chimeric 22E2 100 9, 178 chimeric 24A5 404 95, 102 chimeric 6B8 26, 37, 57 5, 2, 6, 3 Antibody A 5, 5, 5, 15 eleven Antibody B 13, 30, 54, 42 9, 8 Antibody C 53, 71, 162, 89 16, 32 Antibody D 236, 225, 614, 458 133, 125 6Н12 1600, 806, 1300 957, 4400, 1013, 439 QF20 did not define 7, 12 C1273 did not define 93, 44

Example 5 Determination of Functional Specificity for IL-12 Using an Assay in Human Activated T Cells

Anti-IL-23p19 antibodies were tested for functional inhibition of IL-12 using an assay in human activated T cells. Human recombinant IL-12 (1 ng/ml) was pre-incubated with antibodies to IL-23p19 at a concentration of 5 μg/ml. The IL-12/antibody combination was then added to PHA (phytohemagglutinin)-derived blast T cells. A sample containing only recombinant IL-12 was used as a positive control. An anti-IL-12p70 antibody (Bender MedSystems, Vienna, Austria) was used as a control inhibitory antibody. After culturing for 2 days, cell supernatants were collected and analyzed for IFN-γ by ELISA (R&D Systems). The samples were evaluated in triplicate and the average level of IFN-γ was determined. The results (with standard deviations) are shown in Table. 13.

Table 13

Antibody Cytokine stimulation Mean level (pg/mL) IFN-γ ± standard deviation no antibody No 87 ± 7 no antibody 1 ng/ml IL-12 532 ± 51 chimeric 18E5 1 ng/ml IL-12 511±3 chimeric 6B8 1 ng/ml IL-12 523 ± 60 Antibody A 1 ng/ml IL-12 497 ± 30 Antibody B 1 ng/ml IL-12 537±2 Antibody C 1 ng/ml IL-12 495±25 Antibody D 1 ng/ml IL-12 539 ± 38 antibody to IL-12p70 1 ng/ml IL-12 119 ± 12

Example 6 Inhibition of IL-23 Inducible STAT3 Phosphorylation in the Human DB Cell Line.

The DB human cell line (ATCC, Manasas, VA) responds to IL-23 stimulation with

- 46 040834 using the endogenous IL-23R complex (IL-23R and IL-12RP1) and STAT3 phosphorylation occurs in it depending on the dose of IL-23. An assay was performed to evaluate the inhibition of IL-23-induced STAT3 phosphorylation by an anti-IL-23p19 antibody. DB cells were seeded at 1x10E6 cells/well in a 96-well plate. Antibodies to be tested were serially diluted and pre-incubated with recombinant human IL-23 (10 ng/ml) for 1 hour at room temperature. The antibody/IL-23 mixture was then added to the cells for 30 minutes at 37°C. Cells were harvested by centrifugation at 4°C for 10 min and then lysed in ice-cold buffer (Cell Signaling Technology, Beverly, MA). A portion of the lysate was used for phospho-STAT3 ELISA analysis (Invitrogen). IC ₅₀ values for antibodies were calculated as percentage inhibition of STAT3 phosphorylation compared to control wells without antibody. Representative values of IC ₅₀ are given in table. 14.

Table 14

Antibody 1С ₅₀ (pM) Antibody A 25, 15, 38, 23, 13, 18 Antibody B 73, 84 Antibody C 132, 80 Antibody D 158 QF20 26, 26, 27 S-1273 163, 438

Example 7 An in vivo model of IL-23 induced cytokine production in mouse ear.

An in vivo mouse model was used. Recombinant human IL-23 was injected into the ear skin of mice for 4 consecutive days resulting in epidermal thickening and upregulation of IL-17 and IL-22 proteins. Anti-IL-23p19 antibodies were evaluated in this model. Carried out one intraperitoneal injection of antibodies at a dose of 1 mg/kg or 5 mg/kg 1 hour before the introduction of IL-23 into the skin. Recombinant human IL-23 (with linker) was injected once daily for 3 additional days and tissue samples were obtained for cytokine evaluation. Antibodies have been shown to inhibit the production of cytokines. In table. 15 shows the results of three experiments (example 1: lines 1-7, example 2: lines 8-10, example 3: lines 11-14).

Table 15

Ear tissue, IL17 pg/ml mean ± sd Ear tissue, percent inhibition of IL-17 Ear tissue, IL22 pg/ml mean ± sd Ear tissue, percent inhibition of IL-22 0.1% BSA + citrate buffer i.p. (unstimulated control) 3 ± 1 NA 1 ± 0 NA 0.3 µg IL-23 + citrate buffer i.p. (filler as control) 25±3 NA 274 ± 30 NA 0.3 µg IL-23 + 1 mg/kg Antibodies 6B8 7 ± 2 81 57 ± 19 80 0.3 μg IL-23 + 1 mg/kg Antibody A 2 ± 1 101 17±3 94 0.3 µg IL-23 + 1 mg/kg Antibody B 5±1 93 30±2 89 0.3 µg IL-23 + 1 mg/kg Antibody C 11±1 66 108±12 61 0.3 µg IL-23 + 1 mg/kg Antibody D 10±1 67 151 ± 12 45 0.1% BSA + vehicle (unstimulated control) 14±1 NA 1 ± 1 NA 0.3 mcg IL-23 + vehicle 31±4 NA 129 ± 29 NA 0.3 µg IL-23 + 5 mg/kg 24A5 14±1 102 10±5 93 0.1% BSA + mlgG (unstimulated control) 17±1 NA 4 ± 1 NA 0.3 µg IL-23 + mlgG (vehicle as control) 30±2 NA 208±40 NA 0.3 µg IL-23 + 5 mg/kg 24A5 16±0 109 28±5 88 0.3 µg IL-23 + 5 mg/kg 18E5 21 ± 2 70 53 ± 41 80

Example 8 Pharmacokinetic studies in cynomolgus monkeys.

- 47 040834

Humanized antibodies to IL-23p19 were administered by ten-minute intravenous infusion at a dose of 1.0 mg/kg to three cynomolgus monkeys. Serum samples were obtained over a 6 week period and free antibody concentrations were assessed by specific ELISA. Serum concentration versus time profiles for antibodies and corresponding pharmacokinetic parameters are summarized in Table 1. 16.

Table 16

Antibody CL (ml/d/kg) Volume AUC T _1/2 (days) MRT (days)

(ml/kg) (nM*h/ml) Antibody A 5.2 88 32262 12.1 17.2 Antibody B 6.0 87 27030 10.1 14.8 Antibody C 4.7 91 34642 14.1 19.6 Antibody D 3.4 67 47633 12.6 19.8

Example 9 Expression in NSO cells and biophysical data.

Transfection of NSO cells and preparation of stable pools: NSO cells were grown in the presence of 1% FBS prior to transfection. 40 x 10e6 cells were harvested and resuspended in 0.8 ml in media containing 2% FBS with 20 μg of linearized DNA (heavy chain and light chain expression vectors) and then the cells were incubated on ice for about 15 min before cell electroporation at 750 V/ 25 µF (Bio-Rad, Gene Pulser Xcell). Cells were isolated with 2% FBS for about 48 hours at 37° C. and 5% CO ₂ , then plated at 2x10e5 cells/ml in 96-well plates containing G418 and mycophenolic acid, and incubated for 14-21 days before colony formation.

Supernatants from 96-well colony plates were screened by ELISA. ELISA plates were sensitized with 1 μg/ml goat anti-kappa chain antibody (Southern Biotech, Birmingham, AL) in PBS and the diluted supernatant was incubated and then evaluated with goat anti-human IgG Fc-HRP (Jackson ImmunoResearch). Laboratories, West Grove, PA). Positive colonies were pooled for propagation. The titers of the formed antibodies were determined using a ForteBio device using protein A-coated tips according to the manufacturer's protocol. The titers of Antibody A and Antibody D were 250-350 mg/l, with over 80% protein recovery after purification and over 94% of monomers after electrodialysis and ion exchange (IEX) purification. The proteins were resuspended in a final buffer containing 20 mM sodium citrate and 115 mM NaCl, pH 6.0 and were stable at 4° C. for at least 4 months and their solubility in this buffer was up to 100 mg/ml.

Table 17

Protein A column 1EX-column

Titer (mg/l) Yield (mg/l) Regeneration Yield (mg/l) Regeneration Antibody A 345 275 80% 221 80% Antibody D 248 225 90% 175 78%

Table 18

Quality Stability Solubility AUC, freshly isolated (%M) GPC, freshly isolated (%M) ATC 1 month (%M) GFH 1 month (%M) ATC 4 months (%M) GFH 4 months (%M) AUC at 100 mg/mL (%M) Antibody A 98 99 97 99 96 99 99 Antibody D 94 100 98 100 99 99 97

AUC: analytical centrifugation based on the measurement of sedimentation rate at concentrations of 0.5-1 mg/ml;

GPC: gel filtration chromatography;

%M: percentage of monomers.

Example 10 Epitope mapping.

Hydrogen/deuterium exchange mass spectrometry (HXMS) was used to map the Antibody A epitope binding to the human IL-23p19 subunit. This method was used to determine the sensitivity of the hydrogen amide backbone of IL-23p19 to D2O exchange. The experiment was performed using only IL-23 and IL-23 with the addition of Antibody A. In this way,

- 48 040834 identified areas in the sequence of IL-23p19, for which a pronounced protection from exchange was established as a result of binding to Antibody A. The resolution of the method was determined by peptides formed by cleavage with pepsin or protease XVIII. These IL-23p19 derived peptides were identified by additional control experiments with non-exchanged samples using standard precision weight and HPLC-MS/MS techniques.

Recombinant human IL-23 was used. In the case of a sample containing protein + antibody, 50 µl of IL-23 (0.8 mg/ml) was incubated with 10 µl of Antibody A (12.7 mg/ml) for 15 min at room temperature. The final molar ratio of Antibody A/IL-23 was 1.2:1.

To carry out the exchange, 5 μl of IL-23 protein was added to 50 μl of deuterated buffer (50 mM PBS in D ₂ O) and incubated for 100 s at room temperature. Added 50 μl of 2 M urea/0.5 M TCEP and incubated for 60 s at room temperature. Added 5 μl of pepsin or protease XVIII (4 mg/ml in 0.1% formic acid) and the sample was immediately cooled to 4°C.

After 5 min, 50 µl of the sample was injected into a Shimadzu HPLC system (SCL10A controller and two LC10AD pumps) under the following conditions:

Mobile phase A: 99/1/0.1 (water/acetonitrile/formic acid).

Mobile phase B: 95/5/0.1 (acetonitrile/water/formic acid).

Flow rate: 100 µl/min.

Column: Phenomenex Jupiter C5, 5 µm, 50x1.0 mm.

The lines of mobile phases, the column, and the injector loop were in ice baths.

Gradient: Time 0 (3%B), Time 2.2 (3%B), Time 10.1 (90%B), Time 12.0 (90%B), Time 12.1 ( 3%B).

Mass spectrometry was carried out as follows:

Mass spectrometer: Thermo Orbitrap Velos (0900865).

Methods:

A) Fragmentation (to ID peptides): 12 min capture time (3 min delay on start), full scan based on Fourier Transform Mass Spectrometry (FTMS) at 30,000 resolution, seven scanned data images depending on ionic capture (CID).

B) MS shoulder straps: 12 min capture time (3 min start delay), full scan based on FTMS at 60000 resolution.

Peptides obtained after treatment with pepsin and protease XVIII were identified using fragmentation data and the Proteome Discoverer program (Thermoscientific, Waltham, MA). The identified peptides were visualized by comparison (individual protein versus protein in the presence of antibody) using the Xcalibur software (Thermoscientific). When exchanged outside the IL-23p19 region, no significant shifts were found in the assessment of IL-23 individually compared to IL-23 in the presence of Antibody A. For the p19 region of the protein, data were analyzed using the PepMa program (Thermoscientific). This program allows you to calculate the average weight of the modified peptides. The results obtained with PepMar were checked and those peptides for which no confirmed results were obtained were calculated using Microsoft Excel.

Regions of the IL-23 sequence that were markedly protected from exchange by binding to Antibody A were identified as containing amino acid residues 108126 of SEQ ID NO: 181 and amino acid residues 137-151 of SEQ ID NO: 181.

Claims (8)

CLAIM 1. Humanized antibody to IL-23p19 or antigen-binding fragment, where the antibody or antigen-binding fragment contains: a) a light chain variable region containing the amino acid sequence of SEQ ID NO: 19 (CDR1-L); the amino acid sequence of SEQ ID NO: 20 (CDR2-L) and the amino acid sequence of SEQ ID NO: 21 (CDR3-L); And b) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 66 or 67 (CDR1-H); the amino acid sequence of SEQ ID NO: 64 (CDR2-H); and the amino acid sequence of SEQ ID NO: 65 (CDR3-H), wherein the antibody or antigen-binding fragment thereof has a KD value for IL-23 of less than 40 pM. 2. The humanized anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a) a light chain variable region containing the amino acid sequence of SEQ ID NO: 19 (CDR1-L); the amino acid sequence of SEQ ID NO: 20 (CDR2-L) and the amino acid sequence of SEQ ID NO: 21 (CDR3-L); And b) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 66 (CDR1-H); the amino acid sequence of SEQ ID NO: 64 (CDR2-H); and the amino acid sequence of SEQ ID NO: 65 (CDR3-H). - 49 040834 3. The humanized anti-IL-23p19 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof contains: a) a light chain variable region containing the amino acid sequence of SEQ ID NO: 19 (CDR1-L); the amino acid sequence of SEQ ID NO: 20 (CDR2-L) and the amino acid sequence of SEQ ID NO: 21 (CDR3-L); And b) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 67 (CDR1-H); the amino acid sequence of SEQ ID NO: 64 (CDR2-H); and the amino acid sequence of SEQ ID NO: 65 (CDR3-H). 4. A humanized anti-IL-23p19 antibody according to one of claims 1 to 3, wherein said antibody is a monoclonal antibody. 5. The use of an antibody or antigen-binding fragment thereof according to one of claims 1 to 4 for the treatment of an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, or cancer. 6. Use according to claim 5, wherein the disease is psoriasis, inflammatory bowel disease, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, or ankylosing spondylitis. 7. Use according to claim 6 wherein the disease is asthma or chronic obstructive pulmonary disease (COPD). 8. A pharmaceutical composition for the treatment of an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, or cancer, comprising an antibody or antigen-binding fragment according to one of claims 1 to 3 and a pharmaceutically acceptable carrier.