Invention for Treatment of pruritic diseases with IL-31 monoclonal antibody

Invented by Kent Bondensgaard, Roland Beckmann, Merck Serono SA, Zymogenetics Inc

The Merck Serono SA, Zymogenetics Inc invention works as follows

The invention provides mouse anti-human IL31 antibodies, and antibody fragments, that can bind IL31, and thereby neutralize, inhibit, limit, or reduce the proinflammatory, or pro-pruritic, effects of IL31.

Background for Treatment of pruritic diseases with IL-31 monoclonal antibody

When mice are overexpressed with IL-31, a newly-identified cytokine that has recently been identified, dermatitis like symptoms occur. See, Dillon, et al., Nature Immunol. 5:752-760, 2004. Anti-IL-31 antibody and antagonists can alleviate these symptoms. See also U.S. Patent application Ser. No. Patent Publication No. 2003-0224487), now U.S. Pat. The U.S. Patent No. 2003-0224487 is now U.S. Pat. Nos. Nos.

The monoclonal technology has enabled a wide range of diagnostics and therapeutics to be used in the identification and treatment of disease. A number of biosynthetic or recombinant molecules containing rodent antigen binding sites have been described. The molecules with rodent antigen binding sites directly built onto human antibodies have been described. This was achieved by grafting the rodent binding domain rather than the entire variable region into the human immunoglobulin light and heavy chain domains. See, e.g., Riechmann et al. (1988) Nature 332:323-327 and Verhoeyen et al. Science 239, 1534-1536 (1988). Molecules having an antigen-binding site wherein at least one of the complementarity determining regions (CDRS) of the variable domain is derived from a murine monoclonal antibody and the remaining immunoglobulin-derived parts of the molecule are derived from human immunoglobulin have been described in U.K Patent Publication No. GB 2,276,169 published Sep. 21, 1995. The description of a number of polypeptides with antigen-binding sites on single chains and single chain Fv molecules (sFvs) is also included. See, e.g., U.S. Pat. Nos. Nos. 5,132,405 to Huston and co. U.S. Pat. No. No. 4,946,778 to Ladner et al.

Patent application No. No. Patent Publication Number: 2006-02752960 (US Patent No. 11/430,066) was filed on May 8, 2006. It describes mouse monoclonal antibodies that recognize human IL-31 and can be used to generate chimeric antibodies. The patent publication number is 2006-02752960. It describes mouse monoclonal antibodys that recognize human IL-31. They can be used to create chimeric antibodies. Humanized mouse-antihuman IL31 antibodies are preferred over chimeric or mouse antibodies. Humanized antibodies are generally superior to mouse or chimeric antibodies in certain cases. They have three main advantages: (1) The effector portion of the antibody is human and may interact more effectively with other parts of human immunity (e.g. destroy target cells by complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity); (2) Because the humanized antibody’s constant region or framework is human, the immune system will not perceive it as foreign and the antibody response should be lower than that against a completely foreign mouse antibody or a partly foreign Immunol., 138, 4534-4538 (1987)). Humanized antibodies injected into the body will have a longer half-life than naturally occurring antibodies in humans, which allows for smaller doses and less frequent administration.

There is a requirement for molecules that provide humanized variable regions amino acid sequences of the mouse antihuman IL31 antibodies to treat IL31 mediated inflammation.

In one aspect, the invention relates to an isolated antigen that binds human IL-31. This includes: a) a heavy chain variable CDR comprising amino acid SEQID NO: 1, 2, and 3, respectively; b), a light chain variable CDR comprising amino acid SEQID NO: 5, 6, and 7. In another aspect of the invention, antibodies are described wherein: a. said humanized heavy-chain variable domain comprises FR1,FR2,FR3, and FR4 with an amino sequence that is at least 90 percent identical to an amino sequence selected from: 1) The amino sequences of SEQID NO: 8(FR1), 9(FR2), 10(FR3), and 11 ( In one embodiment, the identity is at least 95 percent, and even more preferable at least 99 percent, most preferred at least 99%.

In another aspect, the invention relates to an isolated antigen as described herein. The amino acid in position 29 of FR1 is leucine. And the amino acid in position 32 of FR3 is phenylalanine. Another aspect of the invention is an isolated antibody described here, where amino acid 8 in FR3 is lysine. And amino acid 15 in FR7 is tyrosine.

In a further aspect the present invention pertains to an isolated antibody that binds to human IL-31, comprising: a) a humanized heavy chain variable domain comprising CDR1 and CDR3 having amino acid sequence of SEQ ID NO: 1 and 3 respectively and CDR2 having amino acid sequence of AIYPGDGDTRYSXaa1Xaa2FXaa3G (SEQ ID NO: 22) wherein Xaa1 is glutamine or proline, Xaa2 is serine or lysine and Xaa3 is glutamine or lysine; said humanized heavy chain variable domain comprising framework regions FR1, FR2, FR3 and FR4 having an amino acid sequence at least 90% identical to the amino acid sequence of: SEQ ID NO: 8, 9, 10 and 11 respectively, or SEQ ID NO: 12, 13, 14 and 15 respectively, or SEQ ID NO: 12, 13, 16 and 15 respectively; and with the proviso that amino acid at position 29 in FR1 is leucine and amino acid at position 32 in FR3 is phenylalanine; b) a humanized light chain variable domain comprising CDRs consisting of amino acid sequences of SEQ ID NO: 5, 6 and 7 said humanized light chain variable domain comprising framework regions FR5, FR6, FR7 and FR8 having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17, 18, 19 and 20 respectively, or at least 90% identical to the amino acid sequence of SEQ ID NO: 17, 18, 21 and 20 respectively. In one aspect of the invention, this identity is at least 95 percent, more preferably, at least 99 percent, and most preferably, at least 99%. In another aspect, the invention relates to an isolated antigen as described herein in which amino acid 8 in FR3 is lysine while amino acid 15 in FR7 is tyrosine. The invention relates to an isolated antigen as described herein, wherein amino acids Xaa1 are glutamine, serine, and lysine, or amino acids Xaa1 are proline, glutamine and

In another aspect, the invention pertains an isolated antibody described herein, wherein the CDRs for the heavy-chain variable domain are SEQ NO: 1, 4, and 3, CDRs for the light-chain variable domain are SEQ NO: 5, 6, and 7, framework regions in the heavy-chain variable domain are SEQ NO: 8, 10, and 11, and framework regions in the light-chain variable domain, SEQ NO: 17, 18, 19, and 20.

In another aspect, the invention pertains an isolated antibody described herein, wherein the CDRs for the heavy-chain variable domain are SEQ NO: 1, 2, and 3, CDRs for the light-chain variable domain are SEQ NO: 5, 6, and 7, framework regions in the heavy-chain variable domain are SEQ NO: 12, 14, and 15, and framework regions in the lightchain variable domain, SEQ NO: 17, 18, 19, and 20.

In another aspect, the invention pertains an isolated antibody described herein, wherein the CDRs for the heavy-chain variable domain are SEQ NO: 1, 2, and 3, respectively; CDRs for the light-chain variable domain are SEQ NO: 5, 6, and 7 respectively; framework regions in the heavy-chain variable domain are SEQ NO: 12, 13 and 16 respectively, and framework areas in the light-chain variable domain, SEQ NO: 17, 18, 21, and 20 respectively.

As will be demonstrated in the Examples and Teacings, the presence of a leucine in position 29 of FR1 as well as a phenylalanine in position 32 of FR3 in the humanized antibody described herein has a positive effect on the affinity and potency. In a Biacore test, it was shown that humanized antibody clones with a leucine in position 29 of FR1 or a phenylalanine in position 32 of FR3 have a higher binding affinity than humanized antibodies containing another amino acid in these positions. (Compare for example the clones 7, 10, 13 and 14. in Table 2 in Example 3.) Two other biological tests have confirmed the positive effect of these amino acid at position 29 of heavy chain FR1 (position 29) and 94 in FR3 (position 32).

In one embodiment, the antibody disclosed in this invention contains a constant heavy chain immunoglobulin domain, selected from a group of constant regions of? “In an embodiment of the present invention, the antibody disclosed herein comprises a heavy chain immunoglobulin constant domain selected from a group consisting of?,?,???????????????????????????????????????????????????????????????????????????????????????????? Human immunoglobulin, heavy chain. In one embodiment, the antibody disclosed in this invention contains a heavy-chain immunoglobulin domain that is selected from a group of human IgG1 and human IgG2 domains; human IgG3 domains; human IgG4 domains; human IgM domains; human IgE domains; and human IgA domains. In one embodiment, the human IgG4 is a mutated version that is stable in solution with minimal or no complement activating properties. In one embodiment, the heavy-chain immunoglobulin constant region is a human IgG4 with a Ser/Pro mutation at position 241 (Kabat numbers).

In one embodiment, an immunoglobulin-light chain constant region is selected from a group comprising the constant regions of a? “In an embodiment, the constant region domain of a human immunoglobulin light chain is selected from a group consisting of the constant region of a? human immunoglobulin light chain. Preferably the constant domain of the immunoglobulin-light chain is that of a? human immunoglobulin light chain.

It may be useful to understand the invention better if you define these terms before describing it in detail.

The term “antibodies” is used in this document. “Antibodies” is used here to refer to polyclonal, affinity-purified, monoclonal, and antigen binding fragments such as F(ab)2, Fab proteolytic fragments (scFvs), and single chain variable region fragments. Includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies and antigen-binding fragments such as F(ab? These include genetically engineered antibodies or fragments such as single chain antibodies, Fvs fragments, chimeric antibodies and other fragments. Humanizing non-human antibodies can be achieved by grafting the non-human CDRs on to human constant and framework regions or by incorporating all of the non-human variable areas (optionally “cloaking”). Humanized non-human antibodies can be humanized by grafting non-human CDRs onto the framework and constant regions of humans, or by incorporating all the variable domains (optionally?cloaking? antibody). Humanized antibodies can retain non-human residues in the human variable regions framework domains, which enhances their binding properties. By humanizing antibodies, the biological half-life can be increased and the risk of adverse immune reactions when administered to humans is decreased.

The term “chimeric antibodies” “The term ‘chimeric antibody? Refers to antibodies that have had their light and heavy chains constructed by genetic engineering from genes of immunoglobulin constant and variable regions belonging to different species. The variable segments from the genes of a monoclonal mouse antibody can be combined with human constant segments such as gamma 1, gamma 3, etc. The typical therapeutic chimeric antigen is a protein that combines the variable domain or antigen-binding region of a mouse monoclonal antibody with the constant domains from human antibodies, such as gamma 1 and gamma 3. Other mammalian species can also be used.

As used in this document, the term “immunoglobulin” is a generic term. Refers to a polypeptide that is encoded in large part by the immunoglobulin gene. The basic structural unit for an antibody is a form of immunoglobulin. This form, called a tetramer, consists of two identical immunoglobulin chain pairs. Each pair has one light and one heavier chain. The light and heavy chains’ variable regions in each pair are jointly responsible for binding an antigen. The constant regions, on the other hand, are responsible for antibody effector functions.

Full-length immunoglobulin ?light chains? The variable region gene (about 110 amino acid) at the NH2-terminus and the kappa/lambda constant region at the COOH terminus encode the (about 25 Kd). Full-length immunoglobulin ?heavy chains? The variable region gene (about 112 amino acids) is encoded in the same way as one of the constant region genes (about 332 amino acids). Heavy chains can be classified into gamma or mu and are further divided into alpha, beta, delta or epsilon. These heavy chains define the isotype of an antibody as IgG. IgM. IgA. IgD. and IgE. The variable and constant regions of light and heavy chains are joined together by a “J” Within light and heavy chains, the variable and constant regions are joined by a ‘J? About 10 amino acids are added to the region. Fundamental Immunology, Paul, W. (ed. ), 2nd edition. Raven Press, N.Y. 1989), Ch. “7 (incorporated herein by reference to its disclosure on producing antibody and antibody fragments).

An immunoglobulin heavy or light chain variable region is composed of a framework? Three hypervariable areas interrupt the region. The term “hypervariable regions” is used to describe this. The antigen-binding amino acid residues are referred to as the hypervariable region. The hypervariable area is made up of amino acid residues that are part of a “Complementarity-Determining Region”. The hypervariable region contains amino acid residues from a?Complementarity Determining Region? The residues 24 to 34 (L1), 48-56 (L2), and 89-97 in the light-chain variable domain, and 31 to 35 (H1) and 50-65 in the heavy-chain variable domain are the same (Kabat, et.al., Sequences Of Proteins Of Immunological Interest, Fifth Edition). Public Health Service, National Institutes of Health Bethesda Md. (1991)), and/or residues from a “hypervariable-loop”? The residues 26-32 in the light-chain variable domain, 50-52 in the L2-domain, and 91 to 96 in the H2-domain are examples. Chothia & Lesk, 1987 J. Mol. Biol. 196: 901-917 (both are incorporated by reference herein). ?Framework Region? The term ‘FR’ or?FR? Residues are defined as those residues that have variable domains, but not the hypervariable regions. Within a particular species, the sequences of framework regions are fairly conserved. A ‘human framework region’ is therefore a sequence of the framework regions from different light or heavy chains. A framework region is one that is nearly identical (around 85%, and usually 90-95%) to a naturally occurring immunoglobulin. The CDRs are positioned and aligned by the framework region, which is the combined framework of the light and heavy chains. CDRs are responsible for primarily binding an antigen’s epitope.

Accordingly, “humanized” is a term that describes a product or service. The term immunoglobulin is used to describe an immunoglobulin that contains a human framework and one or more CDRs from a nonhuman immunoglobulin (usually a rat or mouse). The CDR-containing nonhuman immunoglobulin is known as the “donor”. The human immunoglobulin that provides the framework is known as the “acceptor”. Constant regions are not required, but they should be at least 85-90% identical to the constant regions of human immunoglobulin, and preferably 95% identical. All parts of the humanized immunoglobulin except for CDRs are therefore substantially identical to their corresponding sequences in natural human immunoglobulin. A “humanized antibody” A?humanized antibody? A humanized antibody, for example, would not include a typical chimeric antigen as defined above. This is because the entire variable area of a non-human antibody is included in a chimeric antigen.

The term “recombinant antibody” means an antigen whose amino acid sequence has been altered from that of a native antibody. The amino acid sequence of an antibody has been altered from the native antibody. Due to the importance of recombinant-DNA techniques for the generation of antibodies one does not have to be limited to the amino acid sequences found in natural antibodies. Antibodies can be redesigned in order to achieve desired characteristics. There are many variations that can be made, ranging from changing just a few amino acid sequences to redesigning the entire variable or constant region. In general, changes in the constant area will be made to improve or change characteristics such as complement fixing, membrane interaction, and other effector functions. “Changes will be made to the variable region in order improve antigen binding characteristics.

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