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Humanized immunoglubulin reactive with alph4beta7 integrin

Humanized immunoglubulin reactive with alph4beta7 integrin description/claims

This application is a continuation of U.S. application Ser. No. 11/511,164, filed Aug. 28, 2006, which is a divisional of U.S. application Ser. No. 08/700,737, filed Aug. 15, 1996. The entire teachings of the above applications are incorporated herein by reference.

Integrin receptors are important for regulating both lymphocyte recirculation and recruitment to sites of inflammation (Carlos, T. M. and Harlan, J. M., Blood, 84:2068-2101 (1994)). The human α4β7 integrin has several ligands, one of which is the mucosal vascular addressin MAdCAM-1 (Berlin, C., et al., Cell 74:185-195 (1993); Erle, D. J., et al., J. Immunol. 153:517-528 (1994)) expressed on high endothelial venules in mesenteric lymph nodes and Peyer's patches (Streeter, P. R., et al., Nature 331:41-46 (1988)). As such, the α4β7 integrin acts as a homing receptor that mediates lymphocyte migration to intestinal mucosal lymphoid tissue (Schweighoffer, T., et al., J. Immunol. 151:717-729 (1993)). In addition, the α4β7 integrin interacts with fibronectin and vascular cell adhesion molecule-1 (VCAM-1).

Inflammatory bowel disease (IBD), such as ulcerative colitis and Crohn's disease, for example, can be a debilitating and progressive disease involving inflammation of the gastrointestinal tract. Affecting an estimated two million people in the United States alone, symptoms include abdominal pain, cramping, diarrhea and rectal bleeding. IBD treatments have included anti-inflammatory drugs (such as, corticosteroids and sulfasalazine), immunosuppressive drugs (such as, 6-mercaptopurine, cyclosporine and azathioprine) and surgery (such as, colectomy). Podolsky, New Engl. J. Med., 325:928-937 (1991) and Podolsky, New Engl. J. Med., 325:1008-1016 (1991).

Antibodies against human α4β7 integrin, such as murine monoclonal antibody (mAb Act-1), interfere with α4β7 integrin binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) present on high endothelial venules in mucosal lymph nodes. Act-1 was originally isolated by Lazarovits, A. I., et al., J. Immunol. 133:1857-1862 (1984), from mice immunized with human tetanus toxoid-specific T lymphocytes and was reported to be a mouse IgG1/K antibody. More recent analysis of the antibody by Schweighoffer, T., et al., J. Immunol. 151:717-729 (1993) demonstrated that it can bind to a subset of human memory CD4+ T lymphocytes which selectively express the α4β7 integrin. However, a serious problem with using murine antibodies for therapeutic applications in humans is that they are highly immunogenic in humans and quickly induce a human anti-murine antibody response (HAMA), which reduces the efficacy of the mouse antibody in patients and can prevent continued administration. The HAMA response results in rapid clearance of the mouse antibody, severely limiting any therapeutic benefit.

Thus, a need exists for improved therapeutic approaches to inflammatory bowel diseases.

The present invention relates to a humanized immunoglobulin having binding specificity for α4β7 integrin, said immunoglobulin comprising an antigen binding region of nonhuman origin (e.g., rodent) and at least a portion of an immunoglobulin of human origin (e.g., a human framework region, a human constant region of the gamma type). In one embodiment, the humanized immunoglobulin described herein can compete with murine Act-1 or LDP-02 (see, e.g., Example 4) for binding to α4β7 integrin. In a preferred embodiment, the antigen binding region of the humanized immunoglobulin is derived from Act-1 monoclonal antibody (e.g., LDP-02, an immunoglobulin comprising the variable regions of the light and heavy chains shown in FIG. 11 (SEQ ID NO:19) and FIG. 12 (SEQ ID NO:21), respectively).

For example, the humanized immunoglobulin can comprise an antigen binding region comprising a complementarity determining region (CDR) of nonhuman origin, and a framework region (FR) derived from a human framework region. In one aspect, the humanized immunoglobulin having binding specificity for α4β7 integrin, comprises a light chain comprising a CDR derived from an antibody of nonhuman origin which binds α4β7 and a FR derived from a light chain of human origin (e.g., GM607′CL), and a heavy chain comprising a CDR derived from an antibody of nonhuman origin which binds α4β7 and a FR derived from a heavy chain of human origin (e.g., 21/28′CL). In another aspect, the light chain comprises three CDRs derived from the light chain of the Act-1 antibody, and the heavy chain comprises three CDRs derived from the heavy chain of the Act-1 antibody.

The present invention also relates to humanized immunoglobulin light chains (e.g., comprising CDR1, CDR2 and CDR3 of the light chain of the Act-1 antibody, and a human light chain FR), and to humanized immunoglobulin heavy chains (e.g., comprising CDR1, CDR2 and CDR3 of the heavy chain of the Act-1 antibody, and a human heavy chain FR). In a preferred embodiment, the invention relates to humanized heavy and light chains described herein (e.g., a humanized light chain comprising the variable region of the light chain shown in FIG. 7 (SEQ ID NO:12), a humanized heavy chain comprising the variable region of the heavy chain shown in FIG. 9 (SEQ ID NO:15), a humanized light chain comprising the variable region of the light chain shown in FIG. 12 (SEQ ID NO:21), a humanized heavy chain comprising the variable region of the heavy chain shown in FIG. 11 (SEQ ID NO:19)). Also encompassed are humanized immunoglobulins comprising one or more humanized light and/or heavy chains.

The invention further relates to isolated nucleic acids comprising a sequence which encodes a humanized immunoglobulin of the present invention (e.g., a single chain antibody), as well as to isolated nucleic acids comprising a sequence which encodes a humanized immunoglobulin light chain (e.g., SEQ ID NO:20) or heavy chain (e.g., SEQ ID NO:18) of the present invention. For example, the present invention provides a fused gene encoding a humanized immunoglobulin light or heavy chain comprising a first nucleic acid sequence encoding an antigen binding region derived from murine Act-1 monoclonal antibody; and a second nucleic acid sequence encoding at least a portion of a constant region of an immunoglobulin of human origin.

The present invention further relates to a construct comprising a nucleic acid encoding a humanized immunoglobulin having binding specificity for α4β7 integrin or a chain of such an immunoglobulin. For example, an expression vector comprising a fused gene encoding a humanized immunoglobulin light chain, comprising a nucleotide sequence encoding a CDR derived from a light chain of a nonhuman antibody having binding specificity for α4β7 integrin, and a framework region derived from a light chain of human origin, is provided. An expression vector comprising a fused gene encoding a humanized immunoglobulin heavy chain, comprising a nucleotide sequence encoding a CDR derived from a heavy chain of a nonhuman antibody having binding specificity for α4β7 integrin, and a framework region derived from a heavy chain of human origin is another example of such a construct.

The present invention also relates to a host cell comprising a nucleic acid of the present invention, including one or more constructs comprising a nucleic acid of the present invention. In one embodiment, the invention relates to a host cell comprising a first recombinant nucleic acid encoding a humanized immunoglobulin light chain, and a second recombinant nucleic acid encoding a humanized immunoglobulin heavy chain, said first nucleic acid comprising a nucleotide sequence encoding a CDR derived from the light chain of murine Act-1 antibody and a framework region derived from a light chain of human origin; and said second nucleic acid comprising a nucleotide sequence encoding a CDR derived from the heavy chain of murine Act-1 antibody and a framework region derived from a heavy chain of human origin.

The present invention also provides a method of preparing a humanized immunoglobulin comprising maintaining a host cell of the present invention under conditions appropriate for expression of a humanized immunoglobulin, whereby a humanized immunoglobulin chain(s) is expressed and a humanized immunoglobulin is produced. The method can further comprise the step of isolating the humanized immunoglobulin.

The humanized immunoglobulins of the present invention can be less immunogenic than their murine or other nonhuman counterparts. Thus, the humanized immunoglobulins described herein can be used as therapeutic agents in humans, for example to control lymphocyte homing to mucosal lymphoid tissue, thereby, reducing inflammatory responses in the gut.

FIG. 1 is an illustration of a consensus DNA sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) comprising the variable region determined from several independent mouse heavy chain variable region clones.

• Provisional Patent
• Utility Patent

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