Immunoglobulin variants and uses thereof

a technology of immunoglobulin and variants, applied in the field of can solve the problem of limit the use of murine antibodies in human therapy, human anti-mouse antibody (hama) response, etc., to improve effector function, enhance cdc and/or adcc function, and improve stability

Inactive Publication Date: 2006-02-02
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008] The present invention provides CD20 binding antibodies or functional fragments thereof, and their use in the treatment of B-cell associated diseases. These antibodies are monoclonal antibodies. In specific embodiments, the antibodies that bind CD20 are humanized or chimeric. The humanized 2H7 variants include those that have amino acid substitutions in the FR and affinity maturation variants with changes in the grafted CDRs. The substituted amino acids in the CDR or FR are not limited to those present in the donor or recipient antibody. In other embodiments, the anti-CD20 antibodies of the invention further comprise changes in amino acid residues in the Fc region that lead to improved effector function including enhanced CDC and / or ADCC function and B-cell killing (also referred to herein as B-cell depletion). Other anti-CD20 antibodies of the invention include those having specific changes that improve stability. In a specific embodiment, the humanized 2H7 variants with increased stability are as described in example 6 below. Fucose deficient variants having improved ADCC function in vivo are also provided. In one embodiment, the chimeric anti-CD20 antibody has murine V regions and human C region. One such specific chimeric anti-CD20 antibody is Rituxan® (Rituximab®; Genentech, Inc.).

Problems solved by technology

A major limitation in the use of murine antibodies in human therapy is the human anti-mouse antibody (HAMA) response (see, e.g., Miller, R. A. et al.

Method used

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  • Immunoglobulin variants and uses thereof
  • Immunoglobulin variants and uses thereof
  • Immunoglobulin variants and uses thereof

Examples

Experimental program
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experimental examples

Example 1

Humanization of 2H7 Anti-CD20 Murine Monoclonal Antibody

[0279] Humanization of the murine anti-human CD20 antibody, 2H7 (also referred to herein as m2H7, m for murine), was carried out in a series of site-directed mutagenesis steps. The murine 2H7 antibody variable region sequences and the chimeric 2H7 with the mouse V and human C have been described, see, e.g., U.S. Pat. Nos. 5,846,818 and 6,204,023. The CDR residues of 2H7 were identified by comparing the amino acid sequence of the murine 2H7 variable domains (disclosed in U.S. Pat. No. 5,846,818) with the sequences of known antibodies (Kabat et al., Sequences of proteins of immunological interest, Ed. 5. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The CDR regions for the light and heavy chains were defined based on sequence hypervariability (Kabat et al., supra) and are shown in FIG. 1A and FIG. 1B, respectively. Using synthetic oligonucleotides (Table 1), site-directed mutagenesis (Kun...

example 2

Antigen-binding Determinants (paratope) of 2H7

[0292] Alanine substitutions (Cunningham & Wells, Science 244: 1081-1085 (1989) were made in 2H7.v16 or 2H7.v17 in order to test the contributions of individual side chains of the antibody in binding to CD20. IgG variants were expressed in 293 cells from pDR1 and pDR2 vectors, purified, and assayed for relative binding affinity as described above. Several alanine substitutions resulted in significant decreases in relative binding to CD20 on WIL-2S cells (Table 4).

TABLE 4Effects of alanine substitutions in the CDR regions of humanized 2H7.v16measured using cell-based ELISA (WIL2-S cells). The relative binding isexpressed as the concentration of the 2H7.v16 parent over theconcentration of the variant required for equivalent binding; hence aratio 1 indicateshigher affinity for the variant. Standard deviation in relative affinitydetermination averaged + / −10%. Framework substitutions in thevariable domains are relative to 2H7.v16 according...

example 3

Additional Mutations within 2H7 CDR Regions

[0293] Substitutions of additional residues and combinations of substitutions at CDR positions that were identified as important by Ala-scanning were also tested. Several combination variants, particularly v.96 appeared to bind more tightly than v.16.

TABLE 5Effects of combinations of mutations and non-alanine substitutionsin the CDR regions of humanized 2H7.v16 measured using cell-basedELISA (WIL2-S cells). The relative binding to CD20 is expressed asthe concentration of the 2H7.v16 parent over the concentration of thevariant required for equivalent binding; hence a ratio weaker affinity for the variant; a ratio >1 indicates higher affinityfor the variant. Standard deviation in relative affinity determinationaveraged + / −10%. Framework substitutions in the variabledomains are relative to 2H7.v16 according to the numbering systemof Kabat (Kabat et al., supra).2H7Heavy chainLight chainRelativeversionsubstitutionsSubstitutionsbinding16——-1-9...

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Abstract

The invention provides humanized and chimeric anti-CD20 antibodies for treatment of CD20 positive malignancies and autoimmune diseases.

Description

[0001] This is a continuation application of international patent application No. PCT / US03 / 40426, filed Dec. 16, 2003, which claims benefit of provisional application Ser. No. 60 / 526,163, filed on Dec. 1, 2003 and provisional application Ser. No. 60 / 434,115, filed on Dec. 16, 2002, which applications are incorporated herein by reference in their entirety.FIELD OF THE INVENTION [0002] The invention relates to anti-CD20 antibodies and their use in the treatment of B-cell related diseases. BACKGROUND OF THE INVENTION [0003] Lymphocytes are one of several populations of white blood cells; they specifically recognize and respond to foreign antigen. The three major classes of lymphocytes are B lymphocytes (B cells), T lymphocytes (T cells) and natural killer (NK) cells. B lymphocytes are the cells responsible for antibody production and provide humoral immunity. B cells mature within the bone marrow and leave the marrow expressing an antigen-binding antibody on their cell surface. When a ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K16/28A61KC07H21/04C07K16/00C12N15/63
CPCA61K2039/505C07K16/2887C07K2316/95C07K2317/24C07K2317/41C07K2317/55C07K2317/52C07K2317/565C07K2317/72C07K2317/732C07K2317/734C07K2317/92C07K2317/56C07K2317/73C07K2317/75A61P1/00A61P1/04A61P1/12A61P1/16A61P11/00A61P11/02A61P11/06A61P11/08A61P13/12A61P15/08A61P17/00A61P17/02A61P17/06A61P17/14A61P19/02A61P21/04A61P25/00A61P27/02A61P27/16A61P29/00A61P31/06A61P31/18A61P35/00A61P35/02A61P37/00A61P37/02A61P37/06A61P37/08A61P43/00A61P5/00A61P5/14A61P5/40A61P7/04A61P7/06A61P9/00A61P9/08A61P9/10A61P9/14A61P3/10C07K16/00C07K16/28C12N15/63A61K39/39558C12N5/16C07K2317/522C07K2317/567
Inventor ADAMS, CAMELLIACHAN, ANDREWCROWLEY, CRAIGLOWMAN, HENRYNAKAMURA, GERALDPRESTA, LEONARD
Owner GENENTECH INC
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