CD20 Antibodies and Uses Thereof

a technology of cd20 and antibodies, which is applied in the field of cd20 antibodies, can solve the problems of rituximab-refractory nhl patients that are not active on rituximab, the type i antibodies typically do not have effective pro-apoptotic activity on their own, and many patients do not respond to rituximab, so as to improve the adcc function, improve stability, and increase stability

Inactive Publication Date: 2016-04-28
IMMUNOGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is an anti-CD20 antibody and fragments thereof that can be used to treat medical conditions where B cells expressing CD20 influence the course of the condition. The antibody is capable of inducing antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) in B cells expressing CD20. The antibody can be obtained from CHO or NS0 cells and can be used in the treatment of non-Hodgkin's lymphoma. The antibody specifically binds to amino acids residues located between the third and fourth transmembrane domain of CD20. The invention also includes an anti-CD20 antibody that is capable of efficiently translocating CD20 into the lipid raft compartment of a cell membrane expressing CD20. The antibody has a high potency in killing CD20-expressing cells.

Problems solved by technology

Type I antibodies typically do not have effective pro-apoptotic activity on their own, unless the antibodies are cross-linked.
However, many patients do not respond to rituximab treatment and responding patients taking rituximab eventually relapse and often develop resistance to rituximab treatment.
Although ofatumumab with improved CDC activity is effective and was approved for treating patients with refractory chronic lymphocytic leukemia, ofatumumab was not active on rituximab-refractory NHL patients.
However, until now, no novel molecule having unique physical and functional features addressing the problems known in the art was available.
Although CD20 is known in the art, CD20 is not a favorable target for antibody-drug conjugation, since known anti-CD20 antibodies are very poorly internalized.
Conjugates of CD20 antibodies have been studied previously but have not demonstrated significantly strong potency, especially when non-disulfide or acid stable linkers are used.
Similarly, it has been reported that calicheamicin conjugates of anti-CD20 antibody made with an acid-stable amide linker did not show improved in vivo efficacy over rituximab in a Ramos xenograft model in nude mice.
Acid stable adriamycin conjugates of the same antibody were shown to be completely ineffective.
While these clinical data suggest that anti-CD20 radio-immunoconjugates can be more effective than rituximab, they are not widely used because of additional toxicities and difficulty in administration associated with using radioactive compounds.
While even improved anti-CD20 antibody ofatumumab with better CDC activity than rituximab is effective and was approved for treating patients with refractory chronic lymphocytic leukemia, ofatumumab was not active on rituximab-refractory NHL patients.

Method used

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  • CD20 Antibodies and Uses Thereof
  • CD20 Antibodies and Uses Thereof
  • CD20 Antibodies and Uses Thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Production of Murine CD20 Antibodies

[0483]An expression plasmid pSRa-CD20 was constructed that contained the entire CD20 coding sequence (CDS) flanked by XbaI and BamHI restriction sites that allowed expression of human CD20 (GI 23110989). 300-19 cells, a pre-B cell line derived from a Balb / c mouse (Reth et al., Nature, 317:353-355 (1985)), was transfected with this expression plasmid to stably express high levels of human CD20 on the cell surface and used for immunization of Balb / c VAF mice. Mice were subcutaneously immunized with approximately 5×106 CD20-expressing 300-19 cells per mouse every 2-3 weeks by standard immunization protocols known to those of skill, for example, such as those used at ImmunoGen, Inc. Immunized mice were boosted with antigen three days before being sacrificed for hybridoma generation. Spleens from mice was collected according to standard animal protocols, such as, for example grinding tissue between two sterile, frosted microscopic slides to obtain a si...

example 2

Binding Characterization by Flow Cytometry

[0490]Binding specificity was tested by flow cytometry using purified antibodies. FACS histograms demonstrating the binding of muCD20-4 and muCD20-20 to CD20-expressing 300-19 cells and the absence of binding to the parental 300-19 cells are shown in FIG. 1. Either muCD20-4 or muCD20-20 antibody was incubated for 3 h with either CD20-expressing 300-19 cells or the non-transfected 300-19 cells (1×105 cells per sample) in 100 μL FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum). Then, the cells were pelleted, washed, and incubated for 1 h with 100 μL of FITC-conjugated goat anti-mouse IgG-antibody (such as is obtainable from, for example Jackson Laboratory, 6 μg / mL in FACS buffer). The cells were pelleted again, washed with FACS buffer and resuspended in 200 μL of PBS containing 1% formaldehyde. Samples were acquired using a FACSCalibur flow cytometer with the HTS multiwell sampler or a FACS array flow cytometer and analyze...

example 3

Lipid Raft

[0493]The reorganization of CD20 into lipid rafts following antibody binding has been correlated with the ability of the antibody to effectively recruit complement factors and elicit CDC activity. As a measure of antibody induced CD20 re-distribution to lipid rafts we employed a flow cytometry based method modified from Cragg et al. (2003), supra). The principle of this assay is based on the TritonX-100 insolubility of lipid raft compartments at low temperatures.

[0494]Cells were harvested by centrifugation, washed and re-suspended in RPMI-1640 with 1% BSA. 0.2 mL of cells at 2.5×106 cells were used for each assay in duplicate. Anti-CD20 antibodies were added at 10 μg / mL and samples were incubated for 15 min at 37° C. Samples were centrifuged, washed twice and re-suspended in 0.2 mL of FACS buffer (1×PBS, 1% BSA, 20 mM Na-azide). Samples were chilled on ice and kept cold from this point on. To detect the fraction of antigen associated with lipid rafts in response to antibod...

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Abstract

CD20 is a transmembrane protein of the tetra-spanin family expressed on the surface of B-cells from peripheral blood as well as lymphoid tissues. CD20 expression persists from the early pre-B cell stage until the plasma cell differentiation stage. In addition to expression in normal B-cells, CD20 is expressed in B-cell derived malignancies such as non-Hodgkin's lymphoma (NHL) and B-cell chronic lymphocytic leukemia (CLL). The present invention includes anti-CD20 antibodies and antigen-binding fragments thereof comprising a light chain variable region and a heavy chain variable region, wherein the CDR-L1, CDR-L2, and CDR-L3 of said light chain variable region comprise the amino acid sequences of SEQ ID NOs: 23-25, respectively, and wherein the CDR-H1, CDR-H2, and CDR-H3 of said heavy chain variable region comprise the amino acid sequences of SEQ ID NOs: 26-28, respectively.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]Related applications U.S. application Ser. No. 13 / 024,671, filed Feb. 10, 2011, and U.S. Provisional Patent Application No. 61 / 303,210, filed Feb. 10, 2010, are herein incorporated by reference in their entireties.REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB[0002]The content of the electronically submitted sequence listing (Name: 29210410002_substituteSequenceLisiting.txt, Size: 38,573 bytes; and Date of Creation: Dec. 28, 2015) is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION[0003]CD20 and CD20 isoforms (“CD20”) are generally appreciated to be transmembrane proteins of the tetra-spanin family that are expressed on the surface of B-cells (Valentine et al., J. Biol. Chem., 264(19):11282-11287 (1989); Einfeld et al., EMBO J., 7(3):711-717 (1988)). CD20 is expressed by the vast majority of peripheral blood B-cells as well as B cells from various lymphoid tissues. CD20 expression generall...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/28A61K47/48
CPCC07K16/2887A61K47/48561A61K47/48446C07K2317/565C07K2317/24A61K2039/505C07K2317/622C07K2317/734C07K2317/732C07K2317/73C07K2317/92C07K2317/21A61K51/1027A61K39/39541A61K47/6817A61K47/6819A61K47/6849A61P1/00A61P3/10A61P11/00A61P11/06A61P17/02A61P19/02A61P29/00A61P31/12A61P35/00A61P37/00C07K2317/30C07K2319/55G01N33/57426
Inventor DECKERT, JUTTATAVARES, DANIEL J.PARK, PETER U.
Owner IMMUNOGEN INC
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