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Fc variants with optimized Fc receptor binding properties

a technology of fc variants and receptors, applied in the field of fc variants with optimized fc receptor binding properties, can solve the problems of major challenges in obtaining variants that selectively increase or reduce the affinity of fcr, and the anti-cancer potential is unsatisfactory, so as to achieve the effect of increasing affinity and increasing affinity

Inactive Publication Date: 2008-08-28
DESJARLAIS JOHN R +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The Fc variants demonstrate improved binding properties to FcγRs, enhancing antibody-dependent cellular cytotoxicity and phagocytosis, leading to increased therapeutic efficacy against cancer cells while maintaining reduced affinity for inhibitory receptors, thus improving treatment outcomes.

Problems solved by technology

Despite this arsenal of anti-tumor weapons, the potency of antibodies as anti-cancer agents is unsatisfactory, particularly given their high cost.
Because all FcγRs interact with the same binding site on Fc, and because of the high homology among the FcγRs, obtaining variants that selectively increase or reduce FcγR affinity is a major challenge.
A challenge for development of Fc variants with optimized Fc receptor binding properties is the difference between human and murine Fc receptor biology.

Method used

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  • Fc variants with optimized Fc receptor binding properties
  • Fc variants with optimized Fc receptor binding properties
  • Fc variants with optimized Fc receptor binding properties

Examples

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example 1

Design of Fc Variants with Selective FcγR Affinity

[0174]Sequence and structural analysis of the Fc / FcγR interface was carried out for the different human FcγRs. A central goal was to generate variants with selectively increased affinity for the activating receptors FcγRI, FcγRIIa, FcγRIIc, and FcγRIIIa relative to the inhibitory receptor FcγRIIb, and selectively increased affinity for FcγRIIb relative to the activating receptors. FIG. 4 shows an alignment of the sequences of the human FcγRs, highlighting the differences from FcγRIIb and positions at the Fc interface. The analysis indicates that although there is extensive homology among the human FcγRs, there are significant differences. Particularly relevant are differences at the Fc binding interface that may be capitalized on to engineer selective Fc variants.

[0175]The utility of this analysis is illustrated using the example of FcγRIIa vs. FcγRIIb. Engineering an Fc variant that selectively improves binding to FcγRIIa relative t...

example 2

Screening of Fc Variants

[0177]Amino acid modifications were engineered at these positions to generate variants with selective FcγR affinity. Fc variants were engineered in the context of the anti-CD20 antibody PRO70769 (PCT / US2003 / 040426, hereby entirely incorporated by reference). The genes for the variable regions of PRO70769 (SEQ IDs NO: 1 and NO:2, FIGS. 27a and 27b) were constructed using recursive PCR, and subcloned into the mammalian expression vector pcDNA3.1Zeo (Invitrogen) comprising the full length light kappa (CK) and heavy chain IgG1 constant regions. Amino acid substitutions were constructed in the variable region of the antibody in the pcDNA3.1Zeo vector using quick-change mutagenesis techniques (Stratagene). DNA was sequenced to confirm the fidelity of the sequences. Plasmids containing heavy chain gene (VH-CH1-CH2-CH3) (wild-type or variants) were co-transfected with plasmid containing light chain gene (VL-Cκ) into 293T cells. Media were harvested 5 days after trans...

example 3

Performance of Fc Variants in Cell-Based Assays

[0189]A central goal of improving the activating FcγR vs. inhibitory FcγR profile of an antibody or Fc fusion was to enhance its FcγR-mediated effector function in vitro and ultimately in vivo. To investigate the capacity of antibodies comprising the Fc variants of the present invention to carry out FcγR-mediated effector function, in vitro cell-based ADCC assays were run using human PBMCs as effector cells. ADCC was measured by the release of lactose dehydrogenase using a LDH Cytotoxicity Detection Kit (Roche Diagnostic). Human PBMCs were purified from leukopacks using a ficoll gradient, and the EpCAM+ target gastric adenocarcinoma line LS180. Target cells were seeded into 96-well plates at 10,000 cells / well, and opsonized using Fc variant or WT antibodies at the indicated final concentration. Triton X100 and PBMCs alone were run as controls. Effector cells were added at 40:1 PBMCs:target cells, and the plate was incubated at 37° C. fo...

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Abstract

The present invention relates to Fc variants with optimized Fc receptor binding properties, methods for their generation, Fc polypeptides comprising Fc variants with optimized Fc receptor binding properties, and methods for using Fc variants with optimized Fc receptor binding properties.

Description

[0001]This application is a continuation of U.S. application Ser. No. 11 / 538,406, filed Oct. 3, 2006, which claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 60 / 741,966 filed Dec. 2, 2005, U.S. Provisional Application No. 60 / 779,961 filed Mar. 6, 2006, U.S. Provisional Application No. 60 / 745,078 filed Apr. 18, 2006, U.S. Provisional Application No. 60 / 723,294 filed Oct. 3, 2005, U.S. Provisional Application No. 60 / 723,335 filed Oct. 3, 2005, U.S. Provisional Application No. 60 / 739,696 filed Nov. 23, 2005, U.S. Provisional Application No. 60 / 750,699 filed Dec. 15, 2005, U.S. Provisional Application No. 60 / 774,358 filed Feb. 17, 2006; this application is also a Continuation-in-Part of U.S. patent application Ser. No. 11 / 396,495 filed Mar. 31, 2006, each of which is incorporated by reference in its entirety.FIELD OF THE INVENTION[0002]The present invention relates to Fc variants with optimized Fc receptor binding properties, engineering methods for their gener...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/00C07K16/00
CPCC07K16/00C07K16/2863C07K16/2887C07K2317/77C07K2317/72C07K2317/732C07K16/30C07K2317/41C07K2317/52
Inventor DESJARLAIS, JOHN R.KARKI, SHER BAHADURLAZAR, GREGORY ALANRICHARDS, JOHN O.MOORE, GREGORY L.CARMICHAEL, DAVID F.
Owner DESJARLAIS JOHN R
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