Optimization of antibodies that bind lymphocyte activation gene-3 (lag-3), and uses thereof

Abstract

The present invention provides isolated monoclonal antibodies that specifically bind LAG-3, and have optimized functional properties compared to previously described anti-LAG-3 antibodies, such as antibody 25F7 (US 2011 / 0150892 A1). These properties include reduced deamidation sites, while still retaining high affinity binding to human LAG-3, and physical (i.e., thermal and chemical) stability. Nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells and methods for expressing the antibodies of the invention are also provided, as well as immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies. The present invention also provides methods for detecting LAG-3, as well as methods for treating stimulating immune responses using an anti-LAG-3 antibody of the invention. Combination therapy, in which the antibodies are co-administered with at least one additional immunostimulatory antibody, is also provided.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 14 / 093,867, filed Dec. 2, 2013, which claims priority to PCT / US2013 / 048999, filed Jul. 2, 2013, which claims priority to U.S. provisional application 61 / 667,058, filed Jul. 2, 2012. The contents of the foregoing applications are incorporated herein by reference.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 30, 2013, is named MXI-513CN_Sequence_Listing.txt and 42.064 KB in size.BACKGROUND OF THE INVENTION[0003]Therapeutic antibodies are one of the fastest growing segments of the pharmaceutical industry. To maintain potency (i.e., activity) and minimize immunogenicity, antibodies and other protein drugs must be protected from physical and chemical degradation during manufacturing and storage. Indeed, one of the primary difficulti...

AI Technical Summary

Benefits of technology

[0007]The present invention provides isolated monoclonal antibodies (e.g., human monoclonal antibodies) that bind LAG-3 (e.g., human LAG-3) and have optimized physical stability compared to previously described anti-LAG-3 antibodies. In particular, the invention relates to a modified form of antibody 25F7 (US 2011 / 0150892 A1) which exhibits significantly improved thermal and chemical stability compared to the unmodified antibody. Specifically, by altering the critical binding region of the heavy chain CDR2 domain of antibody 25F7, it was shown that the modified antibody exhibited significantly higher physical and thermal stability, reduced deamidation, higher thermal reversibility, and lower aggregation. At the same time, it was unexpectedly observed that the modified antibody retained the same high binding affinity to human LAG-3 and functional activity of the unmodified antibody, including the ability to inhibit binding of LAG-3 to major histocompatibility (MHC) Class II molecules and stimulate antigen-specific T cell responses. The combined substantial increase in stability and retention of binding / biological activity of the modified antibody was surprising, particularly in view of the criticality of CDRs regions to antibody function.

[0010]In a preferred embodiment the antibody exhibits increased physical properties (i.e., thermal and chemical stability) compared to antibody 25F7, while still retaining at least the same binding affinity for human LAG-3 as 25F7. For example, the antibody exhibits decreased sequence variability in the heavy chain CDR2 region due to deamidation, compared to antibody 25F7, e.g., approximately 2.5% or less modification of the amino acid sequence after 12 weeks at 4 C.° (i.e., under “real-time” stability studies as described herein) and / or approximately 12.0% or less modification of the amino acid sequence after 12 weeks at 40 C.° (i.e., under accelerated stress conditions, as described herein), while still retaining a binding affinity for human LAG-3 of about at least KD of 1×10−7 M or less (more preferably, a KD of 1×10−8 M or less, a KD of 5×10−9 M or less, or a KD of 1×10−9 M or less). In another embodiment, the antibody exhibits thermal reversibility of at least about 40% in PBS at pH8.0.

Problems solved by technology

Indeed, one of the primary difficulties in developing antibody therapeutics is the potential immunogenic response when administered to a subject, which can lead to rapid clearance or even induce life-threatening side effects including anaphylactic shock.

Deamidation removes an amide functional group from an amino acid residue, such as asparagine and glutamine, thus damaging its amide-containing side chains.

This, in turn, causes structural and biological alterations throughout the protein, thus creating heterogeneous forms of the antibody.

In addition, reduction / loss of biological activity due to deamidation has been a recognized problem.

Overall, deamidation poses a significant and unpredictable problem to the pharmaceutical industry.

Efforts associated with monitoring the variability caused by deamidation within antibody therapeutics, in particular, as well as FDA concerns associated with this variability, increase costs and delay clinical trials.

Moreover, modifications to address this issue, including shifting conditions (e.g., temperature, pH, and cell type) associated with recombinant production and / or alteration of amino acids which are susceptible to deamidation (e.g., site-directed mutagenesis) can negatively impact stability and activity, especially when changes are made within the complementarity determining regions (CDRs) of the antibody.

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