Somatic hypermutation systems

a somatic hypermutation and system technology, applied in the field of somatic hypermutation systems, can solve the problems of long development time, inefficiency, and system work against the more rapid development of improved therapeutics, and achieve the effects of preventing non-specific mutagenesis of structural proteins, stable maintenance of a mutagenesis system, and increasing and/or reducing shm

Inactive Publication Date: 2012-02-02
ANAPTYSBIO INC
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

[0011]The present invention is based on the development of a system to design and make or generate SHM susceptible and SHM resistant DNA sequences, within a cell or cell-free, environment. The present invention is further based on the development of a SHM system that is stable over a suitable time period to reproducibly maintain increased and/or decreased rates of SHM without affecti...

Problems solved by technology

Even given the success of monoclonal antibodies, the antibody-as-drug modality is continuing to evolve, and subject to inefficiency.
Further, intrinsic biological bias within the native immune system often works against the more rapid development of improved therapeutics.
These limitations include, i) the long development time for the isolation of biologically active antibodies with affinity constants of therapeutic caliber, ii) the inability to raise antibodies to certain classes of protein targets (intractable targets), and iii) the intrinsic affinity ceiling inherent in immune system based affinity selection.
There are several existing well-established methods of developing monoclonal antibodies; however, many of these technologies have specific disadvantages that limit their ability to rapidly evolve the best clinical candidates.
These technological limitations include: i) mouse immunization and hybridoma technology cannot be used iteratively and often fails to yield an antibody with desired characteristics due to antigen intractability; ii) phage display or panning often fails to yield monoclonal a...

Method used

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Examples

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example 1

Creation of Synthetic Polynucleotides Encoding Blasticidin

[0608]By decreasing the likelihood of somatic hypermutation in a vector element, such as a selectable marker, an enzyme involved in SHM, or a reporter gene, the vector and system for exerting and tracking SHM becomes more stable, thereby enabling somatic hypermutation to be more effectively targeted to a polynucleotide of interest.

[0609]A. Polynucleotide Design

[0610]In general, sequences are engineered for SHM using the teaching described herein, and as elaborated in sections III and IV. In the following examples, the sequence optimization is based on the hot spot and cold spot motifs listed in Table 7, and using the computer program SHMredesign.pl as described above.

[0611]Using this program, every position within the sequence is annotated with either a ‘+’, ‘−’, or ‘.’ symbol to designate whether it is desired to obtain a hotter, colder, or neutral changes in SHM susceptibility at that specific position. Where ‘+’ designates...

example 2

Creation of Synthetic Polynucleotides Encoding Hygromycin

[0630]A. Polynucleotide Design

[0631]The starting sequence for unmodified hygromycin is shown in FIG. 11, together with the initial analysis of hot spot and cold spot frequency.

[0632]As described for Example 1, sequence optimization is completed using the computer program SHMredesign, based on the hot spot and cold spot motifs listed in Table 7.

1. Cold Hygromycin

[0633]From iteration 1 to iteration 2000, an additional 71 cold spots are inserted into the gene, 12 existing hot spots are removed, and 61 CpG sites are removed making the gene sequence less susceptible to somatic hypermutation. No further beneficial changes are observed upon further iterations.

[0634]An optimized sequence for a SHM resistant version of hygromycin created using this approach is shown in FIG. 12, together with the resulting changes in frequency of hot spots and cold spots. Optimization of the hygromycin sequence to make the sequence more resistant to som...

example 3

Creation of Synthetic Polynucleotides Encoding Reporter Genes

[0642]A. Polynucleotide Design

[0643]The starting sequence for unmodified Teal Fluorescent Protein (TFP) is shown in FIG. 14, together with the initial analysis of hot spot and cold spot frequency.

1. Hot TFP

[0644]As described for Example 1, sequence optimization is completed using the computer program SHMredesign, based on the hot spot and cold spot motifs listed in Table 7; the resulting hot and cold versions of TFP are shown in FIGS. 15 and 16, respectively.

[0645]Optimization of the TFP sequence to make the sequence more susceptible to somatic hypermutation resulted in an increase of about 170% in number of hot spots (an increase of 28), and reduced the number of cold spots by about 26% (a decrease of 27). Overall the frequency of hot spots increased to an average density of about 10 hot spots per 100 nucleotides from an initial density of about 6 hot spots per 100 nucleotides, and the overall frequency of cold spots decr...

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Abstract

The present application relates to somatic hypermutation (SHM) systems and synthetic genes. Synthetic genes can be designed using computer-based approaches to increase or decrease susceptibility of a polynucleotide to somatic hypermutation. Genes of interest are inserted into the vectors and subjected to activation-induced cytidine deaminase to induce somatic hypermutation. Proteins or portions thereof encoded by the modified genes can be introduced into a SHM system for somatic hypermutation and proteins or portions thereof exhibiting a desired phenotype or function can be isolated for in vitro or in vivo diagnostic or therapeutic uses.

Description

CROSS-REFERENCE[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 902,414 (Attorney docket no. 33547-705.101), filed Feb. 20, 2007, U.S. Provisional Application No. 60 / 904,622 (Attorney docket no. 33547-706.101), filed Mar. 1, 2007, U.S. Provisional Application No. 61 / 020,124 (Attorney docket no. 33547-706.102), filed Jan. 9, 2008, and U.S. Provisional Application No. 60 / 995,970 (Attorney docket no. 33547-708.101), filed Sep. 28, 2007, each of which applications is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]The market for the use of recombinant protein therapeutics has increased steadily for the last quarter century. In 2005, six of the top 20 drugs were proteins, and overall, biopharmaceutical drugs accounted for revenues of approximately $40 billion, of which approximately $17 billion was based on the sales of monoclonal antibodies.[0003]Monoclonal antibodies represent a distinct class of biotherapeutics with a g...

Claims

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Application Information

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IPC IPC(8): C12P21/00C12N9/00
CPCC07K16/00C07K16/22C07K16/40C07K16/461C07K2317/565C07K2317/92C07K2317/21C40B50/06
Inventor HORLICK, ROBERT A.CUBITT, ANDREW B.BOWERS, PETER M.
Owner ANAPTYSBIO INC
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