Somatic hypermutation systems

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...

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Benefits of technology

[0344]Depending on the amount of information available, a number of distinct library design strategies can be employed, ranging from a very aggressive targeted approach based on the use of WAC or WRC motifs, to a more conservative strategy of using fairly selective amino acid replacements, to a cautious strategy in which only codon usage is changed. An advantage of the present invention is that each approach results in the generation of only a limited number of distinct nucleotide sequences; thus all of these strategies can be subjected to SHM mediated diversity in parallel without significant additional burden.

[0345]Art-recognized methods for monitoring SHM in antibody and non-antibody proteins using various vectors and cell lines are known (Rückerl et al. (Mol. Immunol. 43 (2006); 1645-1652), Bacl et al. (J. Immunol., 2001, 166: 5051-5057), Cumbers et al. (Nat. Biotechnol. 2002; 20(11): 1129-1134), Wang, et al. (Proc Natl Acad Sci USA. 2004; 101(19):7352-7356)). In addition, various methods for directly measuring cytidine deamination are known in the art; see, e.g., Genetic and In vitro assays of DNA deamination, Coker et al., Meth. Enzymol. 408: 156-170 (2006).

[0346]Such methods provide rapid means for evaluating the rate of on-going SHM. The methods include, for example, the use of reporter genes, or selectable marker genes that have been modified to include a stop codon within the coding frame which can be mutated in the presence of AID activity.

[0347]As AID acts on a population, it can produce mutations that restore or improve function (of a selectable marker, for instance), or mutations that reduce or eliminate function. The balance in these two rates generates early and rare mutation events that restore function, followed by secondary and ternary mutation events that destroy function in these proteins. The net effect of these competing rates on the observation of gain-of-function events in a population. Given three different assumptions regarding number of inactivating mutations needed to silence GFP, one would expect to observe three very different profiles of reversion events as a function of time, dependent on the rate of enzymatic activity of the AID.

[0348]Additionally, polynucleotides subjected to SHM activity can be sequenced to determine if the nucleotide sequence of has been modified and to what degree. Polynucleotides can be rescued from culture to determine SHM at various time points. Methods of isolating and sequencing genes are well known in the art, and include, the use of standard techniques such as, for example, Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Briefly, the polynucleotide can be reverse transcribed and subjected to PCR using appropriate primers. Clones can be sequenced using automated DNA sequences from companies such as Applied Biosystems (ABI-377 or ABI 3730 DNA sequencers). Sequences can be analyzed for frequency of nucleotide modifications. The polynucleotide can be compared with the polynucleotide from the starting material and analyzed for sequence modifications.IX. Somatic Hypermutation (SHM) Systems

[0350]The development of a practical system for the use of SHM requires that mutations be directed to specific genes (polynucleotides) or regions of interest (made “hot”), and be directed away from structural or marker genes that are functionally required within the cell or episome, to maintain overall system functionality and / or stability (made “cold”). In certain embodiments, a synthetic gene is one that does naturally undergo SHM when expressed in a B cell (i.e., an antibody gene). In other embodiments, a synthetic gene is one that does not naturally undergo SHM when expressed in a B cell (i.e., a non-antibody gene).

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 antibodies with affinity constants of therapeutic caliber, and cannot easily be used to select and co-evolve entire heavy and light chains; and iii) rational design strategies often provides an incomplete solution, and are based solely on existing knowledge.

Such in vitro mutation approaches can be limited by the inability to systematically search a portion of any given sequence, and by the relative difficulty of detecting very rare improvement mutants out of a large number of mutations.

This fundamental problem arises because the total number of possible mutants for a reasonably sized protein is large.

For example, a 100 amino acid protein has a potential diversity of 20100 different sequences of amino acids, while existing high throughput screening methodologies are, in some cases, limited to a maximum screening capacity of 107-108 samples per week.

Additionally, such approaches are relatively inefficient because of redundant codon usage, in which up to around 3100 of the nucleotide sequences possible for a 100 amino acid residue protein actually encode for the same amino acids and protein, (Gustafsson et al.

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