Continuous in vitro evolution

Details Continuous in vitro evolution Continuous in vitro evolution

2000-12-11

2003-05-13

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

C435S004000, C435S006120

Reexamination Certificate

active

06562622

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method for mutating and selecting target binding proteins in a translation system; and to a polynucleotide construct for use in this method. The method of the present invention may be applied to the generation of molecules of diagnostic and therapeutic utility.
BACKGROUND OF THE INVENTION
In vitro evolution of proteins involves introducing mutations into known gene sequences to produce a library of mutant sequences, translating the sequences to produce mutant proteins and then selecting mutant proteins with the desired properties. This process has the potential for generating proteins with improved diagnostic and therapeutic utilities. Unfortunately, however, the potential of this process has been limited by deficiencies in methods currently available for mutation and library generation.
For example, the generation of large libraries (eg beyond a library size of 10
10
) of unique individual genes and their encoded proteins has proven difficult with phage display systems due to limitations in transformation efficiency. A further disadvantage is that methods which utilise phage-display systems (
FIG. 1
) require several sequential steps of mutation, amplification, selection and further mutation (Irving et al., 1996; Krebber et al., 1995; Stemmer. 1994; Winter et al., 1994).
Examples of procedures which have been used to date for affinity maturation of selected proteins, and particularly for the affinity maturation of antibodies, are set out in Table 1. All these methods rely on mutation of genes followed by display and selection of encoded proteins. The particular mutation that is chosen determines the diversity in the resulting gene library. In vitro strategies (Table 1) are severely limited by the efficiency in transformation of mutated genes in forming a phage display library. In one in vivo cyclical procedure (Table 1 No.1),
E. coli
mutator cells there the vehicle for mutation of recombinant antibody genes. The
E. coli
mutator cells MUTD5-FIT (Irving et al., 1996) which bear a mutated DNAQ gene could be used as the source of the S-30 extracts and therefore allow mutations introduced into DNA during replication as a result of proofreading errors. However, mutation rates are low compared to the required rate. For example, to mutate 20 residues with the complete permutation of 20 amino acid requires a library size of 1×10
26
, an extremely difficult task with currently available phage display methodology.
TABLE 1
Affinity maturation strategies
Mechanism
In vivo
1 Mutator cells
Random point mutations
2 SIP-SAP
Co-selection and infection with
antibody-antigen pairs
In vitro
3 DNA shuffling-sexual PCR
Recursive sequence recombination by
DNA homology
4 Site directed mutagenesis over
Oligonucleotide-coded mutations
selected regions (CDRs)
5 Chain shuffling
Sequential replacement of heavy or
light chain domains using phage
libraries
6 Error-prone PCR
Polymerase replication errors
1) Irving et al. (1996); 2a) Krebber et al. (1995); 2b) Duenas and Borrebaeck (1994); 3) Stemmer (1994), Stemmer et al. (1995); 4) Yang et al. (1995); 5a) Barbas et al. (1994); 5b) Winter et al. (1994); 6) Gram et al (1992).
A selection method which enables the in vitro production of complex libraries of mutants which are continuously evolving (mutating) and from which a desired gene may be selected would therefore provide an improved means of affinity maturation (enhancement) of proteins.
In vitro Coupled Transcription and Translation Systems
It is well known that a DNA plasmid containing a gene of interest can act as template for transcription when controlled by a control element such as the T7 promoter. It is also known that coupled cell-free systems may be used to simultaneously transcribe mRNA and translate the mRNA into peptides (Baranov et al 1993; Kudilicki et al. 1992; Kolosov et al 1992; Morozov et al 1993; Ryabova et al 1989, 1994; Spirin 1990; U.S. Pat. Nos. 5,556,769; 5,643,768; He and Taussig 1997). The source of cell free systems have generally been
E. coli
S-30 extracts (Mattheakis 1994; Zubay 1973) for prokaryotes and rabbit reticulocyte lysates for eukaryotes. Transcription/translation coupled systems have also been reported (U.S. Pat. Nos. 5,492,817; 5,665,563; 5,324,637) involving prokaryotic cell free extracts (Mattheakis et al 1994) and eukaryotic cell free extracts (U.S. Pat. Nos. 5,492,817; 5,665,563) which have different requirements for effective transcription and translation. In addition, there are requirements for the correct folding of the translated proteins in the prokaryotic and eukaryotic systems. For prokaryotes, protein disulphide isomerase (PDI) and chaperones may be required. Generally in prokaryotes translated proteins are folded after release from the ribosome; however, for correct folding of the newly translated protein attached (tethered) to the ribosome a C terminal anchor may also be necessary. An anchor is a polypeptide spacer that links the newly translated protein domain (s) to the ribosome. The anchor may be a complete protein domain such as an immunoglobulin constant region. In complete contrast to this, in eukaryotic systems the protein is folded as it is synthesised and has no requirement for the addition of prokaryote PDI and chaperones. An anchor may however be beneficial in eukaryotic systems for spacing from, and correct folding of, the newly translated protein attached (tethered) to the ribosome.
Polypeptides synthesised de novo in cell-free coupled systems have been displayed on the surface of ribosomes, since for example in the absence of a stop codon the polypeptide is not released from the ribosome. The mRNA ribosome protein complex can be used for selection purposes. This system mimics the process of phage display and selection and is shown in FIG.
1
. Features required for optimal display on ribosomes have been described by Hanes and Pluckthun (1997). These features include removal of stop codons. However, removal of stop codons results in the addition of protease sensitive sites to the C terminus of the newly translated protein encoded by a ssrA tRNA-like structure. This can be prevented by the inclusion of antisense ssrA oligonucleotides (Keiler et al 1996).
RNA-directed RNA Polymerases
Q&bgr; bacteriophage is an RNA phage with an efficient replicase (RNA-dependent RNA polymerases are termed replicases or synthetases) for replicating the single-strand genome of coliphage Q&bgr;. Q&bgr; replicase is error-prone and introduces mutations into the RNA calculated in vivo at 10
3
-10
4
bases. The fidelity of Q&bgr; replicase is low and strongly biased to replicating its template (Rohde et al 1995). These teachings indicate that replication over a prolonged period leads to accumulation of mutated strands not suitable for synthesis of a desired protein. Both + and − strands serve as templates for replicase; however, for the viral genome the + strand is bound by Q&bgr; replicase and used as the template for the complementary strand (−). In order for RNA replication to occur the replicase requires specific RNA sequence/structural elements which have been well defined (Brown and Gold 1995; Brown and Gold 1996). A reaction containing 0.14 femtograms of recombinant RNA produces 129 nanograms in 30 mins (Lizardi et al 1988).
RNA-directed RNA polymerases are known to replicate RNA exponentially on compatible templates. Compatible templates are RNA molecules with secondary structure such as that seen in MDV-1 RNA (Nishihara, T., et al 1983). In this regard, a vector has been described for constructing amplifiable mRNAs as it possesses the sequences and secondary structure (MDV-1 RNA) required for replication and is replicated in vitro in the same manner as Q&bgr; genomic RNA. The MDV-1RNA sequence (a naturally occurring template for Q&bgr; replicase) is one of a number of natural templates compatible with amplification of RNA by Q&bgr; replicase (U.S. Pat. No. 4,786,600); it possesses RNA-like structures at its terminus which are similar to structures that occur at th

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