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WO1998049286A2 - Evolution orientee d'enzymes et d'anticorps - Google Patents

Evolution orientee d'enzymes et d'anticorps Download PDF

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Publication number
WO1998049286A2
WO1998049286A2 PCT/US1998/008714 US9808714W WO9849286A2 WO 1998049286 A2 WO1998049286 A2 WO 1998049286A2 US 9808714 W US9808714 W US 9808714W WO 9849286 A2 WO9849286 A2 WO 9849286A2
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Prior art keywords
cells
antibody
cell
seq
host cell
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PCT/US1998/008714
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English (en)
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WO1998049286A3 (fr
WO1998049286A9 (fr
Inventor
Brent Iverson
George Georgiou
Gang Chen
Mark J. Olsen
Patrick S. Daugherty
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Board Of Regents, The University Of Texas System
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Priority to AU71710/98A priority Critical patent/AU7171098A/en
Publication of WO1998049286A2 publication Critical patent/WO1998049286A2/fr
Publication of WO1998049286A3 publication Critical patent/WO1998049286A3/fr
Publication of WO1998049286A9 publication Critical patent/WO1998049286A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins

Definitions

  • the present invention relates generally to the fields of biochemistry, immunology and molecular biology. More particularly, it concerns the use of rapid selection techniques to identify specific polypeptides having desirable characteristics out of large libraries of polypeptides.
  • Antibodies also are of increasing importance in human therapy, assay procedures and diagnostic methods. Therefore, another area of interest lies in the identification of antibodies with particular binding functions, as well as other activities.
  • methods of identifying antibodies and production of antibodies is often expensive, particularly where monoclonal antibodies are required.
  • Hybridoma technology has traditionally been employed to produce monoclonal antibodies, but these methods are time-consuming and result in isolation and production of limited numbers of specific antibodies.
  • relatively small amounts of antibody are produced; consequently, hybridoma methods have not been developed for a large number of antibodies. This is unfortunate as the potential repertoire of immunoglobulins produced in an immunized animal is quite high, on the order of >10 , yet hybridoma technology is too complicated and time consuming to adequately screen and develop large number of useful antibodies.
  • ⁇ -lactamase mutant genes were transformed into E. coli and cells capable of growing in the presence of ⁇ -lactam antibiotics that are normally poor ⁇ -lactamase substrates were isolated (Venkatachalam et al., 1994; Petrosino and Palzkill, 1996).
  • a variety of techniques including chemical mutagenesis of isolated DNA, gene amplification by error prone PCRTM and oligonucleotide mutagenesis have been employed to generate libraries of mutant genes containing a desired range of nucleotide substitutions. Often multiple rounds of selection and mutagenesis are employed to select increasingly improved enzymes.
  • libraries of mutants have to be screened by direct assay. This involves growing individual clones either as colonies on agar plates or, alternatively in 96-well plates, and measuring enzymatic activity usually by a chromogenic assay.
  • a popular and convenient screening format is to determine the enzymatic activity of single colonies growing on agar plates. Sequential cycles of random mutagenesis and plate screening are increasingly employed for the directed evolution of enzyme activities. Using this approach Moore and Arnold (1996) isolated a mutant paranitrobenzyl esterase that exhibits 16-fold higher activity in 30% DMF relative to the parent enzyme. At least one other enzyme has been engineered successfully by random mutagenesis and screening (Yu and Arnold, 1996).
  • the present invention addresses these and other drawbacks inherent in the prior art by providing new methods of screening of polypeptide libraries. For the first time it is possible to rapidly screen polypeptide libraries for potential enzymes and antibodies; often in a matter of hours.
  • the disclosed methods allow production of large quantities of these polypeptides, potentially on a kilogram scale, from microorganism cultures. And, because selected proteins can be displayed on the surface of a host cell, assays can be conducted with remarkable rapidity.
  • expression libraries are prepared such that an expressed protein is displayed on the surface of a cell.
  • the polypeptides will be surface expressed in a host cell such as bacterial, yeast, insect, eukaryotic or mammalian cells. Surface expression of a polypeptide on a cell surface is achieved using a recombinant vector that promotes display on the outer membrane of a host cell.
  • Vectors are such as those of the general construction described in U.S. Patent No. 5,348,867, incorporated herein by reference.
  • the vectors will be appropriate for a bacterial host cell and will include at least three DNA segments as part of a chimeric gene.
  • One segment is a DNA sequence encoding a polypeptide that targets and anchors a fusion polypeptide to a host cell outer membrane.
  • a second DNA segment encodes a membrane-transversing amino acid sequence, i.e., a polypeptide that transports a heterologous or homologous polypeptide through the host cell outer membrane.
  • the third DNA segment encodes any of a number of desired polypeptides. Such vectors will display fusion polypeptides at the exterior of a host cell. These recombinant vectors include a functional promoter sequence.
  • Screening for antibodies employing the methods of the present invention allows one to select an antibody or antibody fragment from a plurality of candidate antibodies that have been expressed on the surface of a host cell.
  • the host cell will be a bacterial cell, preferably E. coli.
  • the antibodies are obtained from an expression vector library that may be prepared from DNAs encoding antibodies or antibody fragments.
  • One source of such DNAs could be from an animal immunized with a selected antigen; alternatively, antibody genes from other sources can be used, such as those produced by hybridomas or produced by mutagenesis of a known antibody gene.
  • One preferred method of obtaining DNA segments is to isolate mRNA from antibody cells of an immunized animal.
  • the mRNA may be amplified, for example by PCR, and used to prepare DNA segments to include in the vectors.
  • One may also employ DNA segments that have been mutagenized from one or more DNAs that encode a selected antibody or antibody fragment.
  • the present invention provides methods for the rapid screening of enzyme libraries.
  • the libraries represent mutagenized version of an enzyme to permit for the "directed" evolution of the enzyme's sequence, and hence function.
  • vectors will comprise a DNA sequence encoding the enzyme, an anchor fused to the enzyme coding region that results in expression of the host cell outer membrane and any other regulatory sequences necessary for the propagation of the vectors and the expression of the enzyme.
  • Standard mutagenic procedures will be applied to various regions of the enzyme coding region, including those regions that encode binding pockets and active sites. Other sites of interest, including residues critical for conformation and post-translational modification also may be targeted.
  • expression libraries are transferred, by standard methodologies, into appropriate host cells.
  • Expression of the antibodies, enzymes or other polypeptides on the surface of host cells permits the rapid and efficient screening of libraries for the appropriate binding specificity, enzyme function or other desirable characteristic.
  • use of appropriate label systems and substrates permits the sorting of host cells expressing proteins of interest by flow cytometery methodology (e.g., FACS).
  • FIG. 1A Western blot analysis of total membrane fractions from E. coli JM109 cells containing pTXIOl (Lanes 2 and 4); JM109/pTX152 (Lanes 3 and 5) and probed with anti-OmpA antibodies at 1 :5000 dilution; Lanes 4 and 5 were probed with monoclonal anti-HSV antibodies at 1 :5000 dilution.
  • FIG. IB Lysate and whole cell ⁇ LISAs of JM109 cells containing plasmid pTXIOl (solid) or pTX152 (hatched). Samples were incubated on microtiter wells coated with digoxin-conjugated BSA and probed with anti- ⁇ -lactamase (pTXIOl) or anti-HSV (pTX152) antibodies. Absorbance readings were referenced to wells that were untreated with either lysates or whole cells.
  • FIG. 2 A and FIG. 2B Phase contrast micrograph of JM109/pTX152 cells after 1 hr of ⁇ incubation with 10 " M digoxin-FITC.
  • FIG. 2B Micrographs of the same field as in FIG. 2A of JM109/pTX152 cells after a 1 hour incubation with 10 "7 M digoxin-FITC.
  • FIGS. 3A-3G Histogram data from FACS.
  • the bar in each graph represents the sorting gate or the fluorescence intensity defined as a positive event.
  • the sorting gate was chosen to maximize the number of positive events while minimizing the number of negative events within the window. All samples were labeled with 10 " M digoxin FITC.
  • FIG. 3 A JM109/pTXl 52 sample used as a negative control.
  • FIG. 3B JM109/pTX 152 sample used as a positive control.
  • FIG. 3C JM109/pTX152 pretreated with 0.2 mg/ml trypsin.
  • FIG. 3D JM109/pTX152 pretreated with free digoxin.
  • FIG. 3 ⁇ JM109/pTX152
  • FIG. 3F A 100,000:1 mixture after growing cells recovered from the first cell sorting run.
  • FIG. 3G A 100,000:1 mixture after growing cells recovered from the second cell sorting run.
  • FIG. 4 Whole cell immunoassay using 0.5 nM FITC labeled digoxin.
  • FIG. 5A and 5B FIG. 5A. Antibody mutants displaying different affinity for the antigen can be distinguished by display on the cell surface and fluorescence activated cell sorting. A. Fluorescence histogram comparing the fluorescence distribution of bacterial cells displaying mutants of the svFv (digoxin) antibody on their surface. FIG. 5B. Relative binding affinity of the corresponding purified antibodies measured by ELISA.
  • FIG. 6A and 6B Immunoassay for the determination of the equilibrium constant for antigen binding for surface displayed scFv antibodies.
  • Cells displaying scFv(digoxin) antibodies on their surface were incubated with different concentrations of BODIPY-digoxin for one hour with gentle shaking. Following incubation, the fluorescence distribution of the cells (50,000 events) was determined by flow cytometry.
  • FIG. 7A and FIG. 7B Examples of single chain antibody surface display plasmid vector pSD 192.
  • FIG. 8 Procedure for the isolation of single-chain antibodies by surface display and FACS.
  • FIG. 9A-9F Isolation of high affinity antibodies from libraries displayed on the bacterial cell surface and screened by FACS.
  • FIG. 9A-9C Plots of Forward Scatter (a measure of the cell size) as a function of fluorescence intensity for cell populations displaying a library of scFv antibodies and incubated with different concentrations of fluorescent hapten (BODIPY-digoxin).
  • the library was constructed by randomizing the heavy chain residues 99, 100, 100a and 100b as described in Example 7. Cells were incubated with Bodipy-digoxin at different concentrations, shown in FIG. 9A-9C, for one hour. Each point represents one event detected by the flow cytometer and a total of 50,000 events (i.e. cells) are shown for clarity.
  • FIG. 9A-9F Isolation of high affinity antibodies from libraries displayed on the bacterial cell surface and screened by FACS.
  • FIG. 9A-9C Plots of Forward Scatter (a measure of the cell size) as a function of fluorescence intensity
  • FIG. 9D-9F Isolation of scFv antibodies from a library.
  • the library was constructed by randomizing the heavy chain residues 99, 100, 100a and 100b as described in Example 7. Cells were incubated with 70 nM BODIPY-digoxin and high fluorescence clones were isolated by FACS. The corresponding fluorescence histogram is shown in FIG. 9D. Sorted cells were grown overnight, incubated with 15 nM BODIPY-digoxin and sorted. The fluorescence distribution of the cells is shown in FIG. 9E. As can be seen, after one round of sorting and growth, cells having high fluorescence are greatly enriched over the starting cell population. Finally, FIG. 9F shows the fluorescence distribution of the cell population obtained after growing the positive cells isolated in the first round. The large majority of the cells bind the BODIPY-digoxin conjugate and thus have a high fluorescence.
  • FIG. 10 Chemical Structure of the OmpT substrate
  • FIG. 11A-11C show a fluorescence histogram of 20,000 cells from a mixture of OmpT + and OmpT " at a ratio of 1 :5,000. After sorting, 32 cells were collected and the fluorescence of nine clones was examined by FACS. FIG. 11B and FIG. 1 IC show representative fluorescence histograms for two of the isolated OmpT+ clones.
  • FIG. 12 Frequency of rare high-fluorescence cells detected in a heavy-chain CDR3 library per 100,000 cells using flow cytometry. Cells were considered to be target cells if they possessed FSC and FLI signals falling into the high-fluorescence region (RI) in FIG. 13.
  • the background control cell population consisted of a pool of 20 random clones characterized by DNA sequencing and flow cytometry, having Kr> values above 100 nM.
  • FIG. 13A, FIG. 13B and FIG. 13C Enrichment of highly fluorescent bacteria using FACS.
  • the heavy-chain CDR3 library cell population was labeled with BODIPY-digoxin and analyzed by flow cytometry.
  • FIG. 13 A Presort (15 nM BODIPY-digoxin);
  • FIG. 13B post- round 1 (10 nM BODIPY-digoxin);
  • FIG. 13C post-round 2 (10 nM BODIPY-digoxin).
  • FIG. 14 The decay in mean fluorescence as BODIPY-digoxin dissociates from whole cells displaying mutant scFv selected from a heavy-chain CDR3 library. At least 4,000 events were acquired at 1 -2 min time intervals and the mean fluorescence is plotted as an exponential decay.
  • FIG. 15 The decay in mean fluorescence as BODIPY-digoxin dissociates from whole cells displaying mutant scFv selected from a light-chain CDR3 library. Selection conditions are given DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • catalytic antibodies are produced via immunization with a molecule (a hapten) that is designed to mimic certain features of the transition state complex for the reaction of interest (Lerner et al, 1991 ; U.S. Patent 5,658,753, incorporated herein by reference, provides additional disclosure regarding catalytic antibodies).
  • catalytic antibodies display precise substrate selectivity. That is, only substrates that are similar in structure to the hapten used to elicit the catalytic antibodies are accepted in the catalytic reaction.
  • catalytic antibodies have not yet fulfilled initial expectations. A number of reasons are at the root of the slow progression. First, the generation of catalytic antibodies is technically difficult and prohibitively expensive. Second, production costs are uneconomical.
  • rates of reaction and acceleration with catalytic antibodies are between 10 -10 3 , although a few examples of higher rates have been reported (Janda et al, 1988; however also see Hollfelder et al, 1996).
  • transition state mimics must be immunogenic and stable in vivo.
  • Animal immunization with the transition state analog is the first step in the production of monoclonal antibodies with catalytic activity.
  • the need for immunization poses two serious constraints on the transition state analog; it must be recognized by the immune response and it must be stable in the animal.
  • polypeptides are displayed on the surface of filamentous bacteriophage (Smith, 1991).
  • the polypeptides are expressed as fusions to the N-terminus of a coat protein.
  • the fusion proteins are incorporated in the viral coat so that the polypeptides become displayed on the bacteriophage surface.
  • Each polypeptide produced is displayed on the surface of one or more of the bacteriophage particles and subsequently tested for specific ligand interactions. While this approach appears attractive, there are numerous problems, including difficulties of enriching positive clones from phage libraries. Enrichment procedures are based on selective binding and elution onto a solid surface such as an immobilized receptor. Unfortunately.
  • Ladner et al (U.S Patent No 5,403,484, specifically incorporated herein by reference) reported the display of proteins on the outer surface of a chosen bacterial cell, spore or phage, in order to identify and characterize binding proteins. Certain elements of Ladner may be used advantageously, for example, methods of generating and expressing single chain antibodies, proteinaceous binding domains other than a single chain antibody, carrier protein and the like, in combination with the present invention.
  • the methods disclosed herein are particularly advantageous because they allow unprecedented rapid and efficient selection, purification and screening of polypeptide libraries from bacterial host cell surfaces, providing several advantages over phage libraries. Unlike most other methods used for screening and assay, the disclosed methods are well-suited for commercial adaptation. Assay procedures are greatly facilitated because of the cell surface display aspect, permitting the use of simple centrifugation to remove the cells from an assay sample. Assays thus are very rapid and inexpensive as they do not require complex or expensive equipment.
  • the invention comprises the following approach.
  • a target enzyme (or catalytic antibody) which is to be subjected to mutagenesis is first displayed on the surface of E. coli bacteria as a fusion to a surface-targeting vehicle.
  • This technology was developed by Georgiou and coworkers and has been used to display a number of proteins on the bacterial surface (Francisco et al, 1992; 1993a; 1993b; Georgiou et al, 1996).
  • the target enzyme By displaying the target enzyme on the bacterial surface it will be fully accessible to molecules in the extracellular fluid.
  • the enzyme is free to react with any substrate added to the cells without any of the limitations that are imposed when intracellular enzymes are studied.
  • the desired polypeptide is fused to an amino acid sequence that includes the signals for localization to the outer membrane and for translocation across the outer membrane.
  • the amino acid sequences responsible for localization and for translocation across the outer membrane may be derived either from the same bacterial protein or from different proteins of the same or different bacterial species. Examples of proteins that may serve as sources of localization signal domains are shown in Table 1. TABLE 1 - EXAMPLES OF OUTER MEMBRANE TARGETING SEQUENCES
  • OsmB E. coli (or functional equivalent in Salmonella)
  • NlpB E. coli (or functional equivalent in Salmonella)
  • E. coli Lpp-OmpA C-terminal fusion cell surface scFv antibodies E. coli Lpp-OmpA C-terminal fusion cell surface scFv antibodies; ⁇ - peptide/antibody libraries, lactamase; protein A; cellular adsorbents, cellulose binding protein immunoassays
  • Salmonella sandwich fusion cell surface 18 aa epitope from HIV1 vaccines Salmonella sandwich fusion cell surface 18 aa epitope from HIV1 vaccines
  • E. coli FimH (Type sandwich fusion cell surface 52 aa sequence from the vaccines
  • Other groups have used flagellum or pilus subunits to develop expression systems for the surface presentation of antigenic/immunogenic epitopes derived from pathogens, suitable for the development of live recombinant vaccines (Newton et al, 1995; Pallesen et ⁇ /., 1995; Van Die et al, 1990).
  • lipoprotein fusions have been found to be either detrimental to the integrity of the cell envelope causing extensive cell lysis, or to be tethered to the interior face of the outer membrane, in which case they are not exposed to the extracellular fluid (Laukkanen et al, 1993;Cornellis et al, 1996).
  • Lpp-OmpA hybrid display vehicle consisting of the N-terminal outer membrane localization signal from the major lipoprotein (Lpp) fused to a domain from the outer membrane protein OmpA (Franscisco et al, 1992) .
  • OmpA mediates the display of passenger proteins fused to the C-terminal of the Lpp- OmpA hybrid.
  • Lpp-OmpA fusions have been used to successfully display on the surface of E. coli several proteins varying in size between 20 and 54 kDa (Stathopoulos et al, 1996). Among the proteins that have been tested thus far only the dimeric bacterial enzyme alkaline phosphatase (phoA) could not be displayed on the cell surface (Stathopoulos et al, 1996).
  • the IgA proteases of Neisseria gonorrhoeae and Hemophilus influenzae use a variation of the most common, Type II secretion pathway (Salmong et al, 1993), to achieve extracellular export independent of any other gene products (Klauser et al, 1993).
  • the C- terminal domain of the IgA protease forms a channel in the outer membrane that mediates the export of the N-terminal domain across the membrane which in turn becomes transiently displayed on the external surface of the bacteria. This export mechanism is used by a number of extracellular proteins from pathogenic bacteria (Klauser et al, 1993; Jose et al, 1995; Suzuki et al, 1995; St. Geme et al, 1994).
  • the lipoprotein pullulanase (PulA) of Klebsiella pneumoniae which is also exported via a type Ii secretion mechanism, requires 14 genes for its translocation across the outer membrane (Salmong et al, 1993).
  • Pugsley and coworkers have shown that the lipoprotein pullulanase (PulA) can facilitate translocation of the periplasmic enzyme ⁇ - lactamase across the outer membrane.
  • pullulanase hybrids remain only temporarily attached to the bacterial surface and are subsequently released into the medium (Kornacker et al, 1990). Although the lack of permanent association with the cell wall is not detrimental for vaccine development, it is a serious limitation in other applications such as library screening.
  • Protein display applications has also spurred the development of suitable expression systems for yeast cells.
  • Surface display expression systems for yeast have relied primarily on the fusion of passenger proteins to agglutinin, a protein involved in cell adhesion (Schreuder et al, 1993; Schreuder et al, 1996; Schreuder et al, 1996; Boder and Wittrup et al 1997).
  • the AG ⁇ l agglutinin is tightly bound to the cell wall through its C-terminus. N-terminal fusions to the cell wall domain of AGAl are stably anchored on the cell surface. This system has been used for the surface expression of a variety of enzymes and binding proteins (Schreuder et al, 1996).
  • Mating-type a cells use the two subunit agglutinin a for cell adhesion.
  • the second subunit of agglutinin a (Aga2p) was used as a vehicle for the surface display of antibodies and peptides (Boder and Wittrup et al, 1997).
  • the passenger polypeptide is fused to the C-terminus of AGA2 which, in turn, is linked to the AGAl via disulfide bonds.
  • the gene encoding the target enzyme is mutagenized by conventional techniques to generate a library of mutants.
  • the library of mutants will be screened using a highly sensitive single cell assay. Cells exhibiting the desired activity will be isolated. This will involve the following: (i) design of a fluorescent substrate for the desired reaction; and may involve immobilization of the cells onto micron-size particles; and (ii) screening and isolation of fluorescent microparticles either by fluorescent activated cell sorting or by using a micromanipulator.
  • the present invention relies on the same cell surface display of polypeptides as described above, except that the polypeptide to be expressed on the host cell surface is an antibody.
  • the screening methods are practiced by first constructing an antibody library, using any of several well-known techniques for library construction. For example, after selecting an immunogen, one may immunize a mammal by conventional means and collect antiserum. mRNA from spleen may be used as template for PCR amplification; for example employing primers complementary to constant and variable domain framework regions of different antibody subclasses. Alternatively, DNA from polyclonal populations of antibodies may be amplified, fragmented if desired, ligated into pTXIOl or a similar vector as described in U.S. Patent 5,348,867, incorporated herein by reference, and transformed into a host cell.
  • the present invention involves the display of polypeptides on the surface of a bacteria.
  • polypeptide refers to any protein and includes antibodies, antibody fragments, receptors, enzymes, cytokines, transcription factors, clotting factors, chelating agents and hormones. Genes for polypeptides of interest are fused to the 3' of a sequence that encodes a cell surface targeting domain.
  • the cell envelope of E. coli and other gram-negative bacteria consists of the inner membrane (cytoplasmic membrane), the peptidoglycan cell wall and the outer membrane.
  • the surface targeting domain includes the first nine amino acids of Lpp, the major lipoprotein of E. coli fused to amino acid 46-159 of the Outer Membrane Protein A (OmpA).
  • OmpA Outer Membrane Protein A
  • the function of the former is to direct the chimera to the outer membrane whereas the OmpA sequence mediates the display of proteins at the C- terminal of OmpA.
  • Lpp-OmpA(46-159) fusions have been used to anchor a variety of proteins such as ⁇ -lactamase, a cellulose binding protein and alkaline phosphatase on the E. coli surface.
  • other analogous surface targeting domains may be employed to stably anchor the recombinant polypeptides on the cell surface.
  • Exemplary of the surface expression method is an Lpp-OmpA (46-159)-antibody fusion expressed in a gram-negative bacterium. Due to the presence of the Lpp-OmpA(46-159) sequence, the fusion is localized on the outer membrane such that the N-terminal domain is embedded in the bilayer and the antibody sequence is fully exposed on the cell surface. Recombinant antibodies expressed on the cell surface as Lpp-OmpA(46-159) fusions are functional and bind to antigens with high affinity. Such fusions, manipulated as described below, will constitute preferred forms of the expression libraries of the present invention.
  • Mutagenesis will be accomplished by a variety of standard, random mutagenic procedures. Mutation is the process whereby changes occur in the quantity or structure of an organism. Mutation can involve modification of the nucleotide sequence of a single gene, blocks of genes or whole chromosome. Changes in single genes may be the consequence of point mutations which involve the removal addition or substitution of a single nucleotide base within a DNA sequence, or they may be the consequence of changes involving the insertion or deletion of large numbers of nucleotides.
  • Mutations can arise spontaneously as a result of events such as errors in the fidelity of DNA replication or the movement of transposable genetic elements (transposons) within the genome. They are also induced following exposure to chemical or physical mutagens.
  • mutation-inducing agents include ionizing radiations, ultraviolet light and a diverse array of chemical such as alkylating agents and polycyclic aromatic hydrocarbons all of which are capable of interacting either directly or indirectly (generally following some metabolic biotransformations) with nucleic acids.
  • the DNA lesions induced by such environmental agents may lead to modifications of bas e sequence when the affected DNA is replicated or repaired and thus to a mutation.
  • Insertional mutagenesis is based on the inactivation of a gene via insertion of a known DNA fragment. Because it involves the insertion of some type of DNA fragment, the mutations generated are generally loss-of-function rather than gain-of-function mutations. However, there are several examples of insertions generating gain-of-function mutations (Oppenheimer et al. 1991). Insertion mutagenesis has been very successful in bacteria and Drosophila (Cooley et al. 1988) and recently has become a powerful tool in several plant species (corn; e.g., Schmidt et al 1987); Arabidopsis; e.g., Marks et al, 1991 ; Koncz et al. 1990; Antirrhinum: e.g., Sommer et ⁇ /. 1990).
  • Transposable genetic elements are DNA sequences that can move (transpose) from one place to another in the genome of a cell.
  • the first transposable elements to be recognized were the Activator/Dissociation elements of Zea mays (McClintock, 1957). Since then they have been identified in a wide range of organisms, both prokaryotic and eukaryotic.
  • Transposable elements in the genome are characterized by being flanked by direct repeats of a short sequence of DNA that has been duplicated during transposition and is called a target site duplication. Virtually all transposable elements whatever their type, and mechanism of transposition, make such duplications at the site of their insertion. In some cases the number of bases duplicated is constant , in other cases it may vary with each transposition event. Most transposable elements have inverted repeat sequences at their termini, these terminal inverted repeats may be anything from a few bases to a few hundred bases long and in many cases they are known to be necessary for transposition.
  • Prokaryotic transposable elements have been most studied in E. coli and Gram negative bacteria but are also present in Gram positive bacteria. They are generally termed insertion sequences if they are less than about 2 kilobases long or transposons if they are longer. Bacteriophages such as mu and D108 which replicate by transposition make up a third type of transposable element, elements of each type encode at least one polypeptide a transposase, required for their own transposition. Transposons further often include genes coding for function unrelated to transposition and often carry antibiotic resistance genes.
  • Transposons can be divided into two classes according to their structure. Firstly, compound or composite transposons have copies of an insertion sequence element at each end usually in an inverted orientation. These transposons require transposases to be encoded by one of their terminal IS elements. The second class of transposon have terminal repeats of about 30 base pairs and do not contain sequences from IS elements.
  • Transposition is usually either conservative or replicative although in some cases it can be both. In replicative transposition one copy of the transposing element remains at the donor site and another is inserted at the target site. Conservative transposition the transposing element is excised from one site and inserted at another.
  • ⁇ ukaryotic elements can also be classified according to their structure and mechanism of transportation. The primary distinction is between elements that transpose via an RNA intermediate and elements that transpose directly from DNA to DNA. Elements that transpose via an RNA intermediate are often referred to as retrotransposons and their most characteristic feature is that they encode polypeptides that are believed to have reverse transcriptionase activity. There are two types of retrotransposon: some resemble the integrated proviral DNA of a retrovirus in that they have long direct repeat sequences, long terminal repeats (LTRs), at each end. The similarity between these retrotransposons and proviruses extends to their coding capacity.
  • LTRs long terminal repeats
  • Retrotransposons of the second type have no terminal repeats. They also code for gag- and ⁇ /-like polypeptides and transpose by reverse transcription of RNA intermediates but do so by a mechanism that differs from that or retrovirus-like elements. Transposition by reverse transcription is a replicative process and does not require excision of an element from a donor site.
  • Transposable elements are an important source of spontaneous mutations and must have influenced the ways in which genes and genomes have evolved. They can inactivate genes by inserting within them and can cause gross chromosomal rearrangements either directly through the activity of their transposases or indirectly as a result of recombination between copies of an element scattered around the genome. Transposable elements that excise often do so imprecisely and may produce alleles coding for altered gene products if the number of bases added or deleted is a multiple of three.
  • Transposable elements themselves may evolve in unusual ways. If they were inherited like other DNA sequences then copies of an element in one species would be more like copies in closely related species than copies in more distant species. This is not always the case, suggesting that transposable elements are occasionally transmitted horizontally from one species to another.
  • Chemical mutagenesis offers certain advantages, the ability to find a full range of mutant alleles with degrees of phenotypic severity, and is facile and inexpensive to perform.
  • 2-acetyl aminofluorene and aflotoxin Bl cause GC to TA transversions in bacteria and mammalian cells .
  • Benzo[a]pyrene also can produce base substitutions such as AT to TA.
  • N- nitroso compounds produce GC to AT transitions. Alkylation of the 04 position of thymine induced by exposure to n-nitrosoureas results in TA to CG transitions.
  • a high correlation between mutagenicity and carcinogenity is the underlying assumption behind the Ames test (McCann et al, 1975 incorporated herein by reference) which speedily assays for mutants in a bacterial system, together with an added rat liver homogenate , which contains the microsomal cytochrome P450, to provide the metabolic activation of the mutagens where needed.
  • N-nitroso-N-methyl urea induces mammary, prostate and other carcinomas in rats with the majority of the tumors showing a G to A transition at the second position in codon 12 of the Ha-ras oncogene.
  • Benzo[a]pyrene-induced skin tumors contain A to T transformation in the second codon of the Ha-ras gene.
  • Ionizing radiation causes DNA damage and cell killing, generally proportional to the dose rate. Ionizing radiation has been postulated to induce multiple biological effects by direct interaction with DNA or through the formation of free radical species leading to DNA damage (Hall, 1988). These effects include gene mutations, malignant transformation, and cell killing. Although ionizing radiation has been demonstrated to induce expression of certain DNA repair genes in some prokaryotic and lower eukaryotic cells, little is known about the effects of ionizing radiation on the regulation of mammalian gene expression (Borek, 1985). Several studies have described changes in the pattern of protein synthesis observed after irradiation of mammalian cells.
  • ionizing radiation treatment of human malignant melanoma cells is associated with induction of several unidentified proteins (Boothman, et al, 1989).
  • Synthesis of cyclin and co-regulated polypeptides is suppressed by ionizing radiation in rat REF52 cells but not in oncogene-transformed REF52 cell lines (Lambert and Borek, 1988).
  • Other studies have demonstrated that certain growth factors or cytokines may be involved in x- ray-induced DNA damage.
  • platelet-derived growth factor is released from endothelial cells after irradiation (Witte, et al, 1989).
  • the term "ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons).
  • An exemplary and preferred ionizing radiation is an x-radiation.
  • the amount of ionizing radiation needed in a given cell generally depends upon the nature of that cell. Typically, an effective expression-inducing dose is less than a dose of ionizing radiation that causes cell damage or death directly. Means for determining an effective amount of radiation are well known in the art.
  • an effective expression inducing amount is from about 2 to about 30 Gray (Gy) administered at a rate of from about 0.5 to about 2 Gy/minute. Even more preferably, an effective expression inducing amount of ionizing radiation is from about 5 to about 15 Gy. In other embodiments, doses of 2-9 Gy are used in single doses. An effective dose of ionizing radiation may be from 10 to 100 Gy, with 15 to 75 Gy being preferred, and 20 to 50 Gy being more preferred.
  • radiation may be delivered by first providing a radiolabeled antibody that immunoreacts with an antigen of the tumor, followed by delivering an effective amount of the radiolabeled antibody to the tumor.
  • radioisotopes may be used to deliver ionizing radiation to a tissue or cell.
  • Random and In Vitro Scanning Mutagenesis Random and In Vitro Scanning Mutagenesis Random mutagenesis may also be introduced using error prone PCR (Cadwell and Joyce, 1992). The rate of mutagenesis may be increased by performing PCR in multiple tubes with dilutions of templates.
  • Structure-guided site-specific mutagenesis represents a powerful tool for the dissection and engineering of protein-ligand interactions (Wells, 1996, Braisted et al, 1996).
  • One particularly useful mutagenesis technique is alanine scanning mutagenesis in which a number of residues are substituted individually with the amino acid alanine so that the effects of losing side-chain interactions can be determined, while minimizing the risk of large-scale perturbations in protein conformation (Cunningham et al, 1989).
  • mutant protein For each mutant protein, the appropriate gene construct must be made, the DNA must be transformed into a host organism, transformants need to be selected and screened for expression of the protein, the cells have to be grown to produce the protein, and finally the recombinant mutant protein must be isolated.
  • transformants There have been only a handful of studies where one, or at most a few, residues in an antibody have been subjected to saturation mutagenesis. Even in those studies, only some of the mutants were examined in detail (Chen et al, 1996, Brumell et al, 1994).
  • In vitro scanning saturation mutagenesis provides a rapid method for obtaining a large amount of structure-function information including: (i) identification of residues that modulate ligand binding specificity, (ii) a better understanding of ligand binding based on the identification of those amino acids that retain activity and those that abolish activity at a given location, (iii) an evaluation of the overall plasticity of an active site or protein subdomain, (iv) identification of amino acid substitutions that result in increased binding.
  • the surface-displayed antibodies molecules may further be presented as fusion proteins that include reporter molecules, e.g. alkaline phosphatase, luciferase, ⁇ -lactamase, green fluorescent protein and others.
  • reporter molecules e.g. alkaline phosphatase, luciferase, ⁇ -lactamase, green fluorescent protein and others.
  • vectors for enzyme expression require that appropriate signals be provided for the synthesis of mRNA and polypeptides, and include various regulatory elements such as enhancers/promoters from both bacterial and eukaryotic systems that drive expression of the enzymes of interest in the appropriate host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
  • the term "expression construct” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • the nucleic acid encoding a gene product is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (t£) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • prokaryotic gene expression unlike eukaryotic systems the DNA is not separated from the cytoplasm by a nuclear membrane. There are also many other differences in mRNA processing of prokaryotes and eukaryotes.
  • the first control point of prokaryotic gene expression is initiation of transcription. Transcription is initiated at positions defined precisely by promoters. Analysis of more than 100 promoters in E. coli has identified two consensus sequences positioned 350 and 10 base pairs upstream of the point in the DNA sequence where transcription begins. These sequences are involved in polymerase recognition and binding.
  • RNA polymerase is a multi-subunit enzyme; the core enzyme is capable of transcriptional elongation on its own but requires the addition of a further subunit ( ⁇ ) in order to bind specifically to promoter sites and initiate transcription.
  • ⁇ factors in prokaryotes allows the polymerase to be directed to different sets of promoters (Helmann and Chamberlain, 1988).
  • B. subtilis for example, several different ⁇ factors are produced at different stages so that different sets of genes can be turned on at each stage.
  • the rate of initiation varies by up to a factor of at least a 1000 over the whole range of promoters in E. coli.
  • the efficiency of initiation is related to the sequence and topology of the promoter region. Gene expression occurring in the absence of regulatory factors is known as constitutive. At many promoters, transcriptional initiation may be increase by binding to specific regulatory proteins.
  • a feature of gene organization common to prokaryotes but rare in eukaryotes is the grouping of functionally related genes into operons.
  • genes encoding, for example the different enzymes of a metabolic pathway are clustered and are transcribed together into a polycistronic transcript, under the control of a single promoter. This transcript is then translated to give individual proteins.
  • Operons enable the rapid and efficient coordinate expression of a set of genes required to respond to a change in the external or internal environment of the microorganism. Premature termination plays a part in the regulation of expression of operons.
  • the termination of transcription occurs at specific sites. There are two types of termination events. The first (factor independent termination) occurs at sites defined by a series of U residues preceded by an inverted repeat that forms a stem-loop structure at the 3' end of the RNA transcript. This structure interferes with the polymerase action and leads to the release of the RNA. Factor dependent termination is dependent on the interaction of protein factor rho p with the RNA polymerase.
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of direction the expression of the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a promoter By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.
  • Tables 2 and 3 list several elements/promoters which may be employed, in the context of the present invention, to regulate the expression of the gene of interest. This list is not intended to be exhaustive of all the possible elements involved in the promotion of gene expression but, merely, to be exemplary thereof.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.
  • a terminator is also contemplated as an element of the expression cassette. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • the cells contain nucleic acid constructs of the present invention, a cell may be identified in vitro or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
  • a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to ampicillin, tetracycline, neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • enzymes such as herpes simplex virus.
  • IRES internal ribosome binding sites
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picanovirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames.
  • each open reading frame can be transcribed together, each separated by an IRES, creating polycistronic messages.
  • IRES element By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter to transcribe a single message.
  • Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.
  • Antibody and Antibody Fragment Constructs Using the fusion technology described above, the present invention also contemplates the generation of host cells expressing, on their surface, antibodies or antibody fragments representing a library of antibodies produced in response to one or more immunogens.
  • Antibody or “antibody fragment” refers to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE or any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2 , single domain antibodies (DABs), Fv, scFv (single chain Fv) and the like.
  • DABs single domain antibodies
  • Fv single domain antibodies
  • scFv single chain Fv
  • the specificity of an antibody is determined by the complementarity determining regions (CDRs) within the light chain variable regions (V L ) and heavy chain variable regions (V H ).
  • CDRs complementarity determining regions
  • the F ab fragment of an antibody which is about one-third the size of a complete antibody contains the heavy and light chain variable regions, the complete light chain constant region and a portion of the heavy chain constant region.
  • F ab molecules are stable and associate well due to the contribution of the constant region sequences.
  • the yield of functional F ab expressed in bacterial systems is lower than that of the smaller F v fragment which contains only the variable regions of the heavy and light chains.
  • the F v fragment is the smallest portion of an antibody that still retains a functional antigen binding site.
  • the F v fragment has the same binding properties as the F ab , however without the stability conferred by the constant regions, the two chains of the F v can dissociate relatively easily in dilute conditions.
  • V H and V L regions may be fused via a polypeptide linker (Huston et al, 1991) to stabilize the antigen binding site.
  • This single polypeptide F v fragment is known as a single chain antibody (scF v ).
  • the V H and V L can be arranged with either domain first.
  • the linker joins the carboxy terminus of the first chain to the amino terminus of the second chain.
  • heavy or light chain F v or F ab fragments may also be used with this system.
  • a heavy or light chain can be displayed on the surface followed by the addition of the complementary chain to the solution. The two chains are then allowed to combine on the surface of the bacteria to form a functional antibody fragment. Addition of random non-specific light or heavy chain sequences allows for the production of a combinatorial system to generate a library of diverse members.
  • Antibody and Antibody Fragment Gene Isolation To accomplish construction of antibodies and antibody fragments, the encoding genes are isolated and then modified to permit cloning into the expression vector. Although methods can be used such as probing the DNA for V H and V from hybridoma cDNA (Maniatis et al. , 1982) or constructing a synthetic gene for V H and V L (Barbas et al, 1992), a convenient mode is to use template directed methods to amplify the antibody sequences. A diverse population of antibody genes can be amplified from a template sample by designing primers to the conserved sequences at the 3' and 5' ends of the variable region known as the framework or to the constant regions of the antibody (Iverson et al, 1989).
  • restriction sites can be placed to facilitate cloning into an expression vector.
  • the primers By directing the primers to these conserved regions, the diversity of the antibody population is maintained to allow for the construction of diverse libraries.
  • the specific species and class of antibody can be defined by the selection of the primer sequences as illustrated by the large number of sequences for all types of antibodies given in Kabat et al, 1987, hereby incorporated by reference.
  • Messenger RNA isolated from the spleen or peripheral blood of an animal can be used as the template for the amplification of an antibody library.
  • mRNA may be isolated from a population of monoclonal antibodies.
  • Messenger RNA from either source can be prepared by standard methods and used directly or for the preparation of a cDNA template.
  • Generation of mRNA for cloning antibody purposes is readily accomplished by following the well-known procedures for preparation and characterization of antibodies (see, e.g., Antibodies: A Laboratory Manual, 1988; incorporated herein by reference).
  • a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance and collecting antisera from that immunized animal.
  • an immunogenic composition in accordance and collecting antisera from that immunized animal.
  • a wide range of animal species can be used for the production of antisera.
  • the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, rabbits are usually preferred for production of polyclonal antibodies.
  • Immunogenic compositions often vary in immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Recognized means for conjugating a polypeptide to a carrier protein are well known and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimides and bis-diazotized benzidine.
  • the immunogenicity of a particular immunogen composition may be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
  • a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
  • the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, may also be given. The process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated, stored and the spleen harvested for the isolation of mRNA from the polyclonal response or the animal can be used to generate MAbs for the isolation of mRNA from a homogeneous antibody population.
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g. a small molecule hapten conjugated to a carrier, a purified or partially purified protein, polypeptide or peptide.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • Rodents such as mice and rats are frequently used animals; however, the use of rabbit, sheep frog cells is also possible.
  • the use of rats may provide certain advantages (Goding, pp. 60-61, 1986), but mice are preferred, particularly the BALB/c mouse as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol.
  • B cells B lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from blood samples. Spleen cells and blood cells are preferable, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 X 10 7 to 2 X 108 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, 1984).
  • the immunized animal is a mouse
  • rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
  • NS-1 myeloma cell line also termed P3-NS- 1-Ag4-1
  • P3-NS- 1-Ag4-1 Another preferred murine myeloma cell
  • Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20: 1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus have been described by Kohler & Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al, 1977).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate (Goding pp. 71-74, 1986).
  • Fusion procedures usually produce viable hybrids at low frequencies, about 1 X 10 " to 1 X 10 " .
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • Simple and rapid assays include radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas are serially diluted and cloned into individual antibody-producing cell lines from which clones can then be propagated indefinitely to provide MAbs.
  • the cell lines may be exploited for MAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • the mRNA can be isolated using techniques well known in the art and used as a template for amplification of the target sequence.
  • PCR polymerase chain reaction
  • the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides.
  • the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction products and the process is repeated.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of target amplified. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, may also be used as an amplification method.
  • a replicative sequence of RNA which has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence which can then be detected.
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • CPR cyclic probe reaction
  • a probe having a 3' and 5' sequences of non-specific DNA and middle sequence of specific RNA is hybridized to DNA which is present in a sample.
  • the reaction is treated with RNaseH, and the products of the probe identified as distinctive products which are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • PCT Application No. PCT/US89/01025 each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention.
  • modified primers are used in a PCRTM like, template and enzyme dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes is added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et al, 1989.
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer which has target specific sequences.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
  • the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6.
  • a polymerase such as T7 or SP6.
  • the RNAs are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al, PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence.
  • This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "race” and "one-sided PCR” (Frohman, 1990; O'Hara et al, 1989).
  • Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di- oligonucleotide, may also be used in the amplification step (Wu et al, 1989).
  • Amplification products may be analyzed by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (see, e.g., Maniatis et al. 1982). For example, one may use a 1% agarose gel stained with ethidium bromide and visualized under UV light. Alternatively, the amplification products may be integrally labeled with radio- or fluorometrically-labeled nucleotides. Gels can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, respectively.
  • Genes for antibody fragments also may be generated by semisynthetic methods known in the art (Barbas et al, 1992). Using the conserved regions of an antibody fragment as a framework, variable regions can be inserted in random combinations one or more at a time to alter the specificity of the antibody fragment and generate novel binding sites, especially in the generation of antibodies to antigens not conducive to immunization such as toxic or labile compounds.
  • a known antibody sequence may be varied by introducing mutations randomly or site specifically. This may be accomplished by methods well known in the art such as mutagenesis with mismatched primers or error-prone PCR (Innis, 1990).
  • any cell may be used as a host cell, but the ease with which bacterial cell are handled makes these a preferred embodiment, at least where eukaryotic modification events (glycosylation, etc.) are not necessary.
  • mammalian cells, plant cells and yeast cells all may be employed.
  • Preferred bacterial hosts include Gram negative bacterial cells, particularly E. coli, although Salmonella, Klebsiella, Erwinia, Pseudomonas aeruginosa, Haemophilus influenza, Rickettsia rickettsii, Neisseria gonorrhea, etc. also are suitable.
  • Bacterial cells in which polypeptides are expressed may be readily immobilized, thus allowing rapid recovery and efficient removal from the system.
  • One may use membranes, dipsticks or beads through a chemically promoted coupling reaction in addition to other well- known immobilization matrices. In this manner, the cells can be separated from the solution without the need for a centrifugation step.
  • Bacterial cultures may be supplied in forms that have an indefinite shelf life and yet can be readily prepared for use; for example as “stab cultures” or lyophilized preparations; the user may prepare large amounts in liquid culture as needed.
  • the reagent is thus renewable as compared with other polypeptide reagents that are "used up” and must be replaced continually.
  • Bacterial cultures may be prepared fresh without concern about shelf-life of reagents that must be stored until use.
  • Elimination of these negative cells in a presort library is a very important step for a successful sorting experiment.
  • a number of approaches may be used to eliminate negative cells.
  • the blue-white screening system has been widely used for library screening in molecular biology.
  • a ⁇ -galactosidase enzyme is used as a reporter protein (Maniatis, 1989), and a multiple cloning site (MCS) is located in the middle of the ⁇ - galactosidase gene.
  • MCS multiple cloning site
  • An insertion of DNA into the MCS may result in the interruption of the ⁇ - galactosidase gene.
  • these cells with the DNA insert will not be able to produce an active ⁇ -galactosidase, and consequently cannot convert the substrate (X-gal) into a colored product, while the cells without an insert can produce ⁇ -galactosidase and hydrolyze the substrate into the indigo colored product.
  • Positive selection systems The direct selection of an antibody with enhanced catalytic activity or altered specificity is possible when the reaction of interest can be coupled to the growth or survival of a cell, such as the release of an essential nutrient or cofactor.
  • many reactions of interest do not easily lend themselves to such a selection scheme.
  • prokaryote metabolism is very complex, and bacteria are usually capable of adapting to new metabolic pathways, resulting in a large number of false positives (Lesley, 1993).
  • a variety of positive selection vectors have been developed to prevent the existence of the cells carrying non-insert plasmids. All the strategies rely on the inactivation of either a lethal gene (O'Connor 1982, Henrich 1986, Kuhn 1986, Bernard 1994), a lethal site (Hagan 1982), a dominant function conferring cell sensitivity to metabolites (Dean 1981, Burns 1984, Pierce 1992, Gossen 1992) or a repressor of an antibiotic-resistance function (Nikolnikov 1984). Most of these strategies are not well adapted for general use due to their large size, the limited number of cloning sites, or the need of special host strain and special culture medium. Another problem of these systems is that they are not an expression system, and the inserted gene cannot be expressed unless it carries a complete promoter and operator sequence.
  • the inventors have employed new positive ("dead man") selection systems in the present invention using two approaches to eliminate negative cells.
  • the target gene is fused with part of an antibiotic gene and used as an insert. Without the insert, the cloning vector does not process the specific antibiotic resistance, and the cells having the self- ligated vectors are eliminated by antibiotic selection.
  • Two different plasmids were constructed with two antibiotic markers, penicillin resistance and chloramphenicol resistance, as well as the surface expression Lpp-OmpA machinery (same as pTX152 which can express the scFv on the surface of E. coli).
  • a plasmid was constructed to eliminate cells without surface- expressed scFv.
  • a restriction site was introduced in the middle of the chloramphenicol acetyl transferase (CAT) gene and in the middle of scFv gene.
  • the N-terminal part of CAT (200bps) was fused to the end of scFv gene by overlapping PCR, while the cloning vector carried the C- terminal part of antibiotic gene.
  • the cells that contain a plasmid without the appropriate insert for example, antibody gene and N-terminal part of CAT
  • a plasmid is constructed to eliminate cells that carry a advertition stop codon in the middle of the scFv gene.
  • the promoter and ribosome binding site (rbs) for CAT are eliminated, and both surface expression lpp-OmpA-scFv gene and CAT gene are under control of a single promoter and operator.
  • the CAT gene can be transcribed but cannot be translated due to the lack of a rbs.
  • the scFv gene and the CAT gene are fused in such a way that the stop codon of the scFv and the start codon of the CAT are arranged in the order of TAATG.
  • antibiotic resistance genes such as the ampicillin resistance gene and kanamycin resistance gene can be fused to the antibody gene in this system.
  • any gene product that is essential for bacterial growth may be used.
  • a competitive immunoassay takes advantage of anti-analyte binding antibodies immobilized on a bacterial cell surface.
  • advantages of the disclosed immunoassay include generally the convenience, wide applicability and the simplicity, rapidity and sensitivity of the assay.
  • selected host cells displaying an polypeptide of interest may be used to stimulate an immune response.
  • the first step in the development of whole cells suitable for the detection of analytes is the screening of antibody libraries displayed on the cell surface.
  • Cells displaying antibodies having affinity for a desired analyte are isolated.
  • a library of cell surface displayed proteins is prepared as described elsewhere in the specification.
  • a library of surface displayed scFv antibodies can be prepared as described in Example 7.
  • the selected antigen for which one desires to identify and isolate specific antibody or antibodies is labeled with a detectable label.
  • detectable labels There are many types of detectable labels, including fluorescent labels, the latter being preferred in that they are easily handled, inexpensive and non-toxic.
  • the labeled antigen is contacted with the cells displaying the antibody expression library under conditions that allow specific antigen-antibody binding. Conditions can be varied so that only very tightly binding interactions occur; for example, by using very low concentrations of labeled antigen.
  • Identifying the antibody or antibody fragment expressing cells may be accomplished by methods that depend on detecting the presence of the bound detectable label.
  • a particularly preferred method for identification and isolation is cell sorting or flow cytometry.
  • One aspect of this method is fluorescence activated cell sorting (FACS).
  • the production of soluble antibodies can be achieved easily without the need for further subcloning steps.
  • the clones may be maintained under standard culture conditions and employed to produce the selected antibody. Production of antibody is limited only to the scaleup of the cultures.
  • the invention further includes competitive binding assays using cells with antibodies or analyte-combining antibody fragments expressed on the outer cell surface.
  • analyte is defined as a species that interacts with a non- identical molecule to form a tightly bound, stable complex.
  • the binding affinity is usually greater than about 10 M "1 and is preferably in the range of 10 9 -10 15 M "1 .
  • the analyte may be any of several types of organic molecules, including alicyclic hydrocarbons, polynuclear aromatics, halogenated compounds, benzenoids, polynuclear hydrocarbons, nitrogen heterocyclics, sulfur heterocyclics, oxygen heterocyclics, and alkane, alkene alkyne hydrocarbons, etc.
  • Biological molecules are of particular interest, including amino acids, peptides, proteins, lipids, saccharides, nucleic acids and combinations thereof. Of course it will be understood that these are by way of example only and that the disclosed immunoassay methods are applicable to detecting an extraordinarily wide range of compounds, so long as one can obtain an antibody that binds with the analyte of interest.
  • the disclosed whole cell immunoassay methods allow rapid detection of a wide range of analytes and are particularly useful for determination of polypeptides.
  • the methods have been developed to take advantage of the binding characteristics of bacterial cell surface exposed anti- analyte antibodies. Such surface displayed antibodies are stable and bind readily with specific analytes.
  • This unique form of protein expression and immobilization thus has provided the basis of an extremely rapid competitive assay that may be performed in a single reaction vessel in an "add and measure” format.
  • Such assays can be described as "one-pot" reactions that make possible in situ detection of an analyte.
  • a particular advantage of cell surface expressed antigen-binding antibodies is that the antibody is attached to the outer membrane of the cell.
  • the cells therefore act as a solid support during the assay, thereby eliminating many of the manipulations typically required in preparing reagents required for existing immunoassay techniques.
  • cells with the antibody displayed on the surface may themselves be attached to a solid support such as a membrane, dipstick or beads to further facilitate removal of the cells following the assay.
  • the immunoassays of the present invention may be used to quantitate a wide range of analytes. Generally, one first obtains the appropriate host cell culture where the anti-analyte antibody is displayed on the host cell surface, calibrates with standard samples of analyte, then runs the assay with a measured volume of unknown concentration of analyte.
  • a host cell that expresses an analyte binding antibody.
  • the host cell is then contacted with a standard analyte sample that contains a known amount of an analyte linked to a detectable label employing conditions effective for forming an immune complex.
  • a second host cell that has the same antibody or analyte-combining fragment expressed on its surface, except that in addition to the standard labeled analyte sample, a test sample in which an unknown amount of analyte is to be determined is added.
  • One is not limited to using the same host cell in this procedure.
  • a known amount of the antibody-covered cells are placed in a solution of a known concentration of the analyte-conjugate along with an unknown concentration of the analyte (the test solution).
  • the analyte conjugate competes with free analyte in solution for binding to the antibody molecules on the cell surface. The higher the concentration of analyte conjugate in the solution, the fewer molecules of fluorescein analyte conjugate bind on the surface of the cells, and vice versa.
  • the mixture is centrifuged to pellet the cells, and the fluorescence of the supernatant is measured.
  • the assay is quantitative because the amount of observed fluorescence is proportional to the concentration of analyte in the test sample, i.e., if there is a very low concentration of analyte to compete with the fluorescein conjugate, then most of the conjugate will bind to the cells and will be removed from solution. The more molecules of analyte in solution, the more molecules of analyte bind to the antibodies thereby preventing the conjugate from binding. In this case, more fluorescein conjugate remains in the supernatant to give a stronger fluorescence signal.
  • the assay can be calibrated to generate a quantitative measurement of the unknown concentration of analyte. The entire assay requires less than one hour. Fluorescence determinations may be made with a basic fluorimeter.
  • the present invention involves a novel method of carrying out competitive immunoassays using antibodies attached to the surface of cells.
  • the disclosed immunoassays are useful for binding, purifying, removing, quantifying or otherwise generally detecting analytes.
  • Antibodies expressed on bacterial cell surfaces have been shown surprisingly adaptable for use in competitive immunoassay procedures.
  • the analyte digoxin, a cardiac glycoside was determined using fluorescein-digoxin conjugate.
  • the assay was quantitative with a sensitivity in the nanomolar range.
  • Cells expressing an antibody fragment on their surface may also be linked to a solid support, such as in the form of beads, membrane or a column matrix, and the sample suspected of containing the unwanted antigenic component applied to the immobilized antibody. A purged or purified sample is then obtained free from the unwanted antigen simply by collecting the sample from the column and leaving the antigen immunocomplexed to the immobilized antibody.
  • Detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These approaches are typically based upon the detection of a label or marker, such as any of the radioactive, fluorescent, chemiluminescent, electrochemiluminescent, biological or enzymatic tags or labels known in the art.
  • Patents concerning the use of such labels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241 , each incorporated herein by reference.
  • a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.
  • the first added component that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the surface expressed antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the surface expressed antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • the competitive immunoassay discussed above requires one to set up a control sample.
  • This sample comprises a second host cell expressing an analyte binding antibody. This is contacted with a known amount of analyte linked to a detectable label and a known amount of unlabeled analyte, i.e. the analyte to be detected or determined in the assay.
  • a known amount of analyte linked to a detectable label i.e. the analyte to be detected or determined in the assay.
  • unlabeled analyte i.e. the analyte to be detected or determined in the assay.
  • the free label in solution after the cells have been separated. This measures the amount of residual detectable label as the decrease in, e.g., fluorescence emission, from which the amount of unknown analyte may be determined.
  • a preferred fluorescent label is fluorescein.
  • the inventors have found that measurement of the amount of residual label that is not bound to antibodies is proportional to the amount of analyte label in solution. This provides the basis for quantitative measure in that the increase in the amount of label is directly proportional to the analyte.
  • the label may be measured in the complexes. In this type of measurement, the analyte is inversely proportional to the amount of label.
  • fluorescence labeling while preferred, does not preclude the use of other detectable agents such as chemiluminescent agents, electrochemiluminescent agents, radioactive labels, enzymatic labels that form a colored product with a chromogenic substrate as well as other fluorescent compounds.
  • a preferred fluorescent label is fluorescein while
  • Ru(bpy) 3 is preferred for use as an electrochemiluminescent agent.
  • the invention is readily adaptable to the determination of multiple analytes. This is achieved using two or more different analyte-binding antibodies expressed in separate host cells. It is also possible to surface express more than one antibody on the surface of a particular host cell; however, this may cause interference in binding. One will, in these situations, use different detecting agents; for example, two different fluorescent labels, each with distinct emissions, such as fluorescein which emits at 520 nm and Texas Red which emits at 620 nm.
  • cells containing antibodies on the surface are produced as described previously.
  • Antibodies known to bind tightly and specifically to a molecule of interest are employed.
  • the molecule could be a medically relevant molecule, a marker molecule used in a scientific study, a pesticide of environmental concern in groundwater, etc.
  • a covalent conjugate of the molecule with a fluorescent moiety such as fluorescein is synthesized for use as a probe in binding.
  • Other detection agents include radioactive compounds, enzyme conjugates, chemiluminescent reagents such as luciferase and electrochemiluminescent reagents such as Ru(bpy) 3 + . (Yang et al, 1994; Blackburn et al, 1991).
  • the assay may also be carried out using tritium as the labeling agent for the antigen and performing a radioimmunoassay.
  • the radioactivity may be detected using a scintillation counter to measure binding constants up to 10 "8 or lO "9 M " '.
  • a known amount of the antibody-covered cells are placed in a solution of a known concentration of the molecule-fluorescein conjugate along with an unknown concentration of the molecule.
  • the molecule-fluorescein conjugate competes with free molecules in solution for binding to the antibody on the cell surface. The higher the concentration of the molecule in the solution, the fewer molecules of fluorescein-molecule conjugate will bind to the surface of the cells, while lower concentrations of the molecule will result in more of the conjugate bound to the cell surface.
  • the cells are removed from the solution, most conveniently by pelleting, and the fluorescence of the supernatant measured.
  • the assay is quantitative because the amount of observed fluorescence is proportional to the concentration of the molecule in the unknown sample. If there is a very low concentration of the molecule to compete with the fluorescein conjugate, most of the conjugate will bind to the cells and will be removed from the solution. The higher the concentration of the molecule in solution, the more molecules bind to the antibodies thereby preventing the conjugate from binding. In this case, more fluorescein conjugate remains in the supernatant to give a stronger fluorescent signal.
  • the assay can be calibrated to generate a quantitative measurement of the unknown concentration of the molecule. The entire assay takes less than an hour and requires only a basic fluorimeter.
  • immunoassay kits are also envisioned comprising a container having suitably aliquoted reagents for performing the foregoing methods.
  • the containers may include one or more bacterial cells with particular surface expressed analyte- binding antibodies.
  • Suitable containers might be vials made of plastic or glass, various tubes such as test tubes, metal cylinders, ceramic cups or the like.
  • Containers may be prepared with a wide range of suitable aliquots, depending on applications and on the scale of the preparation. Generally, this will be an amount in conveniently handled form, such as freeze-dried preparations, and sufficient to allow rapid growth of the bacterial cells as required.
  • kits may optionally include surface-expressed antibodies in host cells that are immobilized on surfaces appropriate for the intended use.
  • One may for example, provide the cells attached to the surface of microtiter plates, adsorbent resins, cellulose (e.g. filter paper), polymers, glass beads, etc.
  • the present invention discloses general methods for the screening of polypeptide libraries for the isolation of polypeptides that recognize and binding to desired target molecules with high affinity. Cell expressing such high affinity polypeptides can then be readily employed for immunoassays as described in section E. of the specification.
  • the screening of combinatorial libraries of polypeptides is greatly facilitated by the display of the polypeptides on the surface of host cells.
  • a cell population where each cell displays a different polypeptide is contacted with a desired ligand.
  • the ligand is labeled such that it can allow the facile separation of cells that display polypeptides capable of binding the ligand.
  • labels suitable for the purposes of this invention include fluorescent dyes and magnetic particles.
  • the desired target molecule can be immobilized on a suitable solid support. Cells producing surface displayed polypeptides capable of binding the desired target molecule thus adhere to the immobilized support and can be readily separated from cells that do not bind to the immobilized support.
  • FACS Fluorescence activated cell sorting techniques
  • E. coli cells with surface expressed polypeptides can be incubated with paramagnetic particles (e.g. Miltenyi Biotec, Bergisch Gladbach) that themselves are coated with an antigen of interest.
  • paramagnetic particles e.g. Miltenyi Biotec, Bergisch Gladbach
  • Paramagnetic particles are available with a variety of surface derivatization chemistries to allow for the covalent attachment of a wide range of antigens.
  • Bacteria having polypeptides with high affinity for the antigen remain bound to the magnetic particles, and the complexes isolated following washing steps in a strong magnetic field that retains the paramagnetic beads.
  • cells labeled with paramagnetic particles can be separated in a continuous magnetic separator.
  • cells displaying polypeptides that bind to the desired target molecule may be isolated via selective adsorption onto solid matrices.
  • the cell population displaying the polypeptide library is contacted with a solid support in which the antigen is covalently immobilized via standard chemical immobilization methodologies.
  • Cells that display polypeptides capable of interacting with the immobilized target molecules are retained on the solid support and can be separated from non-binding cells. Following several washes with buffer to remove non-specifically adsorbed cells, the cells that are bound via specific interactions are employed for further studies.
  • Such specifically-bound cells can be dissociated from the solid support either by adding large concentrations of the soluble desired molecule to serve as a competitor or, alternatively by adding growth media to allow the cells to grow. In the latter case the progeny of the bound cells is released from the solid support.
  • the immobilization onto micron sized particles generally involves bacteria being entrapped within agarose gel microdroplets (AGMs).
  • AGMs agarose gel microdroplets
  • This method involves entrapping microorganisms in AGMs (10 to lOOmicrons in diameter) which are surrounded by a hydrophobic (low dielectric) fluid, subsequently distinguishing occupied and unoccupied AGMs with colorimetric or fluorescence indicators, counting both occupied and unoccupied AGMs and applying statistical analyses to arrive at enumeration. It is possible to use a single preparation of AGMs containing a range of AGM sizes, to simultaneously provide a viable enumeration of growing and non-growing cells.
  • microdroplets are produced by first emulsifying a solution containing bacteria and melted agarose. This results in the formation of liquid microdroplets suspended in oil. By lowering the temperature at which the agarose is made to polymerize, thus forming micron sized droplets containing bacterial cells.
  • AGMs of approximately lO ⁇ M in diameter have been made using standard procedure. The conditions for making AGMs that are occupied by one cell are well known to those of skill in the art (Weaver et al, 1991).
  • This technique starts with a conventional cell suspension and generates a large number (10 ) of AGMs/ml by adding the cells to molten agarose and dispersing into mineral oil. This suspension of AGMs is then transiently cooled to a gelation state (Weaver et al, 1984; Weaver, 1997; Weaver et al, 1988). Poisson statistics allow the measurement of size of those AGMs that have a high probability of containing zero or one initial cell or colony forming units. The AGMs can be transferred out of the mineral oil into a suitable growth medium and incubated to allow for the formation of microcolonies.
  • nucleic acids stained with propidium iodide
  • proteins stained with FITC
  • flow cytometry generally with single cell resolution.
  • DNA staining may also be used and are well known to those of skill in the art (Powell, 1989; Muller et ⁇ /.,1997; van Tol MJ, et al, 1998).
  • This assay method is applicable to any cell type which is amenable to being cultured in a gel-like matrix.
  • the assay method has been successfully demonstrated on mammalian, fungal and bacterial cells (Weaver et al, 1991).
  • the method is sensitive to subpopulations with different growth rates and may be used for working with a mixed population of cells without the need for strain specific stains and is applicable to any growth based assay.
  • AGMs thereby allowing both monoculture and mixed cell populations to be assayed. Further AGMs provide confinement of progeny of individual cells within AGMs, rapid measurement of large numbers of individual microcolonies by flow cytometry and a hybrid of plating and cell suspension culture. Other fundamental advantages include the extreme permeability of AGMs allowing convenient and rapid changes in culture conditions. AGMs respond rapidly to changes in physical conditions (heat, electric field, light ionizing radiation), and they are small and robust enough to be handled in a manner similar to the handling of cells i.e. suspension, pipetting centrifuging and the like.
  • a variety of substrates can be used to assay the activity of, and ultimately select for, desirable mutant enzymes out of surface-expressed mutant enzyme libraries.
  • the gene for an enzyme to be mutated is expressed on the surface of bacteria such as E. coli by cloning the gene for the enzyme into a surface-expression vehicle such as the LPP-OmpA system (Francisco et al, 1992, 1993).
  • Mutant enzyme libraries can be created from these gene constructs using known methods such as chemical mutagenesis, error-prone PCR or amplification in a mutator strain. Identification of mutant enzymes with desirable properties such as novel substrate selectivity or remarkable catalytic activity can be achieved using substrates that change an assayable property, i.e. fluorescence intensity, ratio of multiple fluorophore emissions, antibody detectable structural changes etc., upon catalytic action of the enzyme.
  • a substrate can be synthesized that has a fluorophore (fluorescein, bodipy, etc.) and a quencher (eosin, tetramethyl rhodamine, etc.) attached on either side of the hydrolyzed bond. Enzymatic cleavage will result in separation of the fluorophore from the quencher leading to an assayable increase in fluorophore fluorescence.
  • fluorophore fluorescein, bodipy, etc.
  • quencher eosin, tetramethyl rhodamine, etc.
  • fluorophores or chromophores can be attached to the different individual substrates.
  • the ratio of the emissions can be determined, and a one-to-one ratio would indicate enzymatic reaction.
  • only one substrate could contain the fluorophore, and the presence of fluorophore emission on a product that is preferentially retained (see below) would indicate enzymatic reaction.
  • Additional formats could be envisioned in which quenchers are attached to one of the substrates and a fluorophore is attached to another, so that reaction leads to quenching of emission.
  • antibodies could be created that bind only products, not reactants, analogous to the detection used for cat-ELISA experiments (Tawfik et al. (1993)).
  • the product-specific antibodies could be labeled with a fluorophore. The retention of the fluorophore emission would indicate the presence of product and hence an enzymatic reaction.
  • This antibody labeling method should be applicable to almost any enzymatic process, regardless of reaction type, as long an antibodies could be found that can discriminate between substrates and products.
  • the mutant enzymes will be displayed on the surface of the bacteria, there is no impediment to diffusion of the assayable substrate to the mutant enzymes.
  • This direct substrate access will allow substrates of virtually any size or shape to be used to assay for catalytic activity, including polymeric species such as nucleic acids or peptides, that would not reliably diffuse into cells.
  • An essential element of the experimental design is that the spectroscopically identifiable product must remain associated with the bacterium having the surface-expressed enzyme. This can be accomplished by either of two methods.
  • the product is electrostatically trapped on the bacterial surface.
  • Bacteria such as E coli have negatively charged surfaces so that molecules with positive overall charge are retained on the cellular surface.
  • Substrate systems in which the product(s) of the enzymatic reactions posses substantially more positive charge than substrate(s) will have product preferentially retained on the bacterium harboring the surface expressed mutant enzyme that carried out the reaction.
  • the gate of the FACS could be set to select for any number of parameters such as the presence of an unquenched fluorescent signal, the proper ratio between two fluorophores of different emission wavelengths, the specific retention of a single fluorophore, or the presence of product specific antibodies with covalently attached fluorophores.
  • One or several rounds of mutagenesis and/or FACS selection could be used for enrichment of bacteria expressing desired mutants, and the selected bacteria can be plated directly for screening of individual colonies, or regrown in liquid media in preparation for further rounds of FACS selection.
  • a solvent is chosen, such as mineral oil, to suspend the AGM's such that the product of the enzyme reaction is only soluble in the aqueous environment of the AGM surrounding the cell, not the mineral oil. This will ensure that the AGM with the most active enzyme will accumulate the most product. If the product fluorescence can be distinguished from substrate, i.e.
  • the AGM's with desired enzyme activities could be isolated by FACS or via fluorescence microscopy using a micromanipulator.
  • the substrates can be changed dramatically all at once, or incrementally over the course of several rounds of selection. Either way, enzymes with dramatically different activities compared to wild type will be isolated.
  • E. coli strain JM109 [endAl recAl gyrA thi-1 hsdRIl (r k -, m k +) relAl supE44
  • pTXIOl codes for an Lpp-OmpA- ⁇ -lactamase fusion (Francisco et al, 1992).
  • pTX152 codes for an Lpp-OmpA-scF v (digoxin) fusion, where the scF v (digoxin) is an anti-digoxin single chain F v consisting of the heavy- and light-chain variable regions (V H and V ).
  • V H and V L joined by a 15 amino acid [(Gly) 4 Ser] 3 linker (Huston et al, 1988), were amplified from messenger RNA isolated from two separate anti-digoxin hybridomas.
  • the presence of the HSV peptide allowed detection of the scF v (digoxin) protein by reaction with a monoclonal antibody specific for the 11 amino acid epitope.
  • the sequence of the single chain F v antibody fragment is disclosed in SEQ ID NO: 2.
  • pTX152 was constructed by first removing the bla from pTXIOl by digestion with EcoRI and BamHI. The amplified gene coding for the anti-digoxin scF v was then digested with EcoRI and BamHI and ligated into pTXIOl. Both pTXIOl and pTX152 carried the chloramphenicol resistance gene. Cultures were grown in LB medium (Difco) supplemented with 0.2% glucose and chloramphenicol (50 ⁇ g/ml) at a temperature of either 24°C or 37°C.
  • Cells were grown in liquid culture at 37°C and used to isolate total membranes. The presence of a band of the expected size (42 kDa) was detected in Western blots of whole cell membranes probed with antibodies specific for OmpA and for the HSV peptide (FIG. 1A). Cells containing pTX152 produced a protein which reacted with both the HSV-specific and the OmpA specific sera, as expected for the Lpp-OmpA-scF v (digoxin) fusion. Control cells containing pTXIOl reacted only with OmpA antiserum. The lower molecular weight band in lanes 1 and 2 corresponds to the intact OmpA protein of E. coli.
  • the intensity of the Lpp-OmpA(46-159)-scF v (digoxin) band in FIG. 1A is comparable to that of the native OmpA band.
  • the latter is a highly expressed protein that is present in the E. coli outer membrane at about 100,000 copies per cell (Lugtenberg & Van Alphen, 1983).
  • the level of expression Lpp-OmpA(46-159)-scF v (digoxin) is on the order of 50,000-100,000 copies per cell.
  • the scF v domain of the Lpp-OmpA-scF v (digoxin) fusion protein encoded by pTX152 was shown by ⁇ LISA to bind specifically to the hapten digoxin.
  • Whole cell lysates from JM109/pTX152 and from JM109/pTX101 as control were incubated on microtiter wells that had been coated with either digoxin-conjugated BSA (digoxin-BSA) or unconjugated BSA.
  • FIG. 1A shows that lysates from JM198/pTX152 bound specifically to wells coated with digoxin-BSA but not to unconjugated BSA, whereas the lysates from the control strain, JM109/pTX101, did not give a signal with either.
  • Lpp-OmpA(46-159)-scF v (digoxin) is active and can bind to the hapten specifically.
  • IB shows the results of ELISAs using intact cells. Samples containing the same number of cells were used in all the studies. Cells containing the control plasmid, pTXIOl, gave the same low signal when incubated on microtiter wells coated with either unconjugated BSA or with digoxin-BSA. A similar weak signal was detected with JM109/pTX152 incubated on BSA-coated wells and is presumably due to non-specific binding. In contrast, a much higher absorbance was evident in wells coated with the digoxin-BSA conjugate indicating that there are active fusion protein molecules on the cell surface.
  • the intensity of the fluorescence signal from JM109/pTX152 was dependent on the cell growth temperature and was much higher for cultures grown at 24°C instead of 37°C. This was consistent with previous results showing that the amount of proteins expressed on the surface of E. coli by fusion to Lpp-OmpA(46-159) increased as the temperature is decreased (Francisco et al, 1992; 1993). Assuming that the efficiency of surface display in this case is similar to that of ⁇ -lactamase (Francisco et al, 1992), then at 24°C virtually all the scF v antibody chains must be accessible on the cell surface.
  • FIG. 3A and FIG. 3B show that the fluorescence intensity of JM101/pTX152 cells expressing a surface displayed recombinant antibody specific for digoxin was substantially higher than the intrinsic background signal of control E. coli cells. JMlOl/pTXIOl expressing Lpp-OmpA(46-159)- ⁇ -lactamase is used as a control.
  • Antibody expressing cells were sorted essentially quantitatively from an excess of control E. coli in a single step. Specifically, in mixtures containing JM109/pTX101 control cells at an excess of either 100:1 or 1,000:1, the fraction of the total population that was sorted in the high fluorescence intensity window was 1.1% and 0.1% respectively (after subtracting the background), as expected from the ratio of input cells.
  • JM109/pTX101 cells displaying an unrelated protein on the cell surface
  • JM101/pTX152 cells displaying the scFv(digoxin) antibodies
  • digoxin-FITC digoxin-FITC
  • 500,000 cells were run through the FACSort flow cytometer.
  • a wide sorting gate i.e., the minimum fluorescence required for acceptance of an individual cell, was selected such that up to 0.2% of the control cells fell within the sorting window. This ensured that all the scF v (digoxin) expressing cells would be recovered.
  • the cells having an allowable fluorescence signal were collected and grown in fresh media at 37°C.
  • FIG. 3 ⁇ , FIG. 3F and FIG. 3G show the cell fluorescence distribution for the sorting runs. After only two rounds of growth and sorting, the fluorescence intensity of 79% of the cell population fell within the positive window. A similar enrichment was reproducibly obtained in three independent studies. These results were not due to a growth advantage of the cells expressing Lpp-OmpA(46-159)-scF v (digoxin), since successive regrowth of the input cell mixture in the absence of sorting did not result in any detectable enrichment.
  • DNA was isolated from eight cm + , amp-colonies and the presence of pTX152 was confirmed by restriction analysis.
  • a construct similar to that used for surface expression of scF v (digoxin) can be modified to incorporate a protease cleavage site.
  • the recognition sequence of enterokinase [(Asp) 4 -Ile-Arg] can be introduced in the Lpp-OmpA(46-159)-scF v between the OmpA(46-159) and the scF v domains.
  • the protease cleavage site at the N-terminal of the scF v antibody domain of the fusion protein is then used to release the scF v antibody in soluble form following treatment of the cells with the appropriate proteolytic enzyme. Because the outer membrane of E.
  • a single colony expressing a desired single chain F v antibody can be grown in liquid media and harvested by centrifugation after overnight growth at 24°C. The cells are resuspended in buffer to maintain the pH approximately neutral. Protease added at appropriate concentrations to the fusion protein to be treated and incubated at least 4 hours at 4°C will release the soluble single chain F v . Subsequently the cell suspension is centrifuged and the supernatant containing the solubilized single chain F v antibody is collected.
  • a His 6 sequence may be introduced at the C-terminus of the scF v using PCR amplification with the appropriate primers.
  • Polyhistidine tails bind strongly to metals so that the fusion protein can be purified by immobilized metal-ion affinity chromatography (IMAC).
  • IMAC immobilized metal-ion affinity chromatography
  • Lpp-OmpA(46-159) fusions are typically expressed at a high level, typically around 5xl0 4 per cell.
  • the yield after protease treatment and IMAC is at least 2-3 mg of antibody per liter of shake flask culture.
  • This example illustrates a solid phase immunoassay using E. coli with anti-digoxin single chain F v displayed on the surface.
  • This immunoassay is demonstrated to be a sensitive and quantitative technique.
  • the cells may be attached to a solid support such as a membrane, dipstick or beads.
  • Digoxin-FITC The Binding Site Inc. (San Diego, CA) was diluted to a concentration of 20nM in PBS. Digoxin (Sigma, St. Louis, MO) was brought to a concentration of l ⁇ M in PBS.
  • the pTX152/JM109 cells were grown in LB overnight at 37°C then subcultured into fresh LB and grown overnight at room temperature. The cells were harvested and resuspended in PBS pH 7.4 at a concentration of 10 cells/ml, based on the O.D. 600 to form a cell stock. Some cells were also resuspended in 15%) glycerol/water and stored at 70°C. Frozen cells yielded the same results as freshly prepared cells. The following reagents were transferred to a 1.5 ml microcentrifuge tube, 25 ⁇ l of 20nM digoxin-FITC solution, 0.5-50 ⁇ l of l ⁇ M digoxin and PBS to a final volume of 950 ⁇ l.
  • the mixture was vortexed briefly and pulsed in an Eppendorf microcentrifuge.
  • a 25 ⁇ l or lOO ⁇ l aliquot of the cell stock (10 cells/ml) was added to the mixture and allowed to incubate for 1 hour at room temperature. Following this incubation the cells were spun in a microcentrifuge for 5 minutes at 5,000 rpm and the supernatant collected. The fluorescence of the digoxin-FITC in the supernatant was measured using a fluorimeter.
  • This immunoassay was also performed with the cells attached to a solid support.
  • the solid support was Fisher filter paper P5 was cut into strips approximately 0.5cm x 2 cm, and dampened in PBS by submerging one end of the filter paper and allowing it to move upward.
  • the filter paper was allowed to dry slightly and lO ⁇ l of a solution containing 6x10 cells/ml was applied to the filter paper.
  • the cells were fixed to the paper with a solution of 6-PLP, 8%PFA and NaIO 4 .
  • the 6-PLP solution is lOmM 6-PLP, 200 mM MES, 700 mM NaCl, 50 mM KC1, 700 mM Lysine-HCl, 50 mM MgCl 2 , and 70 nM EGTA followed by 0.01M MgCl 2 and 8% PFA.
  • the 8% PFA was prepared by addition of 4g of PFA into 50 ml of water, followed by heating the solution to 70 °C with constant stirring. Approximately 2 drops of a 1M solution of NaOH was added to the solution until it became clear. The solution was then filtered through Whatman filter paper #1, allowed to cool and stored at 4°C until use.
  • the final fixative solution was produced by mixing the components A, B and C to a final volume of 10 ml just prior to use, where A is 1 ml 6-PLP, 04 g sucrose and 3.03 ml water; B is 21.4 mg NaIO 4 ; and C is 4.62 ml 8% PFA.
  • the fixative was applied dropwise to one end of the filter paper and allowed to soak upwards. The excess fixative was allowed to drain and the filter paper was washed twice with a solution of 100 mM NH 4 C1.
  • the filter paper can then be stored in a solution of PBS until use.
  • the assay was performed by removing the filter paper with the cells attached and then measuring fluorescence of the solution.
  • Essentially quantitative assays for an antigen can be run using a mixture of a known amount of labeled antigen combined with an unknown amount of unlabeled antigen.
  • pTX152/JM109 with antidigoxin antibody displayed on the surface were grown in LB overnight at 37°C, subcultured into fresh LB and grown overnight at room temperature. The cells were pelleted and resuspended in PBS to form a stock solution of cells at a concentration of 1x10 cells/ml.
  • a 25 ⁇ l or a lOO ⁇ l aliquot of the cell stock solution was added to the mixture to give a total of 0.25x10 or 1x10 cells.
  • the mixture was then allowed to incubate at room temperature for 1 hour.
  • the cells were pelleted and the fluorescence of the supernatant was measured for each sample.
  • FIG. 4 shows a plot of the residual fluorescence observed with the indicated amount of free digoxin, 0.5 nM digoxin-FITC conjugate and either 250 million or 1 billion cells expressing the antidigoxin single chain antibody on their surface.
  • the cells may be attached to a solid support.
  • the solid support consisted of a membrane however many other supports would work as well such as dipsticks or beads.
  • Strips of filter paper were moistened with PBS and allowed to dry slightly. A 10 ⁇ l aliquot of a 6x10 cells/ml of a cell suspension containing pTX152/JM109 cells displaying antidigoxin antibodies on their surface was applied to the strips of pre-moistened filter paper. The cells were then fixed to the paper using a mixture of 6-PLP, PFA and NaIO 4 , as described in the materials and methods, and washed twice with a solution of lOOmM NH 4 C1. Following the incubation no centrifugation is necessary the filter paper is simply removed from the solution and the residual fluorescence measured as described previously.
  • This example demonstrates that the display of antibodies on the surface of microorganisms constitutes the basis for discriminating between antibodies of different affinities. By adjusting the fluorescent antigen concentration, it is possible to discriminate binders of high affinity from those with moderate affinity.
  • the heavy-chain residue Y33 of the scFv(digoxin) antibody is known to be critical for antigen binding (Short et al, 1995).
  • Y33N, Y33C, Y33S, Y33G, Y33STOP were constructed by overlap extension PCRTM (Ho et al, 1989).
  • the large fragment from EcoR I digested pSD192 was purified and religated to yield pSD195 (AmpR).
  • Primers Y33C.S, Y33N.S, Y33S.S Y33G.S, and Y33Stop.5 were used to amplify 3' fragments for mutant constructions.
  • the 5' overlap fragment was produced with primers #4 and #3 and overlapped with the mutant fragments using primers #4 and CMI.5.
  • the resulting products were digested with ⁇ coRI and ligated into pSD195, and electroplated into JM109 to yield Cm resistant mutants pY33N, pY33C, pY33S, pY33G, and pY33STOP. Overnight cultures were grown at 37°C and subcultured 1 :100 at 25 °C for 20 hrs. Cells (200 ⁇ l) were harvested into 1 ml PBS pH 7.1-7.4, pelleted by centrifugation at 3000 x g for 4 min, and resuspended in 1.5 ml PBS.
  • FSC-SSC FSC-SSC
  • 20 ⁇ l of culture were added directly from a 24 hr, 25°C culture to 180 ⁇ l BODIPY-digoxin at 10, 5, 2.5, 1.25, 0.62 and 0 nM control in 96 well plate, and allowed to incubate 1 hr with gentle shaking.
  • Cell samples were diluted fivefold in PBS and analyzed immediately by flow cytometry.
  • the relative affinity of the mutants constructed were, as determined by in vitro ELISA data, Y33N (moderate), Y33S (low), Y33G (background), and Y33Stop (background).
  • the flow cytometry data and previously obtained ELISA results are shown in FIG. 5A and FIG. 5B, respectively.
  • the flow cytometry data for individual Y33 mutants correlate with ELISA data.
  • the mean fluorescence signals vary in the manner WT > Y33N >Y33S > Y33G _ Y33Stop.
  • pSD195 containing only the N-terminal portion of the cat gene immediately downstream of the scFv to be expressed at the cell surface, was constructed.
  • Two Pst I restriction sites were introduced, by silent mutagenesis, into the vector to simplify library construction. The first site was created just upstream of the scFv LCDR3 to be randomized and the second was introduced within the chloramphenicol resistance gene, (CmR).
  • CmR chloramphenicol resistance gene
  • Escherichia coli strain JM109 was used for all cloning steps and library experiments.
  • Oligonucleotides for PCR reaction are shown in Table 4. Overnight cultures were grown at
  • pTX152 was digested with Pvu I and BamU I, and the small fragment from pTX152 was ligated into similarly digested pET-22b to yield pGC183.
  • the chloramphenicol acetyl transferase gene (cat) was amplified from pBR325 with primers CM.1.s and CM.2. as, digested with Ec R I and Sph I and ligated into similarly digested pGC183 to yield pGC185.
  • the digoxin scFv was reintroduced into pGC185 by ligating the EcoR I-if ⁇ mH I fragment from pTX152 with similarly digested pGC185 to yield the pGC182 (ampR, cmR). Subsequently, the Pst I site was removed from pGC182 by replacing the AlwN l/Pvu I fragment with that from pUC18 giving rise to pSD182. Pst I sites were introduced upstream of the light-chain CDR3 and within the cat gene by silent mutagenesis.
  • Primers #4 and PIA.las (Rxnl), PIA.2s and PIB.las (Rxn2), and PIB.2s and Sphl.2.as (Rxn3) were used to amplify the corresponding fragments from pTX152, pSD182, and pBR322 respectively.
  • Products from Rxn2 and Rxn3 were amplified with PIA.2 and Sphl .2as and the resulting product was overlapped with the Rxn 1 product using primers #4 and Sphl.2as, digested with EcoR I and Sph I and ligated into similarly digested pSD182 to yield pSD 192 (FIG. 7A).
  • the digoxin scFv light-chain codons for T91, V94, and P96 were chosen for randomization. Both T91 and P96 form important contacts with digoxin in the Fab crystal structure (Jeffrey et al, 1993), suggesting that they play an important role in determining the antibody affinity.
  • the resulting plasmid library was electroporated into JM109 to yield 2 xlO transformants.
  • light-chain library plasmid DNA was prepared to provide a stock for subsequent library screening experiments.
  • the probability that each amino acid sequence is represented in the library pool may be calculated, using a Poisson distribution (Lowman et al, 1991), to be greater than 85 %.
  • Flow cytometric analysis of ten randomly picked clones showed 1/10 to weakly bind fluorescein-digoxin and 9/10 clones exhibited no binding.
  • pSD182 was digested with Xba I and the large fragment was purified by gel electrophoresis, and religated to yield pSD181.
  • pSD181 was digested with Xba 1 and Apa I, and the small fragment was gel purified and used as a template to produce the 3' mutagenic antibody-cat fragment. The 5' fragment was amplified from pTX152 with primers EcoRI.
  • PCR master mix 150 ml lOx Pfu buffer, 200 ⁇ M dNTPs, 8 ng/uL each primer, 1 ng/uL 5' and 3' template fragments and Pfu (0.03 u/mL and water to 1.5 ml
  • PCR master mix 150 ml lOx Pfu buffer, 200 ⁇ M dNTPs, 8 ng/uL each primer, 1 ng/uL 5' and 3' template fragments and Pfu (0.03 u/mL and water to 1.5 ml
  • HC PCR DNA was digested with Pst I, precipitated, digested with Sal I, gel purified and electroeluted.
  • pSD195 was digested with Sal I and Pst I Gel purified and electroeluted.
  • Ligations 12 ug pSD195 Sal UPst I, 3.6 ug HC PCR Sal II Pst I, 40 ⁇ l ligase, 80 ⁇ l 10X, water to 800 ⁇ l
  • 20 hr 16 °C
  • heat inactivated 10 min 70 °C
  • ethanol precipitated ethanol precipitated
  • resuspended 60 ⁇ l Tris-HCl.
  • Electroporation cuvettes were washed with 3 ml of SOC media, and incubated for 1 hr at 37 °C in a total volume of 60 ml.
  • Clones that bind to the hapten digoxin were isolated from the library in a single step. This procedure is outlined in FIG. 8. Following incubation of a library aliquot with fluorescein- digoxin, cells displaying a total fluorescence greater than threshold value, which the user may gradually decrease to reduce selection stringency, were sorted and recovered by vacuum filtration onto a 0.2 ⁇ m membrane (Millipore, Bedford, MA). The membrane was transferred to an agar plate containing the appropriate antibiotics and incubated overnight at 37° C. Individual colonies were then grown in liquid media and assayed for high-affinity antigen binding by flow cytometry. Light-chain library DNA (3 ⁇ L) was transformed into JM109 by electroporation.
  • the heavy-chain library was first screened in a single pass essentially as described for the light-chain library, to demonstrate that high affinity scFv(dig) variants could be selected from large libraries using only a single FACS step.
  • HCDR3 library DNA (3 ⁇ l) was electroporated into JM109 and grown 8 hr to saturation at 37 °C. After a 14 hr subculture at 25 °C, 160 ⁇ l cells were harvested into 640 ⁇ l of 100, 15, and 0 nM BODIPY-digoxin in PBS. Cells were incubated with gentle shaking for 45 min. Propidium iodide was added to a final concentration of 5 ⁇ g/ml and cell were incubated 15 min.
  • the heavy-chain library was also screened using a multiple-step FACS process to enrich high-affinity antibody expressing cells. A library aliquot was labeled with BODIPY-digoxin at
  • NNS G or C
  • the mutagenized DNA was treated with Ncol, which is unique to the wild-type DNA sequence, such that the wild-type is eliminated without affecting the total amino acid diversity encoded by the library.
  • the wild-type amino acid sequence was encoded by a single, non-wild-type DNA sequence ( AGTGGGCGA TG SEQ ID NO:55j.
  • the presence of rare clones expressing high-affinity scFv(dig) antibodies in the heavy- chain library was evaluated by labeling the cells with 100, 10, 1, 0.1, or 0.01 nM BODIPY- digoxin (FIG. 12).
  • High-fluorescence events, defined as those occurring in region 1 (RI, FIG. 13), were detected at a frequency of about 0.01% using 1 nM BODIPY-digoxin and about 0.001%) with 0. 1 nM BODIPY-digoxin.
  • the 20 randomly selected transformants that had been sequenced to assess the diversity of the library were pooled and analyzed in the same manner. In this case, high fluorescence clones were not detected for antigen concentrations below 10 nM.
  • the library cell population was also labeled with propidium iodide to preferentially label non-viable cells (Lopez-Amoros et al, 1995).
  • the library was first screened after labeling with 10 nM BODIPY-digoxin and washing once in PBS (FIG. 13). A sort window was defined such that cells were recovered only if they exhibited high BODIPY fluorescence (FL1 channel), low propidium iodide fluorescence (FL2 channel) and an appropriate range of forward scatter values to account for cell size.
  • More than 10' cells were sorted in recovery mode in less than two hours. Recovery mode enables the collection of all clones in the target window regardless of coincident non-target cells. Highly-fluorescent clones were enriched from a frequency of 0.01%) in the pre-sort population to about 20% following sorting (FIG. 13 A and FIG. 13B, respectively), an enrichment factor of approximately 2000-fold.
  • the sorted population was regrown in LB media and plated on selective agar plates. 96 colonies were picked at random, grown in a 96-well microtiter plate, and subsequently analyzed by flow cytometry.
  • the entire collection of cells selected in the first round were regrown in liquid culture and a second round of enrichment was performed using conditions favoring clones with slower dissociation kinetics. Specifically, the cells were incubated with 10 nM BODIPY-digoxin, washed two times with PBS, and resuspended in 1 ⁇ M unlabelled digoxin in PBS. The number of washing steps and the time of incubation in the presence of unlabelled digoxin (30 min) were optimized by monitoring the fluorescence distribution of the cells after each step. Cells were sorted in exclusion mode, which rejects coincident events, to achieve high purity (FIG. 13C).
  • the sorted cells were regrown and subjected to a final round of selection with 0.2 nM BODIPY- digoxin, a concentration equal to 20% of the equilibrium dissociation constant of the purified, wild-type scFv(dig) in solution.
  • a significant percentage of the population remained fluorescent and was sorted in exclusion mode.
  • selected clones were analyzed by flow cytometry (FIG. 14), and their amino acid sequences were determined by DNA sequencing (Table 9). Clones isolated in the first round displayed a consensus sequence of (K/R)RAL (SEQ ID NO:74), which evolved as the selection stringency was increased in rounds two and three.
  • His-tagged scFv(dig) mutants were expressed without the Lpp-OmpA' sequence and purified to >95% by refolding from inclusion bodies (Burks et al, 1995). Analytical gel filtration confirmed that greater than 95% of the purified protein was present as monomeric scFv.
  • the binding kinetics of soluble purified scFv were analyzed by SPR using a BIAcore instrument. A high flow rate and low coupling density were used to minimize mass transport and rebinding effects (Karlsson and Fait, 1997).
  • the K D values for the purified antibodies were all at, or below, the antigen concentrations originally used to isolate the corresponding clones by FACS.
  • association rate constants, k ⁇ or all isolated clones varied by 1.6-fold, whereas the dissociation rate constants, k dissoc , improved by 3-4 fold between the first and last round of selection (from 2.6-3.6 x 10 " s " to 0.83 X lO"- s " ).
  • the dissociation rate constants of the purified scFvs correlated with the values obtained by flow cytometric analysis of the corresponding antibodies displayed on E. co//_(Table 9).
  • a second scFv(dig) library was created by randomizing light-chain residues T91, V94, and P96.
  • DNA sequencing analysis did not reveal any codon bias. Given the number of transformants obtained, the Poisson probability that all sequences are represented in the library is greater than 85%.
  • the light-chain library was screened using two increasingly stringent sorting steps resulting in the isolation of clones designated LC2.1 and LC2.2. The dissociation rates were again measured by flow cytometry (FIG. 15, Table 10). The average apparent dissociation rates decreased by 3.5-fold between rounds 1 and 2.
  • T91 was conserved among the highest affinity clones isolated.
  • the substitution P96A occurred in 6/8 isolated LC2 clones.
  • V94 displayed a large tolerance to substitution but with a preference for moderately sized hydrophobic residues; V, I, and L.
  • the light-chain library was also screened using conditions to favor the isolation of only the highest affinity clones using just a single round of sorting.
  • Cells were labeled with 5 nM BODIPY-digoxin, washed twice in PBS, and then incubated with 1 ⁇ M digoxin in PBS for 30 min prior to sorting.
  • Highly-fluorescent clones were enriched from 0.01% to greater than 10%) of the population.
  • Three clones having high MFLI signals at 5 nM BODIPY-digoxin were selected at random and their dissociation rate constants were measured by flow cytometry. All
  • This example demonstrates that the enzymatic activity, rather than a ligand binding activity, of a surface displayed polypeptide can be used to isolate cells producing such a surface enzymatic activity from a vast excess of cells that do not express enzymatically active polypeptides.
  • the example specifically demonstrates how E. coli cells that express the protease OmpT on their surface can be distinguished from cells that do not produce OmpT or produce inactive OmpT.
  • a substrate that becomes fluorescent upon cleavage by OmpT was designed by conjugating a fluorescent dye (BODIPY) and a quenching group (trimethylrhodamine) at the opposite ends of the secile bond.
  • Enzymatic cleavage releases the quenching group into the medium resulting in the production of a fluorescent product.
  • the fluorescent product was designed to have several positive charges to allow its binding to the surface of the cells via electrostatic interactions with the negatively charged lipopolysaccharide molecules that comprises the outer layer of the E. coli surface.
  • the chemical structure of the substrate is shown in FIG. 10.
  • the peptide moiety was synthesized at the University of Texas, Austin peptide facility using standard FMOC coupling conditions.
  • the thiol of the cysteine was alkylated with trimethylrhodamine iodoacetamide and this product was acylated with BODIPY-FL succinamidyl ester (Molecular Probes).
  • the crude product was purified by preparative HPLC.
  • OmpT negative E. coli mutant UT5600
  • UT5600 cells expressing OmpT from a multicopy plasmid showed a much larger increase in fluorescence, which continued to increase for over 20 minutes.
  • the mean fluorescence intensity of the OmpT + cells was over 30 times higher than that of the cells without the plasmid (i.e., OmpT " cells).
  • OmpT fluorescence is more than sufficient to allow the sorting of cells expressing active enzyme from cells that do not express OmpT.
  • inactive OmpT mutants exhibit increased fluorescence upon incubation with the substrate.
  • an OmpT mutant was produced in which the conserved His212 residue (which is thought to be part of the catalytic triad) was converted to Ala by site-directed mutagenesis (Maniatis et al 1989).
  • the His212->Ala mutant was confirmed to have no OmpT activity.
  • UT5600 cells expressing the His212->Ala produce the same amount of OmpT as cells transformed with a plasmid encoding the wild type enzyme.
  • UT5600 cells expressing the inactive OmpT mutant were incubated with the substrate and examined by FACS they exhibited a background fluorescence that could be clearly distinguished from that of OmpT positive cells.
  • OmpT + cells can be readily isolated from a population containing a huge excess of OmpT " cells. Specifically, OmpT cells were mixed with OmpT " cells at a 5,000-fold excess. The cell mixture was incubated with the substrate, passed through the fluorescence activated cell sorter and cells exhibiting a high fluorescence intensity were isolated. Nine out of nine sorted clones that were isolated produce OmpT. These studies showed that the OmpT cells can be readily isolated by FACS from a huge excess of background cells solely on the basis of the enzymatic activity of the OmpT protease (FIG. 11).
  • This example illustrates how libraries of polypeptides displayed on the cell surface can be screened to isolate clones that produce polypeptides having a desired enzymatic activity.
  • this example teaches the screening of libraries of OmpT mutant polypeptides to isolate novel enzymes that can hydrolyze peptide sequences not recognized by the wild-type
  • the ompT gene will first be subjected to random mutagenesis using established techniques such as error-prone PCR, chemical mutagenesis or mutator strains.
  • error-prone PCR or chemical mutagenesis the ompT gene is first excised from the high copy plasmid pML19 by digestion with appropriate restriction endonucleases and mutagenized according to standard procedures known to those skilled in the art (Maniatis et al. 1989, Innis et al. 1990).
  • the ompT DNA will be ligated back to restriction-enzyme digested pML19 and the ligation mixture will be electroporated into E. coli UT5600.
  • Transformants will be grown in LB broth containing ampicillin (lOO ⁇ g/ml) and glucose at 0.2%> w/v at 37°C. Cultures will be grown to saturation to ensure maximal expression of OmpT. Subsequently, the cells will be harvested by centrifugation, washed with PBS and resuspended in 1 mM Tris buffer, pH 7.0, in the presence of a substrate having a structure similar to the one shown in FIG. 10 except that the Arg-Arg dipetide that is recognized by the wild type OmpT is substituted with other dipeptide sequences, for example Arg-His, Arg-Ala, His-His, etc.
  • Cells expressing OmpT capable of hydrolyzing the substrate allow the release of the trimethylrhodamine quencher into the solution while the N-terminal cleavage product containing the BODIPY fluorophore is electrostatically retained by the cells.
  • FACS fluorescent activated cell sorting
  • a gate is set such that only cells exhibiting high fluorescence are sorted in the positive window. Cells will be sorted at an event rate of at least 1000 s "1 . A total of 10 6 cells will be screened and cells displaying high fluorescence will be collected by FACS. Isolated colonies will be then screened for product hydrolysis by FACS analysis.
  • DNA sequence of the mutant ompT genes encoding enzymes with altered substrate specificity will be determined by DNA sequencing.
  • Burks et al Biotechnol. Prog, 1 1 :112-1 14, 1995. Burks et al, Proc. Natl. Acad. Sci. USA, 94:412-417, 1997.
  • PCT/US87/00880 PCT/US89/01025.
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Abstract

La présente invention concerne des procédés de sélection de protéines, dans de grandes bibliothèques présentant des caractéristiques désirables. Des procédés d'expression d'enzymes et d'anticorps sur la surface de cellules hôtes et de sélection d'activités désirées sont donnés à titre d'exemple. Ces procédés comparés aux procédés actuels présentent l'avantage d'une utilisation rapide et facile et sont une source de quantités importantes de la protéine considérée, sans clonage supplémentaire.
PCT/US1998/008714 1997-05-01 1998-04-30 Evolution orientee d'enzymes et d'anticorps WO1998049286A2 (fr)

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US9221905B2 (en) 2009-02-24 2015-12-29 Esbatech, An Alcon Biomedical Research Unit Llc Methods for producing immunobinders of cell-surface antigens
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