Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

• hybridoma monoclonal antibody, also referred to herein as "mAb”
• mAb monoclonal antibody
• antigen antigen [Kohler, G. and Milstein, C, Nature , 256:52-52 (1975) ]
• hybridoma cultures typically have a low production capacity and are considered to be unstable.
• Antibodies from different species recognize different antigenic epitopes present on one antigen [S. Twining, et al., Biochem . , 191:681-697 (1980)]. Rabbit has been the animal of choice for the production of polyclonal antibodies of high affinity and specificity directed against native or denatured antigens. However, mAb from this species has not been available.
• Enzyme-linked immunosorbent assay is a solid phase immuno-assay which employs antibodies and has become a versatile and practicle tool in biology and medicine. [Endwall, et al., Biochem . Biophys . Acta . , 251:427 (1971)]. These assays are commonly used for rapid diagnosis of disease, and are of great commercial importance in both research and human health care. The major principle of these assays is the direct immobilization of the antibody to plastic [Catt and Tregaer, Science , 158:1570-1572 (1967)]. Subsequently, simple washing of the plastic solid phase permits separation of bound from non-bound reactants that are added step wise in the process of completing the assay.
• Monoclonal antibodies are commonly used in in vitro diagnosis, such as the sandwich ELISA discussed above. Unfortunately, it has been shown that monoclonal antibodies have lower antigen affinity or antigen capture capacity when absorbed directly on the plastic commonly used as the solid phase in the ELISA assay [Butler, et al.. Molecular JJ ⁇ T ⁇ UJI., 23:971-982 (1986)].
• One explanation for the apparent low affinity of MoAbs absorbed hydrophobically to plastic could be alteration of the antigen-binding site due to absorbtion-induced denaturation [Suter and Butler, Immun . Lett. , 13:313-316 (1986)].
• a recombinant rabbit heavy chain variable domain, or single domain antibody having the ability to bind to a solid phase, for example, plastic, without a decrease in antigen capture capacity.
• the present invention further provides an improved immunological sandwich assay, such as an ELISA, in which the rabbit single domain antibody of the present invention is directly bound to the solid phase as the capture antibody.
• the resulting assay is less complex than systems currently utilized without any significant decrease in sensitivity.
• Figure 1 shows a 2% agarose gel comparing PCR amplified VDJ genes from mRNA isolated from rabbit PBL (lane 2) or amplified from a cloned VDJ gene (clone 4K7, lane 3). Lane 1: 123 bp standard and its multimers.
• Figure 2 shows the results of an inhibition experiment using colony plaque lifts.
• Figure 3 is a graph showing the specificity of antigen binding shown by ELISA. Bacterial supernatant produced by clones 21.1 (A), 17.1 (B) , 16.1 (C) and 13.2 (D) was tested for binding to protein C. The resulting OD is shown as a function of time after induction with IPTG.
• a recombinant rabbit single domain antibody having the ability to bind to a solid phase, for example, plastic, without any significant decrease in antigen capture capacity. More specifically, the present invention directed to a recombinant rabbit single domai antibody, its production and use.
• the gene encoding, the rearranged gene for the rabbit heavy chain variable domain or single domain antibody may be produced, for instance, by cloning or gene synthesis, and placing it into a suitable expression vector. The expression vector is then used to transform a compatible host cell which is then cultured to allow the single domain antibody to be expressed and, preferably secreted.
• Nucleic acid sequence containing a substantial portion of the rabbit immunological gene repertoire may be isolated from a heterogeneous population of antibody-producing cells, i.e., B-lymphocytes (B cells) , preferably rearranged B cells such as those found in the circulation or spleen of a rabbit which has been infected, immunized or partially immunized with the antigen of interest.
• B cells B-lymphocytes
• lymphocytes include, for example, peripheral blood lymphocytes (PBLs) .
• the immunization can be carried out conventionally.
• Antibody titer in the animal can be monitored to determine the stage of immunization desired, which stage corresponds to the amount of enrichment or biasing of the repertoire desired.
• Partially immunized animals typically receive only one immunization and cells are collected therefrom shortly after a response is detected.
• Fully immunized animals display a peak titer, which is achieved with one or more repeated injections of the antigen into the host mammal, normally at 2 to 3 week intervals.
• the spleen is removed and the genetic repertoire of the spleenocytes, about 90% of which are rearranged B cells, is isolated using standard procedures.
• Any strain of rabbit can be used depending on the allotype desired.
• the rabbit immunoglobulin genes can be isolated from either genomic material containing the gene expressing the variable region or the messenger RNA (mRNA) which represents a transcript of the variable region.
• mRNA messenger RNA
• the DNA fragment(s) containing the proper exons are isolated, the introns excised and the introns then spliced in proper order and in proper orientation.
• RNA-DNA heteroduplex An alternative technique employing rearranged B cells is preferred because the constant (C) , diversity (D) and joining (J) im unoglobulin gene regions have translocated to become adjacent, so that the sequence is continuous (free of introns) for the entire variable region.
• the cells must be lysed under RNAse inhibiting conditions. mRNA may be separated from other RNA by oligo-dT chromatography. A complementary strand of cDNA may then be synthesized on the mRNA template, using reverse transcriptase and a suitable primer, to yield an RNA-DNA heteroduplex [Gubler, et al., Gene , 25:263-269 (1983)].
• a second strand of DNA can be made one of several ways, for example, by priming with RNA fragments of the mRNA strand (made by incubating RNA-DNA heteroduplex with RNAse H) , and using DNA polymerase, or by priming with a synthetic oligodeoxynucleotide primer which anneals to the 3' end of the first strand and using DNA polymerase.
• the isolation of polyA selected mRNA and first strand cDNA synthesis can also be accomplished using a commercially available kit available from Invitrogen, Madison, Wisconsin.
• first primers are therefore chosen to hybridize to (i.e., complementary to) conserved regions within the J region, CHI region, hinge region, CH2 region or CH3 region of the immunoglobulin genes. Primers to the J, CHI and hinge regions are preferred.
• second primers are chosen to hybridize with a conserved nucleotide sequence at the 5 1 end of the V H -coding immunoglobulin gene, such as in that area coding for the leader or first framework region.
• One or both of the first and second primers can contain a nucleotide sequence defining an endonuclease restriction site.
• the site can be heterologous to the immunoglobulin gene being amplified, and typically appear at or near the 5' end of the primer.
• the use of primers with restriction sites has the advantage that the DNA can be cut with at least one restriction enzyme which leaves 5' or 3' overhanging nucleotides.
• the double-stranded cDNA produced at the end of the cycles is readily insertable into a cloning vector by using an appropriate restriction enzyme, which can be chosen empirically based upon the present disclosure.
• the choice of restriction sites is such that the double stranded (ds) cDNA is cloned directly into an expression vector.
• the heavy chain primer pair consists of a V H primer and a J H primer, each containing convenient restriction sites for cloning.
• VHal specific oligonucleotide primers Table 1
• the 39 base pair 5• V H primer was designed to be degenerate for two different nucleotides at two positions.
• a 24 base pair J H region oligonucleotide can be designed for reverse priming at the 3• end of the heavy chain variable gene.
• the primers can then be tested by PCR using cloned genomic DNA containing VDJ gene segments encoding VHal allotypes [M. Suter, et al. , . Immunol., supra] .
• the primers do not need to have a sequence exactly complementary to the target sequence to which it is annealed. Differences can arise for instance because of nucleotide variations or because of the introduction of a restriction enzyme site.
• the DNA polymerase used in this method may be any DNA polymerase known in the art, for example any of those commercially available such as Taq polymerase, or Vent® polymerase (New England Biolabs, Inc.).
• the conditions to be used for each polymerase are well known.
• the polymerase reaction will be carried out in the presence of the four nucleoside triphosphates. These and the polymerase enzyme may already be present in the sample or may be provided afresh for each cycle.
• Denaturing of the DNA strands may be carried out by any known method, for instance, by heating the sample.
• a suitable cycle of heating comprises denaturing at about 95°C for about one minute, annealing at from 30°C to 65°C for about one minute, and primer extension at about 75°C for about two minutes.
• the mixture after the final cycle is preferably held at about 72°C for about five minutes when Vent® polymerase is used.
• the product double-stranded cDNA may be separated from the mixture, for instance, by gel- electrophoresis using agarose gels.
• double-stranded cDNA may be used in unpurified form and inserted directly into a suitable cishinging or expression vector by conventional methods. This will be particularly easy to accomplish if the primers include restriction enzyme recognition sites.
• the combinatorial bacteriophage ⁇ vector [D. Huse, et al., Science, supra] can be used.
• the system is commercially available from Stracyte, California, USA. Briefly, amplified DNA is purified using, for example, anionic exchange columns (Quiagen, Kontron, CH) and ethanol precipitated. The purified and ethanol precipitated DNA is digested with the appropriate restriction enzymes at 2-3 fold excess and repurified as described above.
• the amount of insert and vector DNA is estimated using a DNA-indicator system (Invitrogen) .
• E. coli XL-1 Blue cells are infected with approximately 10 phage particles and plated at a density to obtain 30 - 35 x 10 3 pfu/150 mm petri dish. Rescreening is done using biotinylated protein C and 125 I-labelled Streptavidin [M. Suter, Mol . Immunol . , supra] .
• the expression vector pSWl-VHpolyTagl can also be used to express rabbit VDJ genes [Ward, et al., Nature , 381:544-546 (1989)].
• a fusion protein using a single Fc binding portion of staphylococcal protein A (SpA, Pharmacia, Uppsala, S) can be constructed.
• the coding region of the Fc binding part is amplified by PCR and cloned in frame into the PstI site resulting in plasmid pSWl-VHSpApolyTagl.
• Fusion proteins expressed by pSWl-VHpSpApolyTagl are detected by ELISA using purified rabbit Ig coated to 96 well microtiter wells as described above to immobilize the unique Fc binding portion of SpA, followed by a suitable detection system.
• a system to select for binding of antibody, e.g. , single domain antibodies to a selected antigen, to identify desired antibodies from a starting population of greater than 10 6 proteins [McCafferty, et al., Nature , 348:552-554 (1990)].
• phage antibodies filamentous bacteriophage carrying fusion proteins between sFv and the viral gene III surface protein
• the gene III protein is responsible for the attachment of the phage to the F pilus of the target bacterium [Kornberg, "DNA Replication” (Freeman, San Francisco) (1980) ] .
• the gene III protein can tolerate the presence of polypeptides (including sFv fragments of antibodies) inserted near the amino terminus without loss of function [Smith, et al., Science , 228:1315-1317 (1985); Parmley and Smith, Gene , 73:305-318 (1988); Scott and Smith, Science , 249:386-390 (1990); Devlin, et al.. Science , 249:404-406 (1990)].
• replication-competent phage displaying functional antibody variable regions on their surface can be generated.
• Phage containing functional antibody fragments with desirable phenotypes can be enriched by multiple rounds of affinity chromatography [McCafferty, et al. , supra] .
• Specificity of antigen - binding can be shown by competitive inhibition and by ELISA.
• the competitive inhibition test is performed as described in D. Huse, et al.. Science, supra .
• predetermined amount of biotinylated protein X is inhibited by increasing amounts of native protein X.
• the remaining amount of biotinylated protein bound to nitrocellulose immobilized single domain antibodies is determined using 125 I-labelled SA followed by autoradiography.
• Autoradiography obtained by competitive inhibition tests can be scanned using, for example, an IMCO 1000 V 2.0 image analysis system (Kontron, Kunststoff, FRG) . The system allows to integrate grey values and surface areas of autoradiographs.
• Supernatant containing secreted proteins are directly coated to 96 well Immulon II plates, followed by biotinylated protein X and SA- peroxidase as described above.
• VDJ genes encoding proteins with binding activity to the protein of interest can be analyzed by comparing the site of the genes by polyacrylamide gel electrophoresis [T. Maniatis, et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, USA (1982)].
• the genes can be further analyzed by temperature shift gels (Diagen, D ⁇ sseldorf, FRG) . This system allows determination of single base pair differences between genes and hence allows identification of duplicate clones [D. Riesner, et al., Electrophoresis , 10:377-389 (1989)].
• Rabbit single domain antibodies obtained by recombinant technologies and prokaryotic expression systems in accordance with the present invention produce correctly folded single domain antibodies that can be used in immunological capture assays, for example, sandwich ELISA assays.
• sandwich ELISA assays the single domain antibody is first coated to the plastic solid phase. After washing, a sample containing the antigen is incubated with a labeled antibody probe. A detection system is then used to determine the presence or absence of the antigen, as well as its concentration.
• the single domain antibodies of the present invention could be used for affinity isolation procedures to purify the antigen of interest from a mixture of molecules.
• the relative low affinity of single domain antibodies may be an advantage in affinity isolation because the release of the antigen from the single domain antibody after washing non- reacted molecules is more effective if low affinity ligand receptor interactions are used.
• the animals were boosted twice in three-week intervals using 50 ⁇ g of protein C emulsified in incomplete Freud's adjuvant and given subcutaneously.
• Ten days after the last injection rabbits were test bled and sera were assayed for binding to protein C using an enzyme-linked immuno sorbent assay (ELISA) .
• ELISA enzyme-linked immuno sorbent assay
• sera were precipitated with ammonium sulfate [M. Suter, Vet . Med. , supra] and 5 ⁇ g/ml of the precipitated protein was bound to 96 well microtiter plates (Im ulon II, Nunc, Denmark) as described in J. Bulter, et al., "Enzyme-Mediated Immunoassay" (T. T. Ngo, H.M. Lenhoff, Eds.) pp.
• the primers were tested by PCR using cloned genomic DNA containing VDJ gene segments encoding VHal allotypes [M. Suter, et al, J. Immunol. , supra]. A 350 bp fragment of defined size was amplified and the molecule was expressed in the SpA modified vector pSWl- VHSpApolyTagl [Ward, Nature, supra]. In ELISA, the expressed molecule (clone 4K7) reacted specifically with VHal specific alloantisera as expected but not with VHa2 specific alloantisera used as control. Hence, the primers could be employed to amplify cloned VDJ genes and the same primers were therefore further used to amplify VDJ gene segments from mRNA obtained from B cells of immunized rabbits.
• PBL peripheral blood
• RNA-cDNA hybrid Fifty ng of the RNA-cDNA hybrid was amplified in 30 cycles by the polymerase chain reaction (PCR) using GeneAmp amplification reagents (Perking Elmer Cetus, Norwalk, Connecticut, USA) and one unit Taq polymerase (Perking Elmer Cetus) in a thermalcycler (Techne PHC-2,
• Oligonucleotide primers 100 pmoles used for the PCR were specific for the framework region I of VH (5* primer) and specific for the JH region (3' primer). The primers are set out in Table 1. The condition for one cycle were: Melting, 94C, 30 s; annealing, 55C, 30 s; extension, 72C for 10 minutes. An aliquot of the reaction mixture was run on a 2% agarose gel. Analysis of the gel indicated that a broad band of approximately 350 bp was amplified ( Figure 1).
• VH molecules Approximately 30% of the cloned and expressed VH molecules were positive on autoradiography when the blots were reacted with 1251 SA using VHal specific allotypic antisera and less than 1 % when VHa2 specific allotypic antisera were used.
• the library shown to contain VHal encoding molecules and hence a single domain antibodies of rabbit VDJ origin was screened for binding to the antigen protein C.
• Ten clones were rescreened using biotinylated protein C and 1251 SA.
• Three positive clones (12.1, 17.1 and 16.1) and one negative clone (13.2) were further analyzed by inhibition tests and ELISA.
• Phagemids of phage from the clones 21.1, 17.1, 16.1 and 13.2 were excised in vivo and their gene product analyzed by ELISA. Single colonies containing excised plasmids from each phage (21.1, 17.1, 16.1 and 13.2) were grown in liquid cultures, induced with IPTG and culture supernatants containing secreted proteins were bound directly to 96 well plates. The washed plates were incubated with 100 ng ml-1 of biotinylated protein C followed by SA - peroxidase. The substrate conversion is shown as a function of induction time ( Figure 3). The results confirm the inhibition experiments indicating specific binding of clones 21.1, 17.1,
• VDJ genes encoding sDAb were sized on polyacrylamide gels and all clones were estimated to be approximately 350 bp long. All clones were slightly different in size when compared to each other and to the previously cloned VDJ gene 4K7 obtained from a transgenic rabbit [M. Suter, et al., J. Immunol supra]. The absence of identical clones was further examined by temperature shift gels confirming the isolation of separate clones from the library. EXAMPLE ⁇
• Example II 100 1 of 0.1 M carbonate buffer (pH 9.6) containing 5 g/ml of the purified rabbit single domain antibody obtained in Example I is added to each well of a 96-well polystyrene microtiter plate. The plate is then incubated at 4C for 12 hours. After incubation, the plate is washed with phosphate buffered saline (PBS) including 0.5% Tween 20 (washing buffer).
• PBS phosphate buffered saline
• ADDRESSEE LOUISE S. PEARSON; BAXTER DIAGNOSTICS, INC.

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