WO1995014710A1 - Chemically modified binding protein - Google Patents
Chemically modified binding protein Download PDFInfo
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- WO1995014710A1 WO1995014710A1 PCT/US1994/013549 US9413549W WO9514710A1 WO 1995014710 A1 WO1995014710 A1 WO 1995014710A1 US 9413549 W US9413549 W US 9413549W WO 9514710 A1 WO9514710 A1 WO 9514710A1
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- binding
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- protein
- antibody
- pka
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
Definitions
- the present invention relates generally to the chemical modification of binding proteins such as antibodies, enzymes, receptors and lectins.
- the modified binding proteins are characterized by having a pH-dependent binding activity while still retaining their specificity of binding for their binding partners, namely, antigens/haptens in the case of antibodies; substrates in the case of enzymes, ligands in the case of receptors and carbohydrate moieties present on, for example, glycoproteins, in the case of lectins.
- the present invention relates specifically to chemically modified antibodies, uses thereof and methods for their preparation.
- the design of regulatory mechanisms into functional proteins is one of the goals of protein engineering.
- the activity of enzymes can be regulated by genetically engineering a metal-switch, by a photo-activated switch introduced via covalent modulation, or by unnatural amino acid mutagenesis of the enzyme.
- Antibodies play an important role not only in vivo but are also widely used tools in diagnostics and therapy.
- modified antibodies such as bi-specific antibodies, toxindrug- or enzyme-antibody conjugates have been constructed for biomedical applications (for review see "Immunological Reviews", 1992).
- the conformation and binding affinity of most antibodies are unaltered within a wide range of pHs, namely, pH 3.5-10.0 (Jiskoot et al. , 1991 and Sawada et al., 1993) .
- the antibody-antigen complex does not dissociate under mild nondenaturing conditions, eg. physiological pH (pH at or near 7.0), and thus cheotropic conditions are required to release the antigens (e.g. proteins) from the antibodies.
- mild nondenaturing conditions eg. physiological pH (pH at or near 7.0)
- cheotropic conditions are required to release the antigens (e.g. proteins) from the antibodies.
- Many proteins are however sensitive to such denaturing conditions, for example, gamma- interferon is sensitive to acidic pH, requiring elution at a basic pH and under mild conditions, i.e., nondenaturing conditions.
- antibodies may also be applied in cell- separation procedures, e.g. cell-sorting methods, in which immobilized antibodies specific for cell-surface antigens are employed that bind to those cells in the mixed cell
- SUBSTITUTE SHEET (BULE 26) population carrying the specific antigens, thereby enabling the specific separation and isolation (either enrichment or elimination) of the desired cells from the mixed population of cells.
- certain problems arise, the chief problem being the detachment or separation of the cells from the antibodies, as cells are sensitive to extreme conditions, i.e., denaturing conditions, for example, highly acidic or basic pH; the cells require gentle conditions, i.e., non-denaturing conditions, for example, pH conditions that are at or near to physiological pH conditions.
- antibodies may also be applied in cell- purging methods, but here too, recovery of the cells from the antibodycell complexes requires gentle conditions.
- an antibody for use in, amongst others, the above noted methods, that is capable of releasing the bound antigen (or cell) under non-denaturing conditions, eg. at or near to the conditions of physiological solutions and pH.
- Such an antibody has heretofore not been described.
- the technical problem underlying the present invention was to provide modified antibodies which still retain their inherent highly specific antigen-binding charateristics but which are rendered pHsensitive, namely, are capable of releasing the antigen under mild pH conditions i.e, at or near physiological pH.
- tetranitromethane chemically "mutates” or modifies the binding site of antibodies so that the modified antibodies exhibit pH- dependent binding near physiological pH.
- TAM tetranitromethane
- the present invention provides a modified protein selected from the group of binding proteins consisting of antibodies, enzymes, lectins and receptors which bind specifically to their respective binding partners selected from the group consisting of antigens/haptens, substrates, carbohydrates moieties and ligands, said protein being characterized by:
- a modified antibody having at least one modified amino acid residue in the or near to complementarity-determining region (CDR) , said antibody being capable of binding to said antigen/hapten at a pH of less than the pKa, e.g. pH 6.0, and being capable of releasing said antigen/hapten at pH higher than the pKa, e.g. pH 8.0, and said antibody retaining its specificity of binding to said antigen/hapten.
- the present invention also provides a method for preparing the above modified protein of the invention, said method being selected from the group of procedures consisting of:
- said genetic engineering procedure comprises a step in which a chemically-modified amino acid residue is added, in place of its unmodified counterpart, to the medium in which cells (prokaryotic or eukaryotic) are cultured, said cells expressing the gene which encodes said protein, said modified amino acid residue having a different pKa when compared with its unmodified counterpart; or alternatively, said genetic engineering procedure comprises a step in which
- SUBSTITUTESHEET(RULE28) cells are transformed with a modified gene encoding said protein, said modified gene encoding for an at least one amino acid substitution in the region encoding the antigen- binding site/complementarity-determining region, binding domain or active site of said protein, when compared with its normal counterpart, the substitution resulting in the incorporation into the synthesised protein of an amino acid residue having a different pKa than the normally encoded (wild type, or naturally occurring) amino acid residue; either of said alternative genetic engineering procedures resulting in the synthesis of a modified protein containing in its said antigen-binding site/complementarity-determining region, binding domain or active site, at least one modified or different amino acid residue when compared with its normal counterpart, said modified protein having a pH- dependent binding activity, when compared to its normal counterpart, binding its binding partner at a pH below or above pKa, e.g. pH 6, and releasing its binding partner at a pH below or above pKa, e.g. pH 8, and said
- the present invention further provides for uses of the above modified protein of the invention, in particular uses of modified antibodies in various applications as are detailed herein below.
- the modified antibodies in accordance with the present invention may be polyclonal or monoclonal antibodies.
- the unmodified polyclonal or monoclonal antibodies may be obtained by way of any of the well known methods of the art for preparing antibodies, or they may be any of the commercially available antibodies.
- the modification of the antibodies in accordance with the present invention may be chemical modification, eg. modification of tyrosine side chains with agents such as tetranitromethane (TNM) , or it may be by genetic mutagenesis with unnatural amino acids using standard methods (Mendel et al., 1991; Noren et al., 1989), or by total chemical synthesis (see for example Milton et al., 1992), using other tyrosine derivatives having a low pKa, eg., fluorotyrosines.
- TPM tetranitromethane
- modifications of the antibodies in accordance with the present invention include all modifications by any of the above noted procedures which will result in a modified antibody having a complementarity- determining region (CDR) or antigen-binding site with amino acid(s) having a different pKa (i.e. lower or higher pKa) .
- CDR complementarity- determining region
- suitable modifications include substitutions of the aromatic ring with electronattracting groups of a chemical agent that cause a reduction in the pKa of the tyrosine residue and consequently, also a reduced pKa of antigen/hapten binding of the modified antibody.
- the tyrosine residues may be chemically modified with agents such as TNM, as noted above, which results in nitrated tyrosine residues, or with agents which cause the iodination or fluorination of tyrosine, i.e which result in iodinated or fluorinated tyrosine residues, which have lowered pKa values as compared to unmodified tyrosine, eg. iodotyrosine has a pKa of about 8 and fluorotyrosine has a pKa of about 7.
- agents such as TNM, as noted above, which results in nitrated tyrosine residues, or with agents which cause the iodination or fluorination of tyrosine, i.e which result in iodinated or fluorinated tyrosine residues, which have lowered pKa values as compared to unmodified tyrosine, eg. iod
- modifications may be effected by applying the various agents to a solution containing the antibodies to be modified using known iodinating or fluorinating reagents, or by the above noted genetic engineering or total chemical synthesis methods in which tyrosine residues are substituted by nitrated, iodinated or fluorinated tyrosine in the reaction (genetic engineering/ chemical synthesis) mixtures, and as such the modified tyrosines are incorporated into newly synthesised antibody molecules.
- any other amino acid residue within the CDR of antibodies which contributes to antigen binding for example, histidine, aspartic acid glutamic acid, lysine or serine, may also be modified in the same way as described above, namely, by chemical modification, genetic mutagenesis with unnatural amino acids, or by total chemical synthesis using derivatives of the amino acid residue to be modified, which derivatives have a perturbated (i.e. lower or higher) pKa.
- antibodies may also, in accordance with the present invention, be applied to other proteins, for example, enzymes, lectins and receptors, whose binding to substrates, carbohydrate moieties or ligands, respectively, it is desired to modify so as provide such proteins with pHdependent binding.
- the amino acid residues to be somodified will be chosen from amongst those which occur most frequently in the substrate-, carbohydrate- or ligand-binding domains of the enzymes, lectins or receptors, respectively, and which are known to contribute to such substrate-, carbohydrateor ligand-binding activity.
- modified proteins being for example, antibodies, enzymes, lectins or receptors, in accordance with the present invention
- modified, pH "on-off” switched antibodies may find use in various existing biotechnological applications of antibodies in which reversible binding under mild non ⁇ denaturing conditions is required.
- Such applications for the so-modified antibodies are as immunosensors (Blumstein et al., 1990) in which it is desirable to recover the antibody and regenerate the sensor; and as noted above, in affinity chromatography and immunosorbent methods, cell separation and cell-purging methods where it is desired to separate the proteins/antigens or cells from the antibodies under mild non-degenerating conditions.
- Another application of such modified antibodies is in various diagnostic kits using solid-phase antibodies i.e. immunodiagnostic kits in which it is desired to re-use the antibody-bound detector means, e.g. antibody-bound detector-stick or detector-paper.
- Fig. l illustrates schematically the haptenic structures and binding specificities of monoclonal antibodies designated U7.6, PT.20, D2.3 described in Examples 1 and 2;
- Fig. 2 is a bar-graph representation of the results obtained for the binding of anti-DNP antibody to hapten following chemical modification of the antibody with T ⁇ M, as described in Example 2;
- Fig. 3 is a graphic representation of the results obtained for the binding of T ⁇ M chemically-modified (full circles) vs. unmodified anti-D ⁇ P (empty circles) antibody to its hapten at various pHs, as described in Example 3;
- Figs. 4A and 4B are, respectively, graphic representations of the results obtained for the binding of unmodified D2.3 and PT.20 antibodies (empty symbols) and following chemical modification with T ⁇ M (full symbols) at pH 5.8 (circles) and at pH 9.0 (triangles), as described in Example 3;
- FIG. 5A and 5B are, respectively, bar-graph representations of the results of affinity chromatography of T ⁇ M-chemically-modified vs. unmodified TJ7.6 antibody on haptenbound agarose beads at pH 5.8 and at pH 9.0, as described in Example 4; and Fig. 6 is a schematic representation of pH- dependency of antibody-hapten complexation, as described in
- the nitrations were performed by the addition of tetranitromethane (TNM, Aldrich) , freshly diluted in acetonitrile, to the antibody solution (0.35-1 mg/ml in 50 mM TBS, pH 8-9) .
- the concentration of the acetonitrile did not exceed 5%.
- the molar ratio of TNM/antibody, the pH, the temperature and the incubation time are all detailed in Example 2 below.
- the reactions were quenched by the addition of +mercaptoethanol (3-4 volumes of lOOmM jS-mercaptoethanol in PBS pH 7.4) and then dialyzed against PBS pH 7.4.
- NT/Ab 3-nitrotyrosine residues per antibody molecule
- the antibodies diluted in saline buffers (50 mM, MES pH 5.8-6.4, phosphate pH 7.0-7.5, tris pH 8.0-9.0, glycine pH 9.010.0) , were incubated for 1-3 hours at room temperature.
- DNP-agarose was prepared by addition of 2,4- dinitrofluorobenzene (DNFB, 25 ⁇ moles) to a stirred slurry of e-aminoethyl-agarose (Sigma, lml) in 1:1 acetone and 0.1M NaHC0 3 ; after 12 hrs the beads were thoroughly washed with propanol, ethanol and then with PBS.
- DNFB 2,4- dinitrofluorobenzene
- Fig. 1 U7.6,1KLH;PT.20,3-KLH;D2.3,4-KLH
- Fig. 1 2,4- Dinitrophenol (compound 2 in Fig. 1, designated herein DNP or DNPOH) and pnitrobenzyl amide (compound 6 in Fig. 1) were used as competitive inhibitors for antibodies TJ7.6 and D2.3, respectively.
- D2.3 is a catalytic antibody which hydrolyses the ester 5 in Fig. 1 (phosphonate 4 in Fig. 1 is a transition state analog for this reaction) .
- the antibodies were reacted with TNM under varying conditions of: time (0.5-2 hrs.), pH (8.0-9.0), temperature (4°C - room temperature) , and molar excess of TNM (50- 10,000). Following the modifications the antibodies were assayed, by ELISA, for binding of the corresponding hapten- BSA conjugate in the pH range 5.8-9.5. All three antibodies could be selectively modified for inactivation of hapten- binding at basic pH which is reversible at pH ⁇ 7.0.
- Fig. 2 shows the modification of the anti-DNP antibody, U7.6, with increasing concentrations of TNM.
- Fig. 4 there is shown the binding of catalytic antibody D2.3 (Fig. 4A) and of anti-phosphotyrosine antibody, PT.20 (Fig. 4B) modified by TNM (full symbols), and unmodified (empty symbols) which was titrated at pH 5.8 (full circles, empty circles) and at pH 9.0 (full-triangles, empty triangles) .
- the initial antibody concentrations used were 2.3 ⁇ m: D2.3 was modified using 125 molar excess of TNM (pH 8.3; 1.5 hrs at 4°C and then 0.5 hrs at room temperature), and PT-20 was modified using a 10,000 molar excess of TNM (pH 9.0; 0.5 hrs at 4°C and then 1 hr at room temperature) .
- 6-BSA Fig. 4A
- 3-BSA Fig. 4B
- FIG. 5A there is shown the affinity chromatography of TNMmodified antibody U7.6 on DNP-agarose.
- Antibody U7.6 (0.35 mg) was modified by TNM (X500) , dialyzed against MES, pH 5.8, and charged on to DNP-agarose beads (20 ⁇ l; equilibrated in MES 5.8). The beads were washed with MES 5.8 (3 washes, each 1.0ml) and then incubated with TBS pH 9.0 (3 times, each 200 ⁇ l) .
- Fig. 5A Fig. 5A
- MES pH 5.8 wash step 3
- TBS pH 9.0 first and second elutions steps 4 and 5, respectively
- the same procedure was repeated with an equal amount of unmodified U7.6 (Fig. 5B) .
- Binding activities are given in optical absorbance (A ⁇ o ⁇ ) and were all measured at the same antibody dilution (about 1:800).
- TNM-modified anti-DNP antibody U7.6 was efficiently absorbed to an affinity matrix, DNP- agarose, at pH 5.8.
- the antibody remained bound to the immunosorbent during exhaustive washings with a pH 5.8 buffer; the modified antibody was then readily released at pH 9.0 (Fig. 5A) .
- An unmodified preparation of U7.6 (Fig. 5B) was not eluted from DNP-agarose under basic conditions, or by any other pH changes (at 2.8-9.5 range), but only in the presence of lOOmM DNPOH (data not shown) .
- EXAMPLE 5 SUMMARY OF EXPERIMENTAL DATA
- Fig. 6 is a schematic representation of antibody- hapten complexation. Nitration of a binding site tyrosine
- the pH dependency of binding of TNM-modified antibodies is the result of site-specific nitration of tyrosine side chains.
- the pKa of the hydroxyl group of a tyrosine side chain is normally around 10.0, yet the pKa of the 3- nitrotyrosine that results from the reaction of TNM with tyrosine, is 7.2 (Sokolovsky et al. , 1967).
- recovery and loss of binding can be ascribed to the protonation and deprotonation, respectively, of the hydroxyl group of a 3- nitrotyrosine side chain at the binding site.
- Hydrogen bonding is possible to any electronegative site at the haptenic determinant, such as the oxygens of the nitro group or the oxyanion of the phosphonate/phosphate groups, present in the haptens to which the antibodies described herein above are complementary (see Fig. 1) .
- the participation of other binding interactions that are interfered with by formation of the phenolate ion at basic pH cannot be ruled out.
- the observed decrease in affinity of the nitrated PT.20 (Fig. 4B) is probably due to side effects, such as steric hindrance, resulting from the presence of the 3-nitro group on the binding-site tyrosine.
- PROTEINS Structure, function and genetics 2, 112.
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Abstract
The present invention relates to a modified protein selected from the group of binding proteins consisting of antibodies, enzymes, lectins and receptors which bind specifically to their respective binding partners selected from the group consisting of antigens/haptens, substrates, carbohydrate moieties and ligands, said protein being characterized by: (i) having at least one modified amino acid residue with a different pKa, when compared with its unmodified counterpart, in or near to its respective antigen-binding region/complementarity-determining region, binding domain or active site for binding to said binding partners; (ii) having a pH-dependent binding activity when compared with its unmodified counterpart, said protein binding to its said binding partner at a pH lower or higher than the pKa and releasing its said binding partner at a pH lower or higher than the pKa; and (iii) retaining its specificity of binding to its said binding partner. Modified antibodies according to the invention are useful, for example, in affinity chromatography and cell separation methods.
Description
CHEMICALLY MODIFIED BINDING PROTEIN
FIELD OF THE INVENTION
The present invention relates generally to the chemical modification of binding proteins such as antibodies, enzymes, receptors and lectins. The modified binding proteins are characterized by having a pH-dependent binding activity while still retaining their specificity of binding for their binding partners, namely, antigens/haptens in the case of antibodies; substrates in the case of enzymes, ligands in the case of receptors and carbohydrate moieties present on, for example, glycoproteins, in the case of lectins.
The present invention relates specifically to chemically modified antibodies, uses thereof and methods for their preparation.
BACKGROUND OF THE INVENTION
The design of regulatory mechanisms into functional proteins is one of the goals of protein engineering. For example, the activity of enzymes can be regulated by genetically engineering a metal-switch, by a photo-activated switch introduced via covalent modulation, or by unnatural amino acid mutagenesis of the enzyme.
Antibodies play an important role not only in vivo but are also widely used tools in diagnostics and therapy. In addition, modified antibodies, such as bi-specific antibodies, toxindrug- or enzyme-antibody conjugates have been constructed for biomedical applications (for review see "Immunological Reviews", 1992). Antibodies which exhibit enzyme-like properties, i.e., catalysis of chemical reactions with rate enhancement, turnover and substrate specificity, are also known (Lerner et al., 1991). In general, the conformation and binding affinity of most antibodies are unaltered within a wide range of pHs, namely, pH 3.5-10.0 (Jiskoot et al. , 1991 and Sawada et al., 1993) . In most cases, the formation of antibody-antigen complexes is practically irreversible and no dissociation is aβSmilTESHEET(RULE36)
observed under mild, non-denaturing, conditions. It has been previously reported that the immunoprecipitation of nitrated anti-DNP rabbit polyclonal antibodies by DNP-ovalbumin exhibited a pH dependency corresponding to the ionization of the nitrotyrosyl group (Givol and Fuchs, 1968) . However, a pH-dependency of binding of nitrated antibodies, or of any other protein to substances (e.g. antigens in the case of antibodies) to which they bind, has not been reported (Lundblad and Noyes, 1984) . There has been reported a situation in which antibodies elicited against a 4-hydroxy- 3-nitrophenyl hapten were found to exhibit a pH- ependency; the ionized form of the haptenic 3nitrophenol group that is formed at pH>8 being bound with low affinity to the antibodies (Azuma et al., 1987). However, in this case the antibodies were not modified antibodies, rather were unmodified antibodies, the above pH-dependency of binding of the antibodies to the hapten being a result of changes in the structure of the hapten when the pH was altered. Antibodies have many applications in biotechnology. One such application is the use of antibodies in affinity chromatography methods for the purification of proteins. However, when the antibody-bound proteins are required to be released from the antibodies, i.e. during the elution step to obtain the protein, problems are often encountered with respect to the type of solvent/eluant used. As noted above, the antibody-antigen complex does not dissociate under mild nondenaturing conditions, eg. physiological pH (pH at or near 7.0), and thus cheotropic conditions are required to release the antigens (e.g. proteins) from the antibodies. Many proteins are however sensitive to such denaturing conditions, for example, gamma- interferon is sensitive to acidic pH, requiring elution at a basic pH and under mild conditions, i.e., nondenaturing conditions. Further, antibodies may also be applied in cell- separation procedures, e.g. cell-sorting methods, in which immobilized antibodies specific for cell-surface antigens are employed that bind to those cells in the mixed cell
SUBSTITUTE SHEET (BULE 26)
population carrying the specific antigens, thereby enabling the specific separation and isolation (either enrichment or elimination) of the desired cells from the mixed population of cells. However, when it is desired to recover and use these isolated cells, certain problems arise, the chief problem being the detachment or separation of the cells from the antibodies, as cells are sensitive to extreme conditions, i.e., denaturing conditions, for example, highly acidic or basic pH; the cells require gentle conditions, i.e., non-denaturing conditions, for example, pH conditions that are at or near to physiological pH conditions.
Similarly, antibodies may also be applied in cell- purging methods, but here too, recovery of the cells from the antibodycell complexes requires gentle conditions. There is, therefore, a long felt need to provide an antibody, for use in, amongst others, the above noted methods, that is capable of releasing the bound antigen (or cell) under non-denaturing conditions, eg. at or near to the conditions of physiological solutions and pH. Such an antibody has heretofore not been described. Accordingly, the technical problem underlying the present invention was to provide modified antibodies which still retain their inherent highly specific antigen-binding charateristics but which are rendered pHsensitive, namely, are capable of releasing the antigen under mild pH conditions i.e, at or near physiological pH. SUMMARY OF THE INVENTION
In accordance with the present invention, it has been surprisingly found that tetranitromethane (TNM) chemically "mutates" or modifies the binding site of antibodies so that the modified antibodies exhibit pH- dependent binding near physiological pH. A number of antibodies were selectively modified using different conditions, which resulted in all of them having a loss of binding activity at pH>8, which is recovered at pH<6, i.e. the modifications afforded the antibodies with pH-dependent binding activity or so-called pH "on-off" switching of binding activity.
SUBSTITUTE SHEET (f.UL£ 26)
Accordingly, the present invention provides a modified protein selected from the group of binding proteins consisting of antibodies, enzymes, lectins and receptors which bind specifically to their respective binding partners selected from the group consisting of antigens/haptens, substrates, carbohydrates moieties and ligands, said protein being characterized by:
(i) having at least one modified amino acid residue with a different pKa, when compared with its unmodified counterpart, in or near to its respective antigen-binding region/complementarity-determining region, binding domain or active site for binding to said binding partners;
(ii) having a pH-dependent binding activity when compared with its ummodified counterpart, said protein binding to its said binding partner at a pH lower or higher than the pKa and releasing its said binding partner at a pH lower or higher than the pKa; and
(iii) retaining its specificity of binding to its said binding partner.
According to one embodiment of the invention, there is provided a modified antibody having at least one modified amino acid residue in the or near to complementarity-determining region (CDR) , said antibody being capable of binding to said antigen/hapten at a pH of less than the pKa, e.g. pH 6.0, and being capable of releasing said antigen/hapten at pH higher than the pKa, e.g. pH 8.0, and said antibody retaining its specificity of binding to said antigen/hapten. The present invention also provides a method for preparing the above modified protein of the invention, said method being selected from the group of procedures consisting of:
(i) chemically modifying said protein by applying to a solution containing said protein a chemical agent capable of modifying at least one amino acid residue in the or near to antigen-binding region/complementaritydetermining region, binding domain or active site of said protein, in a
way that said modified amino acid residue has a different pKa when compared with its unmodified counterpart, said protein containing said modified amino acid residue having a different pKa resulting in a pHdependent binding activity when compared to its unmodified counterpart, binding its binding partner at a pH lower or higher than pKa, e.g. pH 6, and releasing its binding partner at a pH lower or higher than pKa, e.g. pH 8, and said protein containing said modified amino acid residue retaining its specificity of binding to its binding partner;
(ii) modifying said protein by chemically synthesising, using standard procedures, a modified counterpart of said protein, the chemical synthesis comprising a step in which a chemically-modified amino acid residue is introduced in place of its unmodified counterpart at the stage in which the antigen-binding region/ complementarity-determing region, binding domain or active site of said protein is synthesised, said chemically- modified amino acid residue having a different pKa when compared with its natural counterpart, said synthesised protein containing said modified amino acid residue having a pH-dependent binding activity when compared to its unmodified counterpart, binding its binding partner at pH lower or higher than pKa and releasing its binding partner at pH below or above pKa, and said protein containing said modified amino acid residue retaining its specificity of binding to its binding partner; and
(iii) modifying said protein by synthesising, with standard genetic engineering procedures, a genetically- engineered modified counterpart of said protein, the genetic engineering synthesis procedure comprising a step in which a chemically-modified amino acid residue is added, in place of its unmodified counterpart, to the medium in which cells (prokaryotic or eukaryotic) are cultured, said cells expressing the gene which encodes said protein, said modified amino acid residue having a different pKa when compared with its unmodified counterpart; or alternatively, said genetic engineering procedure comprises a step in which
SUBSTITUTESHEET(RULE28)
cells are transformed with a modified gene encoding said protein, said modified gene encoding for an at least one amino acid substitution in the region encoding the antigen- binding site/complementarity-determining region, binding domain or active site of said protein, when compared with its normal counterpart, the substitution resulting in the incorporation into the synthesised protein of an amino acid residue having a different pKa than the normally encoded (wild type, or naturally occurring) amino acid residue; either of said alternative genetic engineering procedures resulting in the synthesis of a modified protein containing in its said antigen-binding site/complementarity-determining region, binding domain or active site, at least one modified or different amino acid residue when compared with its normal counterpart, said modified protein having a pH- dependent binding activity, when compared to its normal counterpart, binding its binding partner at a pH below or above pKa, e.g. pH 6, and releasing its binding partner at a pH below or above pKa, e.g. pH 8, and said modified protein retaining its specificity of binding to its binding partner.
The present invention further provides for uses of the above modified protein of the invention, in particular uses of modified antibodies in various applications as are detailed herein below. The modified antibodies in accordance with the present invention may be polyclonal or monoclonal antibodies. As starting materials, the unmodified polyclonal or monoclonal antibodies may be obtained by way of any of the well known methods of the art for preparing antibodies, or they may be any of the commercially available antibodies.
The modification of the antibodies in accordance with the present invention may be chemical modification, eg. modification of tyrosine side chains with agents such as tetranitromethane (TNM) , or it may be by genetic mutagenesis with unnatural amino acids using standard methods (Mendel et al., 1991; Noren et al., 1989), or by total chemical synthesis (see for example Milton et al., 1992), using other tyrosine derivatives having a low pKa, eg., fluorotyrosines.
In general, modifications of the antibodies in accordance with the present invention include all modifications by any of the above noted procedures which will result in a modified antibody having a complementarity- determining region (CDR) or antigen-binding site with amino acid(s) having a different pKa (i.e. lower or higher pKa) . In respect of the above noted modification of tyrosine side chains, suitable modifications include substitutions of the aromatic ring with electronattracting groups of a chemical agent that cause a reduction in the pKa of the tyrosine residue and consequently, also a reduced pKa of antigen/hapten binding of the modified antibody. Thus, the tyrosine residues may be chemically modified with agents such as TNM, as noted above, which results in nitrated tyrosine residues, or with agents which cause the iodination or fluorination of tyrosine, i.e which result in iodinated or fluorinated tyrosine residues, which have lowered pKa values as compared to unmodified tyrosine, eg. iodotyrosine has a pKa of about 8 and fluorotyrosine has a pKa of about 7. These modifications may be effected by applying the various agents to a solution containing the antibodies to be modified using known iodinating or fluorinating reagents, or by the above noted genetic engineering or total chemical synthesis methods in which tyrosine residues are substituted by nitrated, iodinated or fluorinated tyrosine in the reaction (genetic engineering/ chemical synthesis) mixtures, and as such the modified tyrosines are incorporated into newly synthesised antibody molecules.
While it may be desirable to modify antibodies at tyrosine residues in view of the frequent occurrence of tyrosine residues within the complementarity-determining regions (CDR) of antibodies and the contribution of these residues to hapten, or antigen, binding (Padlan, 1990; Mian et al., 1991), it should be understood that, in accordance with the present invention, any other amino acid residue within the CDR of antibodies which contributes to antigen binding, for example, histidine, aspartic acid glutamic acid, lysine or serine, may also be modified in the same way
as described above, namely, by chemical modification, genetic mutagenesis with unnatural amino acids, or by total chemical synthesis using derivatives of the amino acid residue to be modified, which derivatives have a perturbated (i.e. lower or higher) pKa.
Moreover, it is also to be understood that the above described modifications to antibodies may also, in accordance with the present invention, be applied to other proteins, for example, enzymes, lectins and receptors, whose binding to substrates, carbohydrate moieties or ligands, respectively, it is desired to modify so as provide such proteins with pHdependent binding. Here too, the amino acid residues to be somodified will be chosen from amongst those which occur most frequently in the substrate-, carbohydrate- or ligand-binding domains of the enzymes, lectins or receptors, respectively, and which are known to contribute to such substrate-, carbohydrateor ligand-binding activity. The above mentioned pH-dependent binding activity or pH "on-off" switching of binding activity of the modified proteins, being for example, antibodies, enzymes, lectins or receptors, in accordance with the present invention, may be utilized in many biotechnological applications. For example, the modified, pH "on-off" switched antibodies may find use in various existing biotechnological applications of antibodies in which reversible binding under mild non¬ denaturing conditions is required. Particular examples of such applications for the so-modified antibodies are as immunosensors (Blumstein et al., 1990) in which it is desirable to recover the antibody and regenerate the sensor; and as noted above, in affinity chromatography and immunosorbent methods, cell separation and cell-purging methods where it is desired to separate the proteins/antigens or cells from the antibodies under mild non-degenerating conditions. Another application of such modified antibodies is in various diagnostic kits using solid-phase antibodies i.e. immunodiagnostic kits in which it is desired to re-use the antibody-bound detector means, e.g. antibody-bound detector-stick or detector-paper.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. l illustrates schematically the haptenic structures and binding specificities of monoclonal antibodies designated U7.6, PT.20, D2.3 described in Examples 1 and 2;
Fig. 2 is a bar-graph representation of the results obtained for the binding of anti-DNP antibody to hapten following chemical modification of the antibody with TΝM, as described in Example 2; Fig. 3 is a graphic representation of the results obtained for the binding of TΝM chemically-modified (full circles) vs. unmodified anti-DΝP (empty circles) antibody to its hapten at various pHs, as described in Example 3; Figs. 4A and 4B are, respectively, graphic representations of the results obtained for the binding of unmodified D2.3 and PT.20 antibodies (empty symbols) and following chemical modification with TΝM (full symbols) at pH 5.8 (circles) and at pH 9.0 (triangles), as described in Example 3; Figs. 5A and 5B are, respectively, bar-graph representations of the results of affinity chromatography of TΝM-chemically-modified vs. unmodified TJ7.6 antibody on haptenbound agarose beads at pH 5.8 and at pH 9.0, as described in Example 4; and Fig. 6 is a schematic representation of pH- dependency of antibody-hapten complexation, as described in
Example 5.
DETAILED DESCRIPTION OF THE INVENTION
The invention will now be described in more detail in the following non-limiting examples with reference to the figures:
EXAMPLE 1: GENERAL PROCEDURES (a) Preparation of monoclonal antibodies:
Generation and characterization of the monoclonal antibodies (MABs) and preparation of the corresponding haptenbovine serum albumin (BSA) conjugates were carried out as previously described: D2.3 (Tawfik et al. , 1993), PT.20 (Shisheva et al. , 1991) and U7.6 was generated as described
for other anti-DNP mouse monoclonal antibodies (Eshhar et al., 1980), the contents of these references being included herein by reference.
The antibodies were purified from ascites by protein A (Pharmacia) affinity chromatography and dialyzed against Tris buffered saline (TBS, 50mM tris (hydroxymethyl) aminomethane/0.14M NaCl, pH 8.0-9.0) . Homogeneity of the purified preparations was judged by SDS/PAGE under reducing conditions with Coomassie blue staining, which yielded only heavy and light chains. Antibody concentrations were determined by measuring the optical density at 280nm (e=1.45 mg.ml"1.cm"1 at pH 8.0) .
(b) Nitrations of antibodies:
The nitrations were performed by the addition of tetranitromethane (TNM, Aldrich) , freshly diluted in acetonitrile, to the antibody solution (0.35-1 mg/ml in 50 mM TBS, pH 8-9) . The concentration of the acetonitrile did not exceed 5%. The molar ratio of TNM/antibody, the pH, the temperature and the incubation time are all detailed in Example 2 below. The reactions were quenched by the addition of +mercaptoethanol (3-4 volumes of lOOmM jS-mercaptoethanol in PBS pH 7.4) and then dialyzed against PBS pH 7.4. The controls, i.e., unmodified antibodies, were treated as described above except that acetonitrile without TNM was added. The number of 3-nitrotyrosine residues per antibody molecule (NT/Ab) was determined by measuring the optical absorbance at 428nm (e=4100 M"1.cm'1 at pH 8.3) (Riordan et al. , 1966) .
(c) Determination of antibody-binding activities: The binding activities were determined by ELISA.
Microtiter plates (Nunk, Maxisorb) were coated with the hapten-BSA conjugates (see Fig. 1 where: 1=BSA, 5 μg/ml; 3=BSA, 0.5 μg/ml; 6=BSA, l μg/ml; 4=BSA, 1 μg/ml; were all incubated for 1 hour at room temperature) and then blocked with BSA (1 mg/ml for 30 mins) . The antibodies, diluted in saline buffers (50 mM, MES pH 5.8-6.4, phosphate pH 7.0-7.5, tris pH 8.0-9.0, glycine pH 9.010.0) , were incubated for 1-3 hours at room temperature. The plates were then washed with
PBS + 0.04% Tween 20 (PBS/T) and peroxidase-linked goat anti-mouse immunoglobulin antibodies (Jackson ImmunoResearch, diluted 1:5000 in PBS) were added. After washing with PBS/T, the substrate, 2,2' -azinobis- (3- ethylbenzthiazoline-6-sulfonic acid) (ATBS) , was added and the absorbance at 690 nm was measured. Esterolytic activity of D2.3 was determined with ester 5 (see Fig. 1, Example 2) by catELISA (Tawfik et al. , 1993). (d) Preparation of DNP-agarose: DNP-agarose was prepared by addition of 2,4- dinitrofluorobenzene (DNFB, 25 μmoles) to a stirred slurry of e-aminoethyl-agarose (Sigma, lml) in 1:1 acetone and 0.1M NaHC03; after 12 hrs the beads were thoroughly washed with propanol, ethanol and then with PBS. EXAMPLE 2: ANTIBODY MODIFICATIONS
The results of antibody modification by TNM as described in 1(b) above, was examined with three different monoclonal antibodies: TJ7.6, PT.20 and D2.3. The haptenic structures and binding specificities of these antibodies are shown schematically in Figure 1.
All three antibodies were elicited against the hapten-KLH (keyhole limpet hemocyanin) conjugates (Fig. 1: U7.6,1KLH;PT.20,3-KLH;D2.3,4-KLH) and their binding was determined using the corresponding BSA conjugates. 2,4- Dinitrophenol (compound 2 in Fig. 1, designated herein DNP or DNPOH) and pnitrobenzyl amide (compound 6 in Fig. 1) were used as competitive inhibitors for antibodies TJ7.6 and D2.3, respectively. D2.3 is a catalytic antibody which hydrolyses the ester 5 in Fig. 1 (phosphonate 4 in Fig. 1 is a transition state analog for this reaction) .
The antibodies were reacted with TNM under varying conditions of: time (0.5-2 hrs.), pH (8.0-9.0), temperature (4°C - room temperature) , and molar excess of TNM (50- 10,000). Following the modifications the antibodies were assayed, by ELISA, for binding of the corresponding hapten- BSA conjugate in the pH range 5.8-9.5. All three antibodies could be selectively modified for inactivation of hapten- binding at basic pH which is reversible at pH<7.0.
Fig. 2 shows the modification of the anti-DNP antibody, U7.6, with increasing concentrations of TNM. The U7.6 antibody (7.6 μM) , was reacted with increasing concentrations of TNM (X50-2000 molar excess - see Table under bar-graph in Fig. 2; pH 8.3; 1.5 hours at 4°C and then 0.5 hours at room temperature). After quenching and extensive dialysis, the number of 3-nitrotyrosines per antibody (NT/Ab) were determined for each sample. The antibodies were then diluted and binding to DNP-BSA (1=BSA) at pH 5.8 (solid bars in Fig. 2) and at pH 9.0 (empty bars in Fig. 2) were determined by ELISA. Sample no. 1 is control (1-BSA) . Samples no. 2, no. 5 and no. 9 were obtained by modification of the antibodies in the presence of ImM DNPOH by standard methods. As shown in Fig. 2, optimal results for TJ7.6 were obtained using a 200 molar excess of TNM at pH 8.3 (sample no. 6). Under more extreme conditions, e.g., a larger excess of TNM (Fig. 2, sample no. 10 and Fig. 3), or higher pH and temperature, the antibody was irreversibly inactivated, i.e., hapten-binding ability was not recovered at acidic pH.
In order to demonstrate that modifications occur in the binding site, monoclonal antibody MAB U7.6 was reacted with TNM in the presence of the haptenic group DNPOH. Binding activity of the antibody was not affected under these conditions (Fig. 2, sample no. 5). Quantitative determination of the number of 3-nitrotyrosines per antibody observed after treatment with TNM, and comparison to the results of the same modifications in the presence of the hapten (Fig. 2) , indicate that the pHdependent binding activity of U7.6 is the result of a sitespecific modification of one tyrosine residue (i.e., two tyrosines per antibody molecule; Fig. 2, sample no. 4 vs. no. 5). Even in the presence of a larger excess of TNM (a 1000 molar excess = 7.6 mM; Fig. 2, sample no. 8) the presence of the hapten 2 (ImM; Fig. 2, sample no. 9) affords 60% protection.
Protection by the haptenic group is also observed with MAB D2.3 reacted with TNM in the presence of the hapten 6 in Fig. 1 (ImM) .
EXAMPLE 3: pH-DEPENDENT BINDING OF MODIFIED ANTIBODIES
For two of the antibodies studied, the conditions under which the binding activity after nitration is fully regained at low pH were readily found (see Fig. 3 in respect of antibody U7.6 and Fig. 4A in respect of antibody D2.3). In Fig. 3 there is illustrated the binding of anti-DNP antibody U7.6, either modified by TNM, (full- circles) or unmodified, (empty-circles) , which was measured at various pHs. U7.6 (7.6 μM) was reacted with TNM (1.5 mM) . After quenching and dialysis, the antibody was diluted in buffers at the indicated pHs and binding to 1=BSA was determined by ELISA (data given at about 1:1000 dilution).
In Fig. 4 there is shown the binding of catalytic antibody D2.3 (Fig. 4A) and of anti-phosphotyrosine antibody, PT.20 (Fig. 4B) modified by TNM (full symbols), and unmodified (empty symbols) which was titrated at pH 5.8 (full circles, empty circles) and at pH 9.0 (full-triangles, empty triangles) . The initial antibody concentrations used were 2.3μm: D2.3 was modified using 125 molar excess of TNM (pH 8.3; 1.5 hrs at 4°C and then 0.5 hrs at room temperature), and PT-20 was modified using a 10,000 molar excess of TNM (pH 9.0; 0.5 hrs at 4°C and then 1 hr at room temperature) . After quenching and dialysis, the antibodies were diluted in 50mM MES pH 5.8 or in 50mM TBS pH 9.0 buffers, and binding to the hapten-conjugate. 6-BSA (Fig. 4A) and 3-BSA, (Fig. 4B) were determined by ELISA.
In the case of antibody D2.3, modification under the conditions described (Fig. 4A) resulted in a pH- dependent binding to p-nitrobenzyl amide 6-BSA. Binding to the phosphonate hapten (4-BSA) remained pH-independent (data not shown) and this may be ascribed to the significantly higher affinity of D2.3 to phosphonate 4 hapten vs. amide 6 (Tawfik et al., 1993).
Esterolytic activity (towards p-nitrobenzyl ester 5) of the TNM-modified D2.3 at pH<6.5 (i.e., where substrate binding is regained; Fig. 4A) was found to be negligible. Yet, since unmodified D2.3 is also inactive at pH<6.5, it was not possible to determine whether catalytic activity of
D2.3 is affected by the nitration. For the anti- phosphotyrosine antibody, PT.20, more extreme conditions were required to observe any tyrosine modification; as a result, binding was not fully recovered at acidic pH (Fig. 4B) .
EXAMPLE 4: AFFINITY CHROMATOGRAPHY WITH THE MODIFIED ANTIBODIES
Affinity chromatography of TNM-modified antibodies was performed on the basis of their pH-dependent binding activity. In Fig. 5A there is shown the affinity chromatography of TNMmodified antibody U7.6 on DNP-agarose. Antibody U7.6 (0.35 mg) was modified by TNM (X500) , dialyzed against MES, pH 5.8, and charged on to DNP-agarose beads (20μl; equilibrated in MES 5.8). The beads were washed with MES 5.8 (3 washes, each 1.0ml) and then incubated with TBS pH 9.0 (3 times, each 200μl) . The various fractions were dialyzed and diluted in MES 5.8 and TBS 9.0 and binding to l-BSA was determined by ELISA at pH 5.8 (solid bars) and at pH 9.0 (empty bars): TNM-modified U7.6, before and after incubation with DNP-agarose (steps 1 and 2 respectively in
Fig. 5A) ; MES pH 5.8 wash (step 3); and TBS pH 9.0 first and second elutions (steps 4 and 5, respectively) . The same procedure was repeated with an equal amount of unmodified U7.6 (Fig. 5B) . Binding activities are given in optical absorbance (A^o^) and were all measured at the same antibody dilution (about 1:800).
It is shown that TNM-modified anti-DNP antibody, U7.6, was efficiently absorbed to an affinity matrix, DNP- agarose, at pH 5.8. The antibody remained bound to the immunosorbent during exhaustive washings with a pH 5.8 buffer; the modified antibody was then readily released at pH 9.0 (Fig. 5A) . An unmodified preparation of U7.6 (Fig. 5B) was not eluted from DNP-agarose under basic conditions, or by any other pH changes (at 2.8-9.5 range), but only in the presence of lOOmM DNPOH (data not shown) . EXAMPLE 5: SUMMARY OF EXPERIMENTAL DATA
Fig. 6 is a schematic representation of antibody- hapten complexation. Nitration of a binding site tyrosine
SUBSTITUTE SHEET (BOLE 26)
(R=H) gives 3-nitrotyrosine (R=N02) and thereby lowers its pKa from about 10 to about 7. Deprotonation of the nitrated tyrosine which occurs at pH>pKa results in disassociation of the haptenantibody complex. Such disassociation with the unmodified antibody (R=H) can occur only at much higher pH values.
As has been set forth in the preceding Examples 2- 4, the pH dependency of binding of TNM-modified antibodies is the result of site-specific nitration of tyrosine side chains. The pKa of the hydroxyl group of a tyrosine side chain is normally around 10.0, yet the pKa of the 3- nitrotyrosine that results from the reaction of TNM with tyrosine, is 7.2 (Sokolovsky et al. , 1967). Thus, recovery and loss of binding can be ascribed to the protonation and deprotonation, respectively, of the hydroxyl group of a 3- nitrotyrosine side chain at the binding site. These results suggest that interaction of tyrosine hydroxyl groups with the haptenic group may involve hydrogen bonding and that the phenolate anion, present at pH>pKa, cannot donate to such interaction.
Hydrogen bonding is possible to any electronegative site at the haptenic determinant, such as the oxygens of the nitro group or the oxyanion of the phosphonate/phosphate groups, present in the haptens to which the antibodies described herein above are complementary (see Fig. 1) . The participation of other binding interactions that are interfered with by formation of the phenolate ion at basic pH cannot be ruled out. The observed decrease in affinity of the nitrated PT.20 (Fig. 4B) is probably due to side effects, such as steric hindrance, resulting from the presence of the 3-nitro group on the binding-site tyrosine.
The difference of affinities exhibited by nitrated antibodies in acidic vs. basic conditions is significant enough to be exploited for practical applications as noted above. This was demonstrated, by way of example only, by the affinity purification of the nitrated anti-DNP antibody U7.6 in Fig. 5A, in which it is shown that a sample of the
nitrated U7.6 antibody was absorbed ( about 95%) on to DNP- agarose at pH 5.8 and was rapidly eluted with close to 100% yield, just by increasing the pH to 9.0. As shown in Fig. 5B, such pH changes, or even more drastic ones, cannot release the unmodified antibodies from the affinity matrix. It should be noted that not in all antibodies, or the other above noted binding proteins, is it expected that the above mentioned modification of tyrosine will afford a complete pH "on-off" effect. However, for practical applications, a reduction in binding affinity of only two or three orders of magnitude should be useful for the various above mentioned applications of the present invention.
Thus, in accordance with the present invention, there is provided a way in which chemical modification of tyrosines may be applied for engineering a pH "on-off" binding switch (or pHdependency of binding affinity) in antibodies. While the preceding non-limiting examples relate to chemical mutagenesis of antibodies, it is to be understood that any of the other herein above mentioned methods may be used to modify the antibodies. Further, it is also to be understood that while the above non-limiting examples concern antibody modifications, the same modifications may also be applied to other proteins, e.g. enzymes, lectins and receptors, as also mentioned herein above. Moreover, the uses of the above noted modified antibodies are also to be understood as not being limited to affinity chromatography applications, but rather, such modified antibodies have many other utilities as described herein above.
REFERENCES
Azuma, T. ; Sakato, N; Fujio,H. (1987) Molecul.Immunol. 2_4, 287.
Blumenstein, B.I.; Walczak, I.M.; Chen, S-Y. (1990) Trends Biotechnol. 8., 161.
Eshhar, Z.; Ofarim, M. ; Waks, T. (1980) J.Immunol. 124. 775.
Givol, D.; Fuchs, S. (1968) FEBS Lett. 2 , 45. Immunological Reviews (1992) Moller, G. (Ed) , Vols.129 and 130.
Jiskoot, W. ; Bloemendel, M. ; Haeringen, B.; Grondelle; R. , Beuvery C.E., Herron, J.;Crommelin D.J.A. (1991), Eur.J.Biochem. 201. 223.
Lerner, R.A. ; Benkovic, S.J.; Schultz, P.G. (1991) Science 252, 659.
Lundblad, R.L.; Noyes, CM. (1984) Chemical Reagents for Protein Modification, CRC Press, Boca Raton, Florida, Vol.2, pp.239-265.
Mendel, D., Ellman, J.A.; Schultz, P.G. (1992) J.Amer.Chem.Soc. 113. 2758.
Mian, I.S.; Bradwell, A.R.; Olson, A.J. (1991) J.Mol.Biol. 2T7, 133.
Milton, R.C. deL.; Milton, S.C.F.; Kent, S.B.H. (1992) Science 251, 1445. Noren, C.J. et al. (1989) Science 244. 182.
Padlan, E.A. (1990) PROTEINS: Structure, function and genetics 2, 112.
Riordan, J.F.; Sokolovsky, M. ; Valee, B.L. (1966) J. A er. Chem. Soc. 8J3, 4104. Sawada, J. ; Yamazaki, T. ; Terao, T. (1993)
Molecul.Immunol. 3j0, 77 (and references therein).
Shisheva, A.; Leitner, 0.; Shechter, Y. (1991) J. Biochem. Biophys. Meth. 23., 307.
Sokolovsky, M. ; Riordan, J.F.; Valee, B.L. (1967) Biochem. Biophys. Res. Comm. 2_7, 20.
Tawfik, D.S.; Green, B.S.; Chap, R.; Sela; M. ; Eshhar, Z. (1993) Proc.Natl.Acad.Sci.USA 9_0, 373.
SUBSπτUIE SHEET (RULE 26)
Claims
1. A modified protein selected from the group of binding proteins consisting of antibodies, enzymes, lectins and receptors which bind specifically to their respective binding partners selected from the group consisting of antigens/haptens, substrates, carbohydrate moieties and ligands, said protein being characterized by:
(i) having at least one modified amino acid residue with a different pKa, when compared with its unmodified counterpart, in or near to its respective antigen-binding region/complementarity-determining region, binding domain or active site for binding to said binding partners; (ii) having a pH-dependent binding activity when compared with its ummodified counterpart, said protein binding to its said binding partner at a pH lower or higher than the pKa and releasing its said binding partner at a pH lower or higher than the pKa; and (iii) retaining its specificity of binding to its said binding partner.
2. A modified protein according to claim 1, wherein said binding protein is an antibody, said binding partner is an antigen/hapten, said modified amino acid residue is in the complementarity-determining region (CDR) , said antibody being capable of binding to said antigen/hapten at a pH lower or higher than'the pKa and being capable of releasing said antigen/hapten at a pH lower or higher than the pKa, and said antibody retaining its specificity of binding to said antigen/hapten.
3. A modified antibody according to claim 2, wherein said antibody is a monoclonal antibody.
4. A modified antibody according to claim 2 or claim 3, wherein said antibody has a modified tyrosine residue in or near to its CDR, said modified tyrosine residue having a reduced pKa when compared with its unmodified counterpart, said modified tyrosine residue being selected from the group consisting of nitrated, iodinated or
SUBSTITUTE SHEET (i.ϋ E 26) fluorinated tyrosine residues.
5. A modified antibody according to claim 4, wherein said modified tyrosine residue is a nitrated tyrosine residue.
6. A method for preparing a modified protein according to any one of claims 1-5, said method being selected from the group of procedures consisting of:
(i) chemically modifying said protein by applying to a solution containing said protein a chemical agent capable of modifying at least one amino acid residue in the or near to antigen-binding- egion/complementaritydetermining region, binding domain or active site of said protein, in a way that said modified amino acid residue has a different pKa when compared with its unmodified counterpart, said protein containing said modified amino acid residue having a pH-dependent binding activity when compared to its unmodified counterpart, binding its binding partner at a pH lower or higher than pKa and releasing its binding partner at a pH lower or higher than pKa, and said protein containing said modified amino acid residue retaining its specificity of binding to its binding partner;
(ii) modifying said protein by chemically synthesising, using standard procedures, a modified counterpart of said protein, the chemical synthesis comprising a step in which a chemically-modified amino acid residue is introduced in place of its unmodified counterpart at the stage in which the antigen binding region/ complementarity-determining region, binding domain or active site of said protein is synthesised, said chemically- modified amino acid residue having a different pKa when compared with its natural counterpart, said synthesised protein containing said modified amino acid residue having a pH-dependent binding activity when compared to its unmodified counterpart, binding its binding partner at a pH lower or higher than the pKa, and releasing its binding partner at a pH lower or higher than the pKa, and said protein containing said modified amino acid residue retaining its specificity of binding to its binding partner;
SUBSTITUTESHEET(RULES) and
(iii) modifying said protein by synthesising, with standard genetic enineering procedures, a genetically- engineered modified counterpart of said protein, the genetic engineering synthesis procedure comprising a step in which a chemically-modified amino acid residue is added, in place of its unmodified counterpart, to the medium in which cells are cultured, said cells expressing the gene which encodes said protein, said modified amino acid residue having a different pKa when compared with its unmodified counterpart; or alternatively, said genetic engineering procedure comprises a step in which cells are transformed with a modified gene encoding said protein, said modified gene encoding for an at least one amino acid substitution in the region encoding the antigenbinding site/complementarity-determining region, binding domain or active site of said protein, when compared with its normal counterpart, the substitution resulting in the incorporation into the synthesised protein of an amino acid residue having a different pKa than the normally encoded amino acid residue; either of said alternative genetic engineering procedures resulting in the synthesis of a modified protein containing in its said antigenbinding site/complementarity-determining region, binding domain or active site, at least one modified or different amino acid residue when compared with its normal counterpart, said modified protein having a pH-dependent binding activity, when compared to its normal counterpart, binding its binding partner at a pH below or above pKa and releasing its binding partner at a pH below or above pKa, and said modified protein retaining its specificity of binding to its binding partner.
7. A method according to claim 6, wherein the modified protein is an antibody, said antibody having at least one modified or different amino acid in its complementarity-binding region, said antibody being capable of binding to its antigen/hapten at a pH lower or higher than pKa and releasing said antigen/hapten at a pH lower or higher than pKa, and said antibody retaining its specificity of binding to said antigen/hapten.
8. A method according to claim 7, wherein said antibody is a monoclonal antibody.
9. A method according to claim 7 or claim 8, wherein said antibody has at least one modified tyrosine residue in its CDR, said modified tyrosine residue having a reduced pKa when compared to its unmodified counterpart, said modified tyrosine residue being selected from the group consisting of nitrated, iodinated or fluorinated tyrosine residues.
10. A method according to claim 9, wherein said modified antibody has a nitrated tyrosine residue in its CDR.
11. The method of any one of claims 7 to 10 which is carried out according to the procedure of (i) of claim 6, wherein a solution containing the antibody to be modified is modified by said chemical agent and the resulting modified antibody has at least one modified amino acid residue with a reduced pKa in its CDR, has said pH-dependent binding activity for its antigen/hapten, and has retained its specificity of binding to its antigen/hapten.
12. A method according to claim 11, wherein said chemical agent is an agent capable of modifying tyrosine residues by chemical interaction with the aromatic ring of said residues which results in modified tyrosine residues having reduced pKa's, said agent being selected from the group consisting of nitration, iodination and fluorination agents, and said modified antibody containing at least one nitrated, iodinated or fluorinated tyrosine residue in its CDR.
13. A method according to claim 12, wherein the chemical agent is a nitration agent.
14. A method according to claim 13, wherein said agent is tetranitromethane (TMN) .
15. An affinity chromatography method for the isolation or purification of antigens sensitive to denaturing elution conditions of acidic or basic pH in which the pH is less than pH 5 and greater than pH 9, comprising attaching a modified pHdependent antibody according to any one of claims 2 to 5 to the solid phase of the chromotographic separation means, adding thereto a solution containing the antigen to be isolated or purified, said solution having a pH of between about pH 5 and pH 6 to facilitate binding of said antigen to said modified antibody, and eluting the bound antigen with an elution buffer having a pH of between about pH 8 and pH 9.
16. A cell-separation method for the isolation and recovery of a specific cell-type from a mixed population of cells, comprising attaching a modified pH-dependent antibody according to any one of claims 2 to 5 to the solid phase of a cellsorting apparatus or to a cell-culture container, adding said mixed population of cells to said apparatus or container, the medium of said cells being adjusted to about pH 6 to enable attachment of specific cells to said antibody, and recovering said attached cells by adding an elution/separation buffer having a pH of about pH 8.
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