Phosphorylated polypeptides and uses related thereto Number:7,129,331 from the United States Patent and Trademark Office (PTO) owispatent

Title: Phosphorylated polypeptides and uses related thereto

Abstract: Methods to generate modified polypeptides, modified antibodies, stably phosphorylated modified polypeptides, stably phosphorylated modified antibodies, polynucleotide sequences encoding the polypeptides, and uses thereof are provided. A computer-aided molecular modeling method is also provided to generate modified phosphorylatable polypeptides, particularly monoclonal antibodies (MAbs) for use in the diagnosis and treatment of cancers and other diseases. The corresponding MAbs contain heterologous recognition sites for polypeptide kinases and can be labeled by an identifiable label, such as radio-isotope .sup.32P. The phosphate group(s) attached to the phosphorylated polypeptide is unusually stable due to engineered intramolecular interactions between the phosphate group and its neighbouring groups. Polynucleotide sequences which encode a monoclonal antibody containing sequences encoding a putative phosphorylation site, and methods for analyzing the biochemical properties of a polypeptide by using molecular modeling tools, are also disclosed.

Patent Number: 7,129,331 Issued on 10/31/2006 to Pestka

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Inventors:	Pestka; Sidney (North Caldwell, NJ)
Assignee:	Pestka Biomedical Laboratories, Inc. (Piscataway, NJ)
Appl. No.:	09/872,349
Filed:	May 31, 2001

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Current U.S. Class:	530/387.3 ; 530/387.1
Current International Class:	A61K 38/00 (20060101)
Field of Search:	530/387.1,387.3 424/130.1

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References Cited [Referenced By]

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U.S. Patent Documents

	4923802		May 1990		Gallis
	5986061		November 1999		Pestka

Foreign Patent Documents

	0 372 707		Jun., 1990		EP

Other References

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Primary Examiner: Huff; Sheela J.
Attorney, Agent or Firm: Fish & Neave IP Group Ropes & Gray LLP

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Parent Case Text

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CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based, at least in part, on Provisional Application No. 60/208,240, filed May 31, 2000, and Provisional Application No. 60/255,296, filed Dec. 13, 2000, the respective disclosures of which are incorporated by reference herein.

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Claims

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What is claimed is:

1. A phosphorylatable antibody or antigen binding fragment thereof, engineered to include at least one heterologous kinase recognition site located in the hinge region and which does not reduce the ability of the antibody or antigen binding fragment to bind antigen, such that an added phosphate group of a phosphorylated form of the antibody or antigen binding fragment is protected from hydrolysis by intramolecular interactions with other amino acid residues so that at least 80% of all the phosphate groups of the phosphorylated form remain attached in vitro after incubation for 5 days at 37.degree. C. in either (i) human, mouse or fetal serum, or (ii) phosphate buffered saline with 5 mg/ml bovine serum albumin.

2. The phosphorylatable antibody of claim 1, wherein at least 95% of all the phosphate groups remain attached in vitro after incubation for 5 days at 37.degree. C. in either (i) human, mouse or fetal serum, or (ii) phosphate buffered saline with 5 mg/ml bovine serum albumin.

3. The phosphorylatable antibody of claim 2, wherein at least 99% of all the phosphate groups remain attached in vitro after incubation for 5 days at 37.degree. C. in either (i) human, mouse or fetal serum, or (ii) phosphate buffered saline with 5 mg/ml bovine serum albumin.

4. The phosphorylatable antibody of claim 1, wherein the kinase recognition site is a recognition site for kinase which phosphorylates a serine, threonine or tyrosine residue.

5. The phosphorylatable antibody of claim 1, wherein the kinase recognition site is a recognition site for a cyclic AMP dependent kinase, a cyclic GMP dependent kinase, or a cyclic nucleotide independent kinase.

6. The phosphorylatable antibody of claim 1, wherein the kinase recognition site is a recognition site for casein kinase I, casein kinase II, Src tyrosine kinase, mitogen-activated S6 kinase or rhodopsin kinase.

7. The phosphorylatable antibody of claim 1, wherein the antibody is a monoclonal antibody.

8. The phosphorylatable antibody of claim 1, wherein the antibody is a humanized antibody, or chimeric antibody.

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Description

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BACKGROUND OF THE INVENTION

This invention relates to improved methods for generating phosphorylatable polypeptides, polypeptides generated using those methods, DNA sequences encoding those polypeptides, and their use in diagnosis and treatment of cancer and other diseases.

Labeled polypeptides are used in a variety of applications. For instance, labeled monoclonal antibodies (MAbs) have been widely used in radio-immunotherapy, diagnostic imaging and staging of tumors.

Labeled monoclonal antibodies (MAbs) have great applicability for the diagnosis and treatment of cancer for several reasons. First, most tumor populations express tumor antigens in a heterogeneous pattern. Some of the cells in the population will not be expressing the target tumor antigen and therefore will not be recognized by the monoclonal antibody. With the use of MAbs to deliver drugs or toxins to tumor cells, the cells which lack the tumor antigen remain untouched. In contrast, radio labeled MAbs provide the advantage of destroying cells within a radius of a few cell diameters around the tumor cell to which the MAb binds. It has been shown that an .sup.131I-labeled MAb can deliver a therapeutic dose of radiation to antigen negative cells. Second, in the case of carcinomas, the tumor antigens are stable on the cell surface and are not internalized. For a drug or toxin to be effective, it is necessary to have it enter the cell. In contrast, radio labeled MAbs kill the tumor cells after binding to the surface and do not require entry into the cell. Therefore, this technique has applicability to great variety of cancers. Furthermore, the use of interferons and other cytokines can be used to enhance the expression of tumor associated antigens on cells providing a better target for monoclonal antibodies and minimize or even eliminate tumor cells previously not expressing the tumor antigen.

In radio-immunotherapy, .sup.131I has been commonly used for cancer therapy. However since iodine labeling is not site specific, it results in a heterogeneous population of labeled MAbs with various affinities for antigen and significant inactivation of the Mab. Iodine-labeled polypeptides can also undergo dehalogenation, which can eliminate .sup.131I from tumors before it starts to function. Another disadvantage of iodine labeling is that iodine can concentrate in the thyroid, salivary glands and stomach, which can pose health problems for patients and health care personnel.

Compared to .sup.131I, .sup.32P has been considered to be a better option for radio-immunotherapy. Being a pure .beta.-emitter, it has high energy (Emax 1700 keV, compared to .sup.131I, 182 keV) which is strong enough for cancer therapy. However the utilization of this radioisotope was greatly limited due to the difficulties in .sup.32P labeling of MAbs. A .sup.32P labeled peptide can also be chemically coupled to the polypeptide via lysine residues. However, the peptide-Ab conjugation is not site specific, which, like iodine labeling, can also compromise the Ag binding ability of the MAb.

This .sup.32P labeling problem was not satisfactorily solved until the development of a simple and rapid labeling procedure and the construction of a phosphorylatable fusion polypeptide by the introduction of a peptide kinase recognition site into the polypeptide. See, for example, U.S. Pat. No. 5,986,061, the disclosure of which is incorporated by reference herein in its entirety. This is a simple, efficient way to label polypeptides using radio-nucleotides, and is applicable to virtually any polypeptide. Many polypeptide kinase recognition sites can be introduced into polypeptides and serve as useful tags for a variety of purposes. The introduction of polypeptide kinase recognition sites into polypeptides can be achieved without modifying the essential structure or function of the polypeptides. Because polypeptides modified by these procedures retain their activity after phosphorylation, they can be used in many applications.

Phosphorylatable MAbs (MAb-chB72.3-P, MAb-chCC49K1, MAb-chCC49CKI, MAb-chCC49CKII and MAb-chCC49Tyr) can be created by inserting the predicted consensus sequences for phosphorylation by the cAMP-dependent polypeptide kinase and other polypeptide kinases, such as casein kinase I, casein kinase II and the Src tyrosine kinase, at the carboxyl terminus of the heavy chain constant region of MAb-chB72.3-P or MAb-chCC49. These MAbs are purified and phosphorylated by the appropriate polypeptide kinase with [.gamma.-.sup.32P]ATP to high specific activity. These [.sup.32P]MAbs bind to cells expressing TAG-72 antigens with high specificity. In all these cases, the phosphate is stable in vitro in various sera so that less than 8% of the phosphate is hydrolyzed in 24 hours.

However, it has been found that the attached .sup.32P in the above phosphorylatable antibodies is not sufficiently stable in buffer or serum to be useful for in vivo applications in animals and humans. Several methods have been suggested to improve the stabilities of the phosphorylatable MAbs. Since RRX(S/T) is a PKA recognition site, changing the amino acid residue X or the amino acid residues downstream of this site changes the stability of the phosphorylatable MAbs. It has also been found that using threonine, instead of serine, in the PKA recognition site increases the stability of the phosphorylatable Mabs, although this would compromise the efficiency of the phosphorylation dramatically. Alternatively, the stability of the phosphorylatable MAbs might also be changed if other phosphorylation enzymes are used. There is no assurance that these approaches would be satisfactory.

The choice of putative phosphorylation sites can at times be tricky since many point mutations, insertions or deletions may dramatically change the conformation of the entire molecule or at least render the polypeptide less functional. In addition, those sites might be potentially unaccessible to the intended kinases due to steric hinderance. In the past, these problems were dealt with using such inefficient and time-consuming methods as trial-and-error.

Accordingly, what is needed is a reasonably accurate yet highly efficient means to carry out this process, not only for labeling phosphorylatable monoclonal antibodies, but also as a general method for generating any phosphorylatable polypeptides.

SUMMARY OF THE INVENTION

The instant invention provides improved methods, such as computer-aided molecular modeling, to locate phosphorylation sites in polypeptide of interest (i.e. MAb such as MAb-chCC49). An advantage of these methods is that a myriad of potential phosphorylation sites in the target polypeptide can be quickly surveyed and the optimum choices identified by predicting potential intramolecular stabilizing interactions. Hydrogen bonding between the attached phosphate groups and their neighboring groups provides a simple method to locate regions where surrounding residues protect the phosphate from hydrolysis. Therefore, stability of the attached phosphate groups can be reliably predicted within a short period of time, thus representing a vast improvement over the time-consuming and rather inefficient trial-and-error approach.

In a broad sense, the invention contemplates computer-aided molecular modeling to generate phosphorylatable polypeptides, e.g. to radio-label polypeptides, especially monoclonal antibodies (MAbs), and polynucleotide molecules encoding the radio-labellable polypeptides.

In one aspect, the invention provides improved methods to generate radio-labeled polypeptides. In one embodiment, the instant invention provides methods to generate, inter alia, MAbs and Ag binding polypeptides which can be stably phosphorylated to high radio-specific activity with retention of biological activity (affinity for their intended antigens); MAbs modified with various isotopes of phosphorus (e.g., .sup.32P, .sup.33P), or with sulfur (e.g., .sup.35S, .sup.38S); and MAbs labelled with phosphorus or analogs. In accordance with the invention, the MAbs and modified polypeptides may have single or multiple radioactive labels.

The invention also provides a method to generate polypeptides other than MAbs, which are modified by the addition of phosphorylation sites which allow for and are labeled to higher radio-specific activities than the corresponding unmodified polypeptide with a single phosphorylation site. By the "addition" of phosphorylation sites, there is also intended in accordance with the invention, to include polypeptides in which a phosphorylation site heretofore unavailable or inaccessible, has been modified to make the phosphorylation site available.

The invention further provides a method to generate polypeptides, especially MAbs and Ag binding polypeptides, phosphorylated by appropriate kinases on amino acid residues other than on the serine residue, like on threonine and/or tyrosine residues, and the DNA sequences which code for one or more putative phosphorylation sites, which sequences code for these polypeptides.

The invention additionally provides a method to generate polypeptides, such as interferons, cytokines, growth factors, receptor binding proteins and peptides with phosphorylation sites to bind to receptors or other cellular targets.

In accordance with the invention, it is sufficient that a portion of the phosphorylation recognition sequence, as opposed to the entire sequence, be added when the natural polypeptide sequence contains the remaining (or other complementary) amino acids of said recognition sequence (e.g., Arg-Arg-Ala-Ser, (SEQUENCE ID NO. 1)). In such embodiment of the invention, from 1 through 4 amino acids of the sequence (in the case of Arg-Arg-Ala-Ser-Val, (SEQUENCE ID NO. 2)) can be supplied to the polypeptide, thereby constituting the Ser-containing recognition sequence. This illustrates the versatility of the invention for positioning the nucleotide sequence which encodes the amino acid recognition sequence containing a putative phosphorylation site.

Further, the availability of the 3-dimentional structure of a template molecule for computer-aided modeling can precisely predict the consequences of altering natural amino acid sequences in generating putative phosphorylation sites in the test polypeptide, the consequences of introducing phosphate groups, and the possibility of forming stabilizing intramolecular interactions loacted by identifying regions where the phosphate is protected by neighboring residues (i.e. hydrogen-bonding serves as a surrogate marker for the facile location of such regions). This will significantly speed up the trial-and-error engineering process, thus achieving more accurate and predictable results.

The phosphorylated MAbs generated using the methods provided by the instant invention are unexpectedly stable. In one preferred embodiment of the invention, monoclonal antibodies are generated to posess optimized phosphorylation sites, so that phosphate groups attached to those sites are unusually resistant to hydrolysis, either in vitro or in vivo. In a preferred embodiment, at least 80%, more preferably 95%, and most preferably 99% of the phosphate groups remain attached after at least 5 days, more preferably 10 days, and most preferably 18 days in sera or buffer. In a most preferred embodiment, 95% of the phosphate groups remain attached after 18 days in bufffer.

In addition, it was unexpectedly found that those stable monoclonal antibodies had much more improved plasma clearance and biodistribution properties when compared with other phosphorylated MAbs generated by conventional methods. In a preferred embodiment, only 70% (as compared to 90% of control phosphorylated Mabs) of phosphorylated Mabs were cleared from blood in a plasma clearance assay. In another preferred embodiment, phosphorylated Mabs were accumulated in significantly higher amounts in tumor than those in all of the other organs.

The kinase recognition sequence may be positioned at either termini or other positions of the DNA coding sequence, irrespective of the specific phosphorylated amino acid.

The invention also provides labellable and labeled polypeptides, such as hormones and modified streptavidin. The modified streptavidin can be bound to individual biotinylated antibodies, each streptavidin being modified by single or multiple phosphorylated groups, which results in greatly enhanced radiation and therefore diagnostic and therapeutic potential.

The invention also provides phosphorylatable polypeptides which contain at least one phosphorylation recognition site for protein kinase(s), and which, upon phosphorylation at the said site by kinase(s), contain a particularly stable phosphate group by virtue of its ability to form intramolecular stabilizing interactions with neighboring groups (i.e. amino acids side chains). The intramolecular stabilizing interaction can be charge, hydrophobic and/or other covalent interactions that prevent hydroxy groups from attacking or reaching the phosphate residues. Evaluation of regions of hydrogen bonding serves as a way to locate such regions where phosphates are protected from hydrolysis.

The invention also provides phosphorylated polypeptides which contains at least one phosphate group attached to engineered phosphorylation recognition site(s) for protein kinase(s), and which phosphate group is particularly stable by virtue of its ability to form intramolecular stabilizing interactions with neighboring groups (i.e. amino acids side chains). The intramolecular stabilizing interaction can be charge, hydrophobic, and/or other non-covalent interactions that prevent hydroxy groups from attacking or reaching the phosphate residues. Evaluation of regions of hydrogen bonding serves as a way to locate such regions where phosphates are protected from hydrolysis.

The invention also encompasses recombinant DNA sequences which encode functional polypeptides having one or more putative phosphorylation sites; expression vectors for expressing the functional polypeptide; transformed host cells; methods of expressing the modified polypeptides; and the modified polypeptides.

The invention also provides such MAbs and polypeptides made by recombinant DNA techniques, including MAbs radio-labeled with phosphorus or with sulfur, and recombinant DNA-produced radio-labeled polypeptides and polypeptides.

The invention further provides DNA sequences encoding a functional MAb which possesses one or more labelling sites and is sufficiently duplicative of the unmodified MAb to possess substantially similar affinity for its intended Ag. Further, there is provided a recombinant-DNA containing a coding sequence for a putative recognition site for a kinase; the recombinant expression vector; the host organisms transformed with the expression vector that includes the DNA sequence; and an expressed modified polypeptide. A method involving site-specific mutagenesis for constructing the appropriate expression vector, a host transformed with the vector and expressing the modified polypeptides, in particular the modified human interferons, is also provided.

The invention provides in one of its several embodiments DNA sequences which encode one or more putative phosphorylation sites, which sequences encode functional MAbs each of which possesses at least one putative phosphorylation site and each of which possesses at least substantially similar affinity for its intended Ag; expression vectors for expression of the functional modified MAb under the control of a suitable promoter such as the lambda P.sub.L promoter or others described hereinafter; and the biologically active phosphorylated MAb.

The invention also provides a kit comprising at least one phosphorylatable polypeptide with at least one engineered phosphorylation site, or polynucleotide sequence encoding the said phosphorylatable polypeptide; at least one protein kinase, or polynucleotide sequence encoding the protein kinase, capable of phosphorylating the polypeptide at the engineered phosphorylation site; and at least one kind of nucleic acid or its derivative that is capable of being used as a substrate by the protein kinase to label the phosphorylatable polypeptide.

Thus, in accordance with the invention, a nucleotide sequence is constructed that codes for the necessary number and specific amino acids required for creating the putative phosphorylation site.

The invention also provides phosphorylatable or phosphorylated polypeptides, either as separate products or as one of the components of certain kits.

The invention also provides a method to analyse biochemical properties of molecules by using molecular modeling tools.

An "internal sequence" of a polypeptide, as used herein, generally denotes that there is at least one amino acid N-terminal corresponding to the first amino acid of said internal polypeptide sequence, and that there is at least one amino acid C-terminal corresponding to the last amino acid of said internal polypeptide sequence.

By "biological activity" is generally meant the intrinsic biochemical and/or biological activities of any given polypeptide, including, but not limited to, such properties as the catalytic activity of enzymes, the ability to bind certain molecules (i.e. other polypeptides, polynucleic acids, metal ions, steroid hormones, lipids, polysaccharides, etc), and ability to activate or inhibit the function of other molecules.

By "engineered" is generally meant that a moleucle is purposefully changed according to certain predetermined criteria, usually by way of site-directed mutagenesis of the polynucleotide sequence encoding the target amino acid sequence, using conventional molecular biology techniques such as PCR and/or subcloning.

The foregoing is not intended to have identified all of the aspects or embodiments of the invention nor in any way to limit the invention. The accompanying drawings and examples, which are incorporated and constitute part of the specification, illustrate various embodiments of the invention, and together with the specification and claims, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a model of the MAb-chCC49 antibody. The light chains are shown in yellow, and the heavy chains in green. The violet regions represent the sites where the polypeptide kinase recognition site can be introduced. Altogether, nine sites on the heavy chains and three potential sites on the light chains are shown.

FIG. 2 depcits a comparison of the modeled MAb-chCC49 and MAb231 antidobies. The light chains of MAb-chCC49 are shown in yellow, the heavy chains in green. MAb231 is shown in white.

FIG. 3 depicts the nucleotide (SEQ ID NOs 71 72) and amino acid (SEQ ID NO 73) sequences of the synthetic fragment K2. The two phosphorylation sites recognized by the cAMP-dependent protein kinase is underlined. The cloning site, XmaI, is shown in italics.

FIG. 4 illustrates a model of the MAb-chCC49 antibody. This figure shows the complete 3D model of MAb-chCC49. The light chains are shown in yellow, while the heavy chain on the left is in cyan, and the one on the right in royal-blue. The red-orange regions shown in space-filling models represent the sites where protein kinase recognition sites were considered: nine sites on the heavy chains and three on the light chains.

FIG. 5 depicts a comparison of the structures of the MAb-chCC49 and MAb231 antibodies. MAb-chCC49 is shown in magenta, and MAb231 is shown in green.

FIG. 6 illustrates models of mutant MAbs. The light chains of the MAbs are shown in yellow, while the heavy chain on the left is in cyan, and the one on the right in royal-blue. The red-orange regions shown in the space-filling models represent the region where the protein kinase recognition sites are introduced. A: the model of Mab-chCC49K1; B: the model of MAb-CC49CKI; C: the model of MAB-CC49CKII; D: the model of MAb-CC49Tyr.

FIG. 7 also illustrates models of mutant MAbs. The light chains of the MAbs are shown in yellow, while the heavy chain on the left is in cyan, and the one on the right in royal-blue. The red-orange regions shown in the space-filling models represent the regions where the protein kinase recognition sites were introduced. A: the model of MAb-chCC49-6P; B: the model of MAb-WW1; C: the model of MAb-WW2; D: the model of MAb-WW3; E: the model of MAb-WW4; F: the model of MAb-WW5; G: the model of MAb-WW6; H: the model of MAb-WW7; I: the model of MAb-WW8.

FIG. 8 illustrates models of mutant [.sup.32P]MAbs. The light chains of the MAbs are shown in yellow, while the heavy chains are in royal-blue. The white regions shown in the space-filling models represent the regions where the protein kinase recognition sites are introduced. The green regions that represent the phosphates attached to the serine or tyrosine residues are barely visible. The oxygens attached to the phosphates are in red. A: the model of [.sup.32P]MAb-chCC49K1; B: the model of [.sup.32P]MAb-CC49CKI; C: the model of [.sup.32P]MAb-CC49CKII; D: the model of [.sub.32P]MAb-Tyr.

FIG. 9 depicts models of mutant [.sup.32P]MAbs. The light chains of the MAbs are shown in yellow, while the heavy chains are in royal-blue. The white regions shown in the space-filling models represent the regions where the protein kinase recognition sites were introduced. The green regions that represent the phosphates attached to the serine or threonine residues are barely visible. The oxygens attached to the phosphates are in red. A: the model of [.sup.32P]MAb-chCC49-6P; B: the model of [.sup.32P]Mb-WW1; C: the model of [.sup.32P]MAb-WW2; D: the model of [.sup.32P]MAb-WW3; E: the model of [.sup.32P]MAb-WW4; F: the model of [.sup.32P]MAb-WW5; G: the model of [.sup.32P]MAb-WW6; H: the model of [.sup.32P]MAb-WW7; I: the model of [.sup.32P]MAb-WW8.

FIG. 10 is a comparison of the structures of MAb-chCC49 and MAb-WW5. MAb-WW5 is shown in cyan, while MAb-chCC49 is in magenta. The magenta is not visible because the two structures are virtually identical. The inset (lower left) shows a magnification of the hinge region with side chains between the CH1 and CH2 domains where the protein kinase recognition site was introduced (boxed area).

FIG. 11 shows the hydrogen bond of the serine phosphate group with the adjacent amino acid in MAb-chCC49K1. The serine carbons are: C, carbonyl carbon; CA, alpha carbon; CB, beta carbon to which the phosphate (P) is attached through the serine oxygen (OG). Other symbols are: OXT, first oxygen of the phosphate in hydrogen bond on the right; O1P, second oxygen on phosphate; O2P, third oxygen on phosphate; H, hydrogen in a hydrogen bond from amino acid nitrogen (N) to phosphate oxygen OXT. All four serine residues shown in this figure are modified with phosphate groups. Only one of the phosphates forms a hydrogen bond.

FIG. 12 depicts the hydrogen bond of the phosphate group with the adjacent amino acid. A. Hydrogen bond of the Thr-phosphate group with the adjacent amino acid in MAb-WW2. B. Hydrogen bond of the Ser-phosphate group with the adjacent amino acid in MAb-WW3. The Ser/Thr carbons are: C, carboxyl carbon; CA, alpha carbon; CB, beta carbon to which the phosphate (P) is attached through the serine oxygen (OG). Other symbols are: OXT, one oxygen of the phosphate; O1P, second oxygen of phosphate; O2P, third oxygen on phosphate. The figure is a ball and stick model as described herein.

FIG. 13 shows the stabilization of the phosphate moiety on serine 224 in MAb-WW5. A. The side chain of Ser224 stabilized the phosphate moiety through hydrogen bonding either between the phosphate and main chain nitrogen on cysteine 225 (on the left), or between the phosphate and main chain nitrogen on serine 224 (on the right). B. The side chain of Ser224 stabilized the phosphate moiety through hydrogen bonding between the phosphate and main chain nitrogen. The serine carbons are: C, main chain carbonyl carbon; CA, alpha carbon; CB, beta carbon to which the phosphate (P) is attached through the serine oxygen (OG). Other symbols are: OXT, first oxygen of the phosphate; O1P, second oxygen of phosphate that forms a hydrogen bond; O2P, third oxygen on phosphate; H, hydrogen in hydrogen bonds from amino acid nitrogens (N) to phosphate oxygens.

FIG. 14 shows the stabilization of the phosphate moiety on serine 224 in MAb-WW6. A. The side chain of Ser224 stabilized the phosphate moiety through hydrogen bonding between the phosphate and main chain nitrogen on cysteine 225. B. The side chain of Ser224 stabilized the phosphate moiety through hydrogen bonding between the phosphate and main chain nitrogen on cysteine 225 on the left, and hydrogen bonding between the phosphate and main chain nitrogen on cysteine 225 on the right. Serine 224 are shown in magenta, and cysteine 225 in green. The serine carbons are: C, main chain carbonyl carbon; CA, alpha carbon; CB, beta carbon to which the phosphate (P) is attached through the serine oxygen (OG). Other symbols are: OXT, first oxygen of the phosphate; O1P, second oxygen of phosphate; O2P, third oxygen on phosphate; H, hydrogen in hydrogen bonds from amino acid nitrogens (N) to phosphate oxygens.

FIG. 15 depicts the stabilization of the phosphate moiety on serine 224 in MAb-WW7. A. The side chain of Ser224 stabilized the phosphate moiety through hydrogen bonding between the phosphate and main chain nitrogen on cysteine 225. B. The side chain of Ser224 stabilized the phosphate moiety through hydrogen bonding between the phosphate and main chain nitrogen on cysteine 225 on the left, and hydrogen bonding between the phosphate and main chain nitrogen on cysteine 225 on the right. Serine 224 are shown in magenta, and cysteine 225 in green. The serine carbons are: C, main chain carbonyl carbon; CA, alpha carbon; CB, beta carbon to which the phosphate (P) is attached through the serine oxygen (OG). Other symbols are: OXT, first oxygen of the phosphate; O1P, second oxygen of phosphate; O2P, third oxygen on phosphate; H, hydrogen in hydrogen bonds from amino acid nitrogens (N) to phosphate oxygens.

FIG. 16 depicts the stabilization of phosphate moiety on serine 224 in MAb-WW8. A. The side chain of Ser224 stabilized the phosphate moiety through hydrogen bonding either between the phosphate and main chain nitrogen on cysteine 225 (on the left), or between the phosphate and main chain nitrogen on both histidine 223 and arginine 221 (on the right). B. The side chain of Ser224 stabilized the phosphate moiety through hydrogen bonding either between the phosphate and main chain nitrogen on serine 224 (on the left), or between the phosphate and main chain nitrogen on both histidine 223 and arginine 221 (on the right). The serine carbons are: C, main chain carbonyl carbon; CA, alpha carbon; CB, beta carbon to which the phosphate (P) is attached through the serine oxygen (OG). Other symbols are: OXT, first oxygen of the phosphate; O1P, second oxygen of phosphate; O2P, third oxygen on phosphate; H, hydrogen in hydrogen bonds from amino acid nitrogens (N) to phosphate oxygens.

FIG. 17 depicts the expression vector, pdHL7-CC49-6P, constructed for the expression of MAb-CC49-6P.

FIG. 18 illustrates the construction of pWW1. Because the construction is extensive, the figure provides the details in sequential parts (FIGS. 18A and 18B).

FIG. 19 shows the construction of pWW2. Because the construction is extensive, the figure provides the details in sequential parts (FIGS. 19A and 19B).

FIG. 20 shows the construction of pWW3. Because the construction is extensive, the figure provides the details in three sequential parts (FIGS. 20A, 20B and 20C).

FIG. 21 shows the construction of pWW4. Because the construction is extensive, the figure provides the details in sequential parts (FIG. 21A and FIG. 21B).

FIG. 22 shows the construction of pWW5. Because the construction is extensive, the figure provides the details in sequential parts (FIGS. 22A and 22B).

FIG. 23 shows the construction of pLgpCXIIHuWW5. Because the construction is extensive, the figure provides the details in sequential parts (FIGS. 23A and 23B).

FIG. 24 shows the construction of pLNCXIIHuCC49HuKV5. The construct pLNCXIIHuCC49HuKV5 expresses the light chain of the MAb-WW7.

FIG. 25 shows the construction of pLgpCXIIHuWW5V8.DELTA.CH2. The final construct pLgpCXIIHuWW5V8.DELTA.CH2 expresses the heavy chain of the MAb-WW7 with the CH2-domain deleted and amino acid substitutions K221R, T222R and T224S in the humanized MAb-CC49.

FIG. 26 shows the construction of pWW8. Because the construction is extensive, the figure provides the details in sequential parts (FIGS. 26A, 26B, 26C and 26D).

FIG. 27 illustrates an SDS-polyacrylamide gel electrophoresis of the modified MAbs. A: MAb-chCC49-6P represents the gel of unlabeled MAb-chCC49-6P. [.sup.32P]MAb-CC49-6P represents the autoradiograph of the phosphorylated MAb-chCC49-6P. STDS represents the molecular weight markers (SDS-PAGE standards, broad range, Bio-Rad, Cat. No. 161-0317). The kDa of the markers is shown to the left of panels A and G. Arrows point to the places where the phosphorylated mutant MAbs migrated as seen on the autoradiograph (right lane of each panel). Similar labels are used to represent the SDS-polyacrylamide gel electrophoresis of the other mutant MAbs in B-H.

FIG. 28 depicts the stability of [.sup.32P]MAb-chCC49-6P in various sera over a 24-hour period. The percentage of .sub.32P remaining on the [.sup.32P]MAb-chCC49-6P in sera and buffer over a 24-hour period at 37.degree. C. is shown.

FIG. 29 depicts the stability of [.sup.32P]MAb-WW5 in various sera over a 24-hour period. The percentage of .sup.32P remaining on the [.sup.32P]MAb-WW5 in sera and buffer over a 24-hour period at 37.degree. C. is shown.

FIG. 30 depicts the stability of [.sup.32P]MAb-WW5 in various sera over a 5-day period . The percentage of .sup.32P remaining on the [.sup.32P]MAb-WW5 in sera and buffer over a 5-day period at 37.degree. C. is shown.

FIG. 31 depicts the stability of [.sup.32P]MAb-WW5 in buffer over a 21-day period. The percentage of .sup.32P remaining on the [.sup.32P]MAb-WW5 in buffer over a 21-day period at 37.degree. C. is shown.

FIG. 32 depicts the stability of [.sup.32P]MAb-WW6 in various sera over a 24-hour period. The percentage of .sup.32P remaining on the [.sup.32P]MAb-WW6 in sera and buffer over a 24-hour period at 37.degree. C. is shown.

FIG. 33 depicts the stability of [.sup.32P]MAb-WW6 in various sera over a 5-day period. The percentage of .sup.32P remaining on the [.sup.32P]MAb-WW6 in sera and buffer over a 5-day period at 37.degree. C. is shown.

FIG. 34 depicts the stability of [.sup.32P]MAb-WW6 in buffer over a 21-day period. The percentage of .sup.32P remaining on the [.sup.32P]MAb-WW6 in buffer over a 21-day period at 37.degree. C. is shown.

FIG. 35 depcuts the stability of [.sup.32P]MAb-WW7 in various sera over a 24-hour period. The percentage of .sup.32p remaining on the [.sup.32P]MAb-WW7 in sera and buffer over a 24-hour period at 37.degree. C. is shown.

FIG. 36 depicts the stability of [.sup.32P]MAb-WW7 in various sera over a 5-day period. The percentage of 32p remaining on the [.sup.32P]MAb-WW7 in sera and buffer over a 5-day period at 37.degree. C. is shown.

FIG. 37 depicts the stability of [.sup.32P]MAb-WW7 in buffer over a 21-day period. The percentage of .sup.32P remaining on the [.sup.32P]MAb-WW7 in buffer over a 21-day period at 37.degree. C. is shown.

FIG. 38 is a comparison of primary sequences of MAb-chCC49, MAb231 and MAb61.1.3 in the hinge region. A: Primary sequences of MAb-chCC49, (SEQ ID NO. 74), MAb231 (SEQ ID NO. 75) and MAb61.1.3 (SEQ ID NO. 76) in the hinge region are aligned. B: Bestfit of primary sequence of MAb-chCC49 (SEQ ID NO. 74) to that of MAb231 (SEQ ID NO. 75) in the hinge region. C: Bestfit of primary sequence MAb-chCC49 (SEQ ID NO. 74) to that of MAb61.1.3 (SEQ ID NO. 76) in the hinge region.

FIG. 39 is a comparison of stabilities of [.sup.32P]MAb-WW5, [.sup.32P]MAb-WW6, [.sup.32P]MAb-WW7 and [.sup.32P]MAb-chCC49K1 in mouse serum. The percentage of 32P remaining on [.sub.32P]MAb-WW5, -WW6, -WW7 and [.sup.32P]MAb-chCC49K1 in mouse serum over a 24-hour period at 37.degree. C. is shown. In the figure, blue symbols represent [.sup.32P]MAb-WW5; green symbols represent [.sup.32P]MAb-WW6; pink symbols represent [.sup.32P]MAb-WW7; black line represents [.sup.32P]MAb-chCC49K1.

FIG. 40 is a comparison of plasma clearance of [.sup.32P]MAb-WW5 and [.sup.32P]MAb-chCC49K1 in mice. The plasma clearance was performed by collecting 10 .mu.l of blood (by tail bleed) at various timepoints. The values are normalized to the bleed taken at about 2 5 minutes after the injection.

FIG. 41 depicts the crystal structure of the catalytic subunit of the cAMP-dependent protein kinase from Bos Taurus with its inhibitor. The catalytic subunit of the PKA is shown in cyan, while its inhibitor is in magenta. Thr197 and Ser338 are shown in white. The green regions that represent the phosphates attached to the serine or threonine residues are also shown. The oxygens attached to the phosphates are in red.

FIG. 42 depicts the stabilization of phosphate moiety on threonine 197 in the catalytic subunit of the cAMP-dependent protein kinase from Bos Taurus. The threonine carbons are: C, main chain carbonyl carbon; CA, alpha carbon; CB, beta carbon to which the phosphate (P) is attached through the serine oxygen (OG). Other symbols are: O1P, first oxygen of phosphate; O2P, second oxygen on phosphate; O3P, third oxygen of the phosphate; NZ3, nitrogen on the side chain of Lys189. HZ3, hydrogen in hydrogen bonds from side chain nitrogen (NZ3) of Lys189 to O1P of Thr197. NH1, first nitrogen on the side chain of Arg165. HH12, hydrogen in hydrogen bonds from side chain nitrogen (NH1) of Arg165 to O2P of Thr197. NH2, second nitrogen on the side chain of Arg165. HH22, hydrogen in hydrogen bonds from side chain nitrogen (NH2) of Arg165 to both O1P and O2P of Thr197.

FIG. 43 depicts the stabilization of phosphate moiety on serine 338 in the catalytic subunit of the cAMP-dependent protein kinase from Bos Taurus. The side chain of Ser338 stabilized the phosphate moiety through hydrogen bondings between O1P and side chain nitrogens on both Asn189 and Lys342, and also between O3P and main chain nitrogen on Ile339. In addition the side chain OG of Ser338 could also form hydrogen bonds with both main chain nitrogen and the first side chain nitrogen on Asn340, and with third side chain nitrogen on Lys342. Other labels are the same as those in the legend to FIG. 42.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides which are normally not phosphorylatable can be modified to render them phosphorylatable (see U.S. Pat. No. 5,986,061, the dislcosure of which is incorporated herein in its entirety). The methodology to achieve this result (especially without loss of the biological activity of the polypeptide of interest) has provided the potential to modify other polypeptides, such as monoclonal antibodies, and render them phosphorylatable. However, selection of ideal putative phosphorylation sites can be tricky, largely due to uncertainties such as unpredictability of the effects of mutagenesis on overall polypeptide structure. Therefore, the improvement described in the instant invention not only helps to alleviate this problem but also has the unexpected advantage of predicting intramolecular interactions between the added phosphate group and its neighbouring groups so that the overall stability of the phosphate group can be predicted. The stability of the attached phosphate group is a critically important parameter for many utilities of the phosphorylatable polypeptide.

One aspect of the present invention concerns three-dimensional molecular models of template polypeptides, and their use for computer-aided modeling of polypeptides of interest. An integral step to this approach to designing phosphorylation sites involves the construction of computer graphics models of the polypeptides of interest and their mutants, which can be used to determine the consequences of introducing those mutations on the overall conformation (and thus, biological activities) of those polypeptides; the effects of phosphate groups on neighbouring groups; and the stability of the attached phosphate groups based on their potential to form intramolecular interactions with neighbouring groups. For instance, for a putative phosphorylation site to be effective, it will generally be desirable that it is exposed on the surface of the polypeptide rather than buried deep within other structures so that there is no steric hindrance and polypeptide kinases can easily have access to the phosphorylation site. Additionally, other factors, including electrostatic interactions, hydrogen bonding, hydrophobic interactions, and desolvation effects, all influence the stability of the attached phosphate group, which is a critical parameter for many utilities of the instant invention. Therefore, all of these factors should be taken into account in attempts to design the ideal putative phosphorylation sites.

As described in the following examples, a computer-generated molecular model of the subject polypeptide can be created. In preferred embodiments, at least the C''-carbon positions of the MAbs are mapped to a particular coordinate pattern, such as the coordinates for MAb231 shown in FIG. 2, by homology modeling. Typically, such a protocol involves primarily the prediction of side-chain conformations in the modeled polypeptide, while assuming a main-chain trace taken from a tertiary structure such as provided in FIGS. 1 and 2. Computer programs for performing energy minimization routines are commonly used to generate molecular models. For example, both the CHARMM (Brooks et al. (1983) J Comput Chem 4:187 217) and AMBER (Weiner et al (1981) J. Comput. Chem. 106:765) algorithms handle all of the molecular system setup, force field calculation, and analysis (see also, Eisenfield et al. (1991) Am J Physiol 261:C376 386; Lybrand (1991) J Pharm Belg 46:49 54; Froimowitz (1990) Biotechniques 8:640 644; Burbam et al. (1990) Polypeptides 7:99 111; Pedersen (1985) Environ Health Perspect 61:185 190; and Kini et al. (1991) J Biomol Struct Dyn 9:475 488). The disclosure of these references are incorporated herein in their entireties.

At the heart of these programs is a set of subroutines that, given the position of every atom in the model, calculate the total potential energy of the system and the force on each atom. These programs may utilize a starting set of atomic coordinates, such as the model coordinates provided in FIG. 1 or 2, the parameters for the various terms of the potential energy function, and a description of the molecular topology (the covalent structure). Common features of such molecular modeling methods include: provisions for handling hydrogen bonds and other constraint forces; the use of periodic boundary conditions; and provisions for occasionally adjusting positions, velocities, or other parameters in order to maintain or change temperature, pressure, volume, forces of constraint, or other externally controlled conditions.

Most conventional energy minimization methods use the input data described above and the fact that the potential energy function is an explicit, differentiable function of Cartesian coordinates, to calculate the potential energy and its gradient (which gives the force on each atom) for any set of atomic positions. This information can be used to generate a new set of coordinates in an effort to reduce the total potential energy and, by repeating this process over and over, to optimize the molecular structure under a given set of external conditions. These energy minimization methods are routinely applied to molecules similar to the subject polypeptides as well as nucleic acids, polymers and zeolites.

In general, energy minimization methods can be carried out for a given temperature, T.sub.i, which may be different than the docking simulation temperature, T.sub.o. Upon energy minimization of the molecule at T.sub.i, coordinates and velocities of all the atoms in the system are computed. Additionally, the normal modes of the system are calculated. It will be appreciated by those skilled in the art that each normal mode is a collective, periodic motion, with all parts of the system moving in phase with each other, and that the motion of the molecule is the superposition of all normal modes. For a given temperature, the mean square amplitude of motion in a particular mode is inversely proportional to the effective force constant for that mode, so that the motion of the molecule will often be dominated by the low frequency vibrations.

After the molecular model has been energy minimized at T.sub.i, the system is "heated" or "cooled" to the simulation temperature, T.sub.o, by carrying out an equilibration run where the velocities of the atoms are scaled in a step-wise manner until the desired temperature T.sub.o is reached. The system is further equilibrated for a specified period of time until certain properties of the system, such as average kinetic energy, remain constant. The coordinates and velocities of each atom are then obtained from the equilibrated system.

Further energy minimization routines can also be carried out. For example, a second class of methods involves calculating approximate solutions to the constrained EOM for the polypeptide. These methods use an iterative approach to solve for the Lagrange multipliers and, typically, only need a few iterations if the corrections required are small. The most popular method of this type, SHAKE (Ryckaert et al. (1977) J Comput Phys 23:327; and Van Gunsteren et al. (1977) Mol Phys 34:1311) is easy to implement and scales as O(N) as the number of constraints increases. Therefore, the method is applicable to macromolecules such as the polypeptides of the present invention. An alternative method, RATTLE (Anderson (1983) J Comput Phys 52:24) is based on the velocity version of the Verlet algorithm. Like SHAKE, RATTLE is an iterative algorithm and can be used to energy minimize the model of the subject polypeptide. These references are incorporated herein in their entireties.

From the above observation, the same-principles are applicable to construct any amino acid sequences other than the particular amino acid recognition sequence illustrated above.

In the situations where the phosphorylation site is other than serine (as illustrated above), the DNA sequence codes for part or all of the appropriate amino acid sequence containing the putative recognition site containing threonine, tyrosine, etc. Thus, where in any particular 40

polypeptide one or more amino acids (at any position of the amino acid sequence) are the same as that of an amino acid recognition sequence for a kinase, it is sufficient to add (or modify) those complementary amino acids of the amino acid recognition sequence to complete that sequence. This is accomplished by constructing a DNA sequence which codes for the desired amino acid sequence. There may indeed be situations where such addition (or modification) is a more desirable procedure as where it is important to retain the integrity of the polypeptide molecule to be modified (for instance, to minimize risks of affecting a particular activity, e.g., biological), or for simplicity of the genetic manipulations, or because either or both termini or other positions are more accessible.

In accordance with the invention, phosphorylation of the phosphorylatable site of the polypeptide can be performed by any suitable phosphorylation means. Phosphorylation and dephosphorylation of polypeptides catalyzed by polypeptide kinases and polypeptide phosphatases is known to affect a vast array of polypeptides. A large number of polypeptide kinases have been described and are available to one skilled in the art for use in the invention. Such polypeptide kinases may be divided into two major groups: those that catalyze the phosphorylation of serine and/or threonine residues in polypeptides and peptides and those that catalyze the phosphorylation of tyrosine residues. These two major categories can be subdivided into additional groups. For example, the serine/threonine polypeptide kinases can be subdivided into cyclic AMP (cAMP)-dependent polypeptide kinases, cyclic GMP (cGMP)-dependent kinases, and cyclic nucleotide-independent polypeptide kinases. The recognition sites for many of the polypeptide kinases have been deduced.

In short synthetic peptides cAMP-dependent polypeptide kinase recognize the sequence Arg-Arg-Xxx-Ser-Xxx, where Xxx represents an amino acid. As noted above, the cAMP-dependent polypeptide kinase recognizes the amino acid sequence Arg-Arg-Xxx-Ser-xxx, but also can recognize some other specific sequences such as Arg-Thr-Lys-Arg-Ser-Gly-Ser-Val, (SEQUENCE ID NO. 3). Many other polypeptide serine/threonine kinases have been reported such as glycogen synthase kinase, phosphorylase kinase, casein kinases I and II, pyruvate dehydrogenase kinase, polypeptide kinase C, and myosin light chain kinase.

Polypeptide kinases which phosphorylate and exhibit specificity for tyrosine (rather than for serine, threonine, or hydroxyproline) in peptide substrates are the polypeptide tyrosine kinases (PTK). Such PTKs are described in the literature. The PTKs are another class of kinases available for use in the invention.

Another available class of kinases are the cyclic GMP-dependent (cGMP-dependent) polypeptide kinases. The cGMP-dependent polypeptide kinases exhibit substrate specificity similar to, but not identical to the specificity exhibited by cAMP-dependent polypeptide kinases. The peptide Arg-Lys-Arg-Ser-Arg-Lys-Glu, (SEQUENCE ID NO. 4) is phosphorylated at serine by the cGMP-dependent polypeptide kinase better than by the cAMP-dependent polypeptide kinase. It has also been shown that the cAMP-dependent polypeptide kinase can phosphorylate hydroxyproline in the synthetic peptide Leu-Arg-Arg-Ala-Hyp-Leu-Gly, (SEQUENCE ID NO. 5).

Casein kinases, widely distributed among eukaryotic organisms and preferentially utilizing acidic polypeptides such as casein as substrates, have been classified into two groups, casein kinases I and II. Casein kinase II phosphorylated the synthetic peptide Ser-Glu-Glu-Glu-Glu-Glu, (SEQUENCE ID NO. 6). Evaluation of results with synthetic peptides and natural polypeptide substrates reveals that a relatively short sequence of amino acids surrounding the phosphate acceptor site provides the basis for the specificity of casein kinase II. Accordingly, the acidic residues at positions 3 and 5 to the carboxyl-terminal side of the serine seem to be the most important. Serine is preferentially phosphorylated compared to threonine. In another study, the peptide Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu, (SEQUENCE ID NO. 7) is found to be a specific substrate for casein kinase II; however, Arg-Arg-Arg-Glu-Glu-Glu-Ser-Glu-Glu-Glu, (SEQUENCE ID NO. 8) is a better substrate; and Arg-Arg-Arg-Asp-Asp-Asp-Ser-Asp-Asp-Asp, is a better substrate than Arg-Arg-Arg-Glu-Glu-Glu-Ser-Glu-Glu-Glu, (SEQUENCE ID NO. 9). Thus, aspartate is preferred over glutamate. Acidic residues on the COOH-terrninal side of the serine (threonine) are as far as known today absolutely required; acidic residues on the amino-terminal side of the serine (threonine) enhance phosphorylation, but are not absolutely required: thus, Ala-Ala-Ala-Ala-Ala-Ala-Ser(Thr)-Glu-Glu-Glu, (SEQUENCE ID NO. 10) served as a substrate for casein kinase II, but is less effective than Ala-Ala-Ala-Glu-Glu-Glu-Ser(Thr)-Glu-Glu-Glu, (SEQUENCE ID NO. 11) (the designation Ser(Thr) means serine or threonine). Casein kinases I and II phosphorylate many of the same substrates although casein kinase I does not phosphorylate any of the decamer peptide substrates noted here. It is concluded from studies with a variety of synthetic peptides that the sequence Ser-Xxx-Xxx-Glu (and by inference Ser-Xxx-Xxx-Asp) may represent one class of sequences that fulfill the minimal requirements for recognition by casein kinase II although some other peptides and sequences may also suffice.

As noted above, other kinases are described. The mitogen-activated S6 kinase phosphorylates the synthetic peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala, (SEQUENCE ID NO. 12) as does a protease-activated kinase from liver. The rhodopsin kinase catalyzes the phosphorylation of the peptide Thr-Glu-Thr-Ser-Gln-Val-Ala-Pro-Ala, (SEQUENCE ID NO. 13). Other polypeptide serine/threonine kinases are described and their sites of phosphorylation elucidated.

Thus, one skilled in the art has quite an adequate selection of available kinases for use in the invention, which have relatively high specificity with respect to the recognition process, but some flexibility to the specific sequence of the amino acid recognition site. Such kinases provide means for phosphorylation of putative phosphorylation sites in the desired polypeptides.

The selection of the position of the molecule best suited for the modification depends on the particular polypeptide (and its configuration). Where multiple putative phosphorylation sites (and phosphorylatable sites) are to be included in the modified polypeptide, one would consider the potential availability of either or both ends and other positions of the molecule for providing the amino acid recognition sequence. Thus, in accordance with the invention, phosphorylation recognition sequences can be introduced at any point in a naturally occurring polypeptide sequence providing such introduced sequences do not adversely affect biological activity where such activity is desired.

Once the recognition site for a particular polypeptide kinase is identified, the invention provides a method for making by recombinant-DNA techniques the DNA sequence which encodes the recognition site for that kinase within, fused or linked to the DNA sequence encoding the functional polypeptide which is to contain the corresponding putative labelling site. Due to the intrinsic advantage of the instant invention, molecular modeling can be used to quickly scan through a number of potential sites so that only those sites, with or without the attached phosphate group, that will not adversely affect the three-dimentional structure and/or biological activity of the target polypeptide will be selected for further consideration.

The invention contemplates and includes any polypeptide which is radio-labellable by the methods of this invention and which possesses at least one of the properties of the corresponding unlabeled (or unlabellable) polypeptide. In accordance with the invention, the non-phosphorylated (or non-phosphorylatable) polypeptide is modified to introduce into the amino acid sequence the putative phosphorylatable site; this is performed after having modified the DNA sequence encoding the amino acid sequence of the polypeptide with the DNA sequence (part or all) which codes for the putative phosphorylated site. In the case of MAb, the invention embraces all MAbs, including such structurally modified MAb species which have been reported in the literature (such as humanized MAbs, hybrid antibodies, chimeric antibodies, and modified MAb Fab or Fc fragments) as discussed above, and other modified MAbs which will be developed in the future.

In a preferred embodiment of the instant invention, recognition sites for the cAMP-dependent polypeptide kinase is introduced into the MAb-chCC49 by site-directed mutation of the coding sequence to make variants of MAb-chCC49 to be able to contain highly stable phosphate groups. To design those MAbs without changing their immunoreactivity or biological properties, molecular modeling is used to locate appropriate regions for introduction of the cAMP-dependent phosphorylation site with desirable properties. With the use of molecular modeling, we chose positions on the heavy chain to mutate. Vectors expressing the mutants are constructed and transfected into mouse myeloma NS0 cells that expressed a high level of the resultant MAb-WW5, -WW6 and -WW7. Those variants contain the cAMP-dependent phosphorylation site at the hinge region of the heavy chain, and can be phosphorylated by the catalytic subunit of cAMP-dependent polypeptide kinase with [.gamma.-.sup.32P]ATP to high specific activity and retains the phosphate stably. Compared to MAb-chCC49K1 (Lin et al., Int. J. oncology, 13, 115 120, 1998), another phosphorylatable variant of MAb-chCC49, the phosphate attached to MAb-WW5, -WW6 and -WW7 show much improved stability: about a ten-fold increase in resistance to hydrolysis. They also exhibit the same binding specificity to the TAG-72 antigen on MCF-7 4C10 breast cancer cells observed with MAb-chCC49K1. The improved stability of the attached phosphate provides a MAb with potential to be used in diagnosis and therapy of adenocarcinomas.

Radio labeled monoclonal antibodies (MAbs) against tumor-associated antigens (TAA) are used clinically for the early detection and staging of the disease as well as for therapy. Chimeric MAb-chCC49 is one of these MAbs which reacts with the TAA expressed on the surface of a wide range of human adenocarcinomas. It consists of the variable region from mouse MAb-CC49 (GenBank Accession No: M95575) and the constant region from the human IgG1 heavy chain (GenBank Accession No: J00228) and the human chain (GenBank Accession No: J00241).

Since molecular modeling is a powerful tool to build 3-D models of polypeptides, an alternative way to obtain structural information about MAb-chCC49 is to build a 3-D model by using the crystal structures of the known MAbs as a template. This report provides a summary of the development of a 3-D model of MAb-chCC49 and its variants, and the use of the 3-D mo