A humanized form of an anti-idiotype antibody to CEA, e.g., hWI2, has conserved immunoreactivity. The clinical benefits of anti-CEA antibodies are maximized by using the humanized anti-idiotype as a clearing agent for anti-CEA antibodies or antibody fragments. The humanized anti-idiotype also can be used as an immunogenic vaccine.

Description of the Invention

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a chimeric anti-idiotype Ab with anti carcinoembryonic-antibody properties, a humanized anti-idiotype Ab with anti carcinoembryonic-antibody properties as an immunological reagent useful in clearing an organism of anti-CEA antibody initially used as a cancer treatment, diagnostic, or vaccine, where the anti-idiotype Ab has the immunogenic properties of a human MAb in a human patient, and to provide an anti-idiotype Ab with anti carcinoembryonic-antibody properties which can serve as a detection agent or vaccine. It is another object of the present invention to provide DNA constructs encoding such antibodies.

To achieve these objectives, in one aspect of the invention, a chimeric anti-idiotype antibody or fragment thereof which specifically binds to the idiotype region of an anti-CEA monoclonal antibody is provided, comprising the rWI2 light chain and heavy chain variable regions, or silent mutations thereof. In a preferred embodiment, the heavy chain variable region comprises the rWI2VH sequence shown in FIG. 1 (SEQ ID NO:18 (see Original Patent)) and the light chain variable region comprises the rWI2VK sequence shown in FIG. 2 (SEQ ID NO:22 (see Original Patent)).

In another aspect of the invention, a humanized anti-idiotype antibody or fragment thereof which specifically binds the idiotype region of an anti-CEA monoclonal antibody is provided, comprising rWI2 CDR regions and humanized FR regions. In a preferred embodiment, the heavy chain variable region comprises the KOLWI2VH-1 (SEQ ID NO:19) or the KOLWI2VH-2 (SEQ ID NO:20) sequence shown in FIG. 1 and the light chain variable region comprises the REIWI2VK (SEQ ID NO:24) or the REIWI2VKRS (SEQ ID NO:23) sequence shown in FIG. 2.

In another aspect of the invention, an isolated polynucleotide encoding the heavy chain or the heavy chain variable region of a chimeric or humanized antibody or antibody fragment which specifically binds the idiotype region of an anti-CEA monoclonal antibody is provided, comprising sequences enclosing at least two rWI2 heavy chain CDRs, selected from the group of CDRs consisting of:

the complementary determining region-1 (CDR-1) sequence NYWMT(SEO ID NO:1),

the complementary determining region-2 (CDR-2) sequence SITSTGGGTYHAESVKG (SEQ ID NO:2), and

the complementary determining region-3 (CDR-3) sequence DDYGGQSTYVMDA (SEQ ID NO:3).

In another aspect of the invention, an isolated polynucleotide encoding the light chain or the light chain variable region of a chimeric or humanized antibody or antibody fragment which specifically binds the idiotype region of an anti-CEA monoclonal antibody is provided, comprising sequences enclosing at least two rWI2 CDRS, selected from the group of CDRs consisting of:

the complementary determining region-1 (CDR1) sequence RASQDIGNYLR (SEQ ID NO:4),

the complementary determining region-2 (CDR2) sequence GATNLAA (SEQ ID NO:5), and

the complementary determining region-3 (CDR3) sequence LHHSEYPYT (SEQ ID NO:6).

In another aspect of the invention, an isolated first expression vector comprising a gene for the chimeric WI2 heavy chain and an isolated second expression vector comprising a gene for the chimeric WI2 light chain are provided.

In another aspect of the invention, an isolated first expression vector comprising a gene for a humanized WI2 heavy chain and an isolated second expression vector comprising a gene for a humanized WI2 light chain are provided.

In another aspect of the invention, a host is provided, comprising an isolated first vector comprising a gene for the chimeric WI2 heavy chain and an isolated second expression vector comprising a gene for the chimeric WI2 light chain, or an isolated first expression vector comprising a gene for a humanized WI2 heavy chain and an isolated second expression vector comprising a gene for a humanized WI2 light chain.

In another aspect of the invention, a method of stimulating an immune response in a patient against cancers expressing carcinoembryonic antigen is provided, which comprises administering to a patient an effective amount of a vaccine comprising a humanized anti-idiotype antibody or fragment which specifically binds the idiotype region of an anti-CEA monoclonal antibody, conjugated to a soluble immunogenic carrier protein, optionally in combination with a pharmaceutically acceptable vaccine adjuvant.

In another aspect of the invention, a method of diagnosis or treatment of a patient is provided, wherein an antibody or antibody fragment that specifically binds CEA is used as a targeting, pre-targeting or therapy agent, either as such or as a component of a conjugate, the improvement to the method consisting of an anti-idiotype antibody used to clear non-targeted antibody or antibody fragment.

In another aspect of the invention, a method of detecting the presence of an antibody or fragment that specifically binds CEA, in a fluid biological sample, comprising contacting the sample with rWI2, or a chimeric anti-idiotype antibody or antibody fragment which specifically binds the idiotype region of an anti-CEA monoclonal antibody, or a humanized anti-idiotype antibody or antibody fragment which specifically binds the idiotype region of an anti-CEA monoclonal antibody, and detecting binding of said anti-idiotype antibody or antibody fragment to an antibody idiotype or antibody idiotype fragment in said sample.

DETAILED DESCRIPTION OF THE INVENTION

An overview. Providing chimeric and humanized WI2 anti-idiotype antibodies.

Rat WI2 (rWI2) is an anti-idiotype monoclonal antibody which binds the CDR of MN-14. See Losman et al., (1994) supra. It was previously proposed that PCR primer pairs may be designed which might isolate the variable regions of rat antibodies from a RNA substrate. See Kutemeier et al., Hybridoma, 11: 23-32 (1992). Similar approaches have been employed to isolate the variable regions of mouse antibodies. See Orlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). In the present invention, a combination of PCR primers as described by Kutemeier et. al., and by Orlandi et al., supra, are required to isolate the rWI2 variable region. These primers could be used to isolate other rat variable regions.

cDNA for the variable regions of both the light and heavy chains of rWI2 are isolated and the sequence from a number of clones is confirmed. The rat variable regions are cloned into plasmid vectors, such that the rat variable K and H regions are attached to respective human IgG constant regions. The plasmids are transfected into SP2/O cells, clones comprising both plasmids are selected, and chimeric WI2 (cWI2) antibody is expressed.

To humanize the variable regions, FR rat regions are replaced by the human IgG counterparts. The rat CDR regions are preserved, in order to maintain the specificity of the antibody. However, to enhance stability and maintain specificity and binding affinity of the antibody, it is best to employ a human sequence which is most similar to the newly identified rat FR sequences. Rat residues that are close to the CDRs, or known from prior experience to be important for interactions with the CDRs, were retained in both humanized sequences.

For both the heavy and light chain, long oligonucleotides corresponding to the variable region are engineered to reflect the humanized design. The oligonucleotides replace the rat variable region in plasmid constructs designated to produce full length human WI2 (hWI2). The effectiveness of cWI2 or hWI2 are compared with that of rWI2 in assays where they compete with CEA for binding to a peroxidase conjugated MN-14.

In order to express the cWI2 and the hWI2s, the rat and the humanized variable regions are inserted into one or separate plasmid vectors in such a manner as to allow coexpression of full length light and heavy antibody chains. The vectors can be transfected into SP2/O cells. Selection of transfected cells, and eventual amplification of the clones, are based on the expression of the dihydrofolate reductase (DHFR) gene which is located on the same plasmid vector. The functionality of various constructs is determined by binding assays. For example, final hWI2 constructs were compared with both rWI2 and cWI2 in their ability to bind MN-14--see FIG. 5 (see Original Patent).

Production of Monoclonal Ab1 and Ab2 Antibodies

Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991) [hereinafter "Coligan"]. Briefly, monoclonal antibodies can be obtained by injecting rats with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. These techniques are applicable for production of a monoclonal antibody from other mammals injected with an antigen.

A wide variety of monoclonal antibodies against tumor associated antigens or infectious agents have been developed. See, for example, Goldenberg et al., international application publication No. WO 91/11465 (1991), and Goldenberg, international application publication No. WO 94/04702 (1994), each of which is incorporated herein by reference in its entirety.

Polyclonal Ab2 can be prepared by immunizing animals with Ab1 or fragments, using standard techniques. See, for example, Green et al., "Production of Polyclonal Antisera," in METHODS IN MOLECULAR BIOLOGY: IMMUNOCHEMICAL PROTOCOLS, Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively, monoclonal Ab2 can be prepared using Ab1 or fragments as immunogen with the techniques described above.

MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al., "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

The Design of a Humanized Antibody

One antibody of the present invention is a "humanized" monoclonal antibody. That is, rat complementarity determining regions are transferred from heavy and light variable chains of the rat immunoglobulin into a variable region designed to contain a number of aa residues found within the FR region in human IgG. Similar conversion of mouse/human chimeric antibodies to a humanized antibody has been described before. General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Orlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989), which is incorporated by reference in its entirety. Techniques for producing humanized MAbs are described, for example, by Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993), each of which is hereby incorporated by reference.

Construction of cWI2 and hWI2.

The engineering of chimeric and humanized WI2 employs standard molecular biology and cellular biology approaches, for example PCR reactions, sequencing of DNA, synthesis of long oligonucleotides, cloning, site-directed mutagenesis, transfection of vectors into cells, expression of chimeric and humanized antibodies, their purification and so on. The techniques employed are standard techniques, well known in the art and well established. A person skilled in the art would have no difficulty carrying out those techniques. All necessary materials are readily available. Reference manuals describing these techniques are widely available. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Press, (1989); Co et al., J. Immunol., 148: 1149 (1992); Ausubel et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, and updates, John Wiley & Sons, NY, (1987); "Protocols--Current Methods and Applications" White, ed., Methods in Mol. Biol. (15), Humana Press, Totawa, N.J. (1993); Uhlmann, Gene 71: 29 (1998); Wosnick et al., Gene 60: 115 (1988); and Huse, in ANTIBODY ENGINEERING: A PRACTICAL GUIDE, Boerrbaeck, ed., W.H. Freeman & Co., 103-120 (1992).

The PCR primers used in the initial cloning of the rat variable region are designed over regions of the sequence that are expected to be relatively conserved between human antibodies. Furthermore, an amino acid (aa) can generally be represented by more than one codon, although the codons often differ only in the third position. Such codons are sometimes referred to as "degenerate." To design the PCR primer one attempts to choose a protein sequence over which degenerate codons are limited in number. The approach is further limited by the need to design over a conserved protein sequence, as discussed above. Therefore, often, the "primer" as designed actually consists of a mix of numerous molecules that differ in sequence at specific positions. Any one primer molecule, which would provide a perfect match for the specific mRNA substrate to be amplified is present as a minuscule fraction of the overall primer mix. To enhance likelihood of success, a few primer pairs, i.e. covering different regions of the mRNA are generally tried.

One skilled in the art recognizes that alternative techniques are readily available. For example, initial changes to the cloned rat variable region could be introduced by any of a number of site specific mutagenesis protocols. Furthermore, it should be realized that additional aa changes in the variable and especially the FR regions may be possible, with the expectation that there will be some changes which would be silent in nature. Silent changes are those replacements, deletion or addition of one or a small number of aa that would not significantly affect the binding affinity or specificity of the antibody or antibody fragments thereof. Most obvious among such silent changes would be the replacement of one aa by an aa of a similar size and chemical properties. Such changes are well known in the art and are generally referred to as a "conservative" aa substitution. For example, a leucine when replaced by an isoleucine would generally not be expected to affect the structure of a protein. All such conserved aa changes, as well as silent changes which do not significantly affect the binding affinity or specificity of the anti-carcinoembryonic antibody, are within the scope of the invention.

Expression of the Engineered cWI2 or hWI2

Genes encoding the antibodies of the invention are introduced via expression vectors into a host cell, for expression. In a preferred embodiment, the genes for both the light and heavy genes are introduced in a single expression vector, which is introduced in a host cell. The expression vectors generally contain drug markers for selection of the transformed cell. A drug marker can furthermore be used to amplify the copy number of nearby genes, resulting in a clone overexpressing the antibody. For example, a vector expressing the light and heavy chains of cWI2 or hWI2 were introduced into SP2/O cells on vectors containing the DHFR gene. The original clones were amplified after selection by growth on methotrexate (MTX).

It should be understood that alternative ways to coexpress the light and heavy chain genes are feasible. A skilled artisan could consider other selection regimens, introduction of both the light and heavy chain genes on one plasmid or cotransformation with separate vectors encoding the light and heavy genes, and transfection of other cell lines. Furthermore, expression of the antibody in yet other systems is possible. For example, expression could occur in yeast. Alternatively, baculoviruses can be engineered with the light and heavy genes and expressed in cultured cells, or used to infect an insect.

The antibodies require purification from their expression system and media by methods that are generally similar to methods described above for purification of MAbs from hybridomas.

Uses for the Antibodies of the Invention

Humanized monoclonal antibodies in accordance with the invention are suitable for use in therapeutic methods. For example, MN-14 has been proposed as a therapeutic agent or as a vaccine to stimulate Ab2. The dosage of MN-14 conjugated to a drug can be enhanced if removal or clearing of unbound MN-14 is made possible by its binding, and aggregate formation, with hWI2. Similarly, MN-14 can be used as a vaccine, optionally by alternative applications with an anti-idiotype antibody vaccine. Delivery of the Ab2 would be counterproductive in the presence of excess, free-floating MN-14 vaccine. Here, hWI2, for example, would clear the MN-14 vaccine, to allow efficient administration of the Ab2 vaccine.

In addition, hWI2 itself can be a vaccine. Generally, the antibodies and fragments of the present invention can be used as vaccines by conjugating the antibodies or fragments to a soluble immunogenic carrier protein. Suitable carrier proteins include keyhole lympet hemocyanin, which is the preferred carrier protein. The antibodies and fragments can be conjugated to the carrier protein using standard methods. See, for example, Hancock et al, "Synthesis of Peptides for Use as Immunogen," in METHODS IN MOLECULAR BIOLOGY: IMMUNOCHEMICAL PROTOCOLS, Manson (ed.), pages 23-32 (Humana Press 1992).

A preferred vaccine composition comprises an antibody conjugate or fragment conjugate, and an adjuvant. Examples of suitable adjuvant include aluminum hydroxide and lipid. Methods of formulating vaccine compositions are well-known to those of ordinary skill in the art. See, for example, Rola, "Immunizing Agents and Diagnostic Skin Antigens," in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, Gennaro (ed.), pages 1389-1404 (Mack Publishing Company 1990).

Additional pharmaceutical methods may be employed to control the duration of action of a vaccine in a therapeutic application. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the antibodies or fragments. For example, bio-compatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release of an antibody or antibody fragment from such a matrix depends upon the molecular weight of the antibody or fragment, the amount of antibody or fragment within the matrix, and the size of dispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid dosage forms are described in Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990).

The anti-idiotype Ab of the invention can be used for the detection of an anti-carcinoembryonic antibody. For example, rWI2, cWI2 and hWI2 can be used in vitro to test the blood sample of a patient for the presence of anti-carcinoembryonic antibody. Detection of MN-14 levels, when MN-14 is used as a therapeutic agent or as a vaccine would be important. Presence of MN-14 can be detected by rWI2, cWI2, or hWI2. Similarly, hWI2 can be used in a patient to determine the presence of natural anti-CEA Abs. To be useful in the detection of an anti-CEA Ab, the anti-idiotype Abs can be conjugated to a label. Suitable labels include, e.g., a radiolabel, an enzyme, or a fluorescent label. Such labeling agents and methods of conjugation are well known to one skilled in the art.

The antibody preparations of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby antibodies or antibody fragments are combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient mammal. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known to those in the art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990).

The antibodies or fragments may be administered to a mammal intravenously or subcutaneously. Moreover, the administration may be by continuous infusion or by single or multiple boluses. Preferably, an antibody vaccine is administered subcutaneously, while an antibody preparation that is not a vaccine is administered intravenously. In general, the dosage of administered antibodies or fragments for humans will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of antibodies or fragments which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate.

For purposes of therapy, antibodies or fragments are administered to a mammal in a therapeutically effective amount. An antibody preparation is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient mammal. In particular, an antibody preparation of the present invention is physiologically significant if its presence invokes a humoral and/or cellular immune response in the recipient mammal.

The present invention, in addition to the specifically described techniques, cell lines, and vectors, relies on descriptions of said techniques, cell lines, and vectors presented in U.S. Pat. Nos. 5,443,953, and 4,624,846 and in U.S. patent application Ser. Nos. 08/318,157, and 08/289,576, all of which are incorporated herein by reference in their entireties.
 

Claim 1 of 9 Claims

1. An isolated expression vector comprising a first nucleic acid sequence that encodes a heavy chain variable region sequence comprising the CDR-1 sequence NYWMT (SEQ ID NO:1), CDR-2 sequence SITSTGGGTYHAESVKG (SEQ ID NO:2), and CDR-3 sequence DDYGGQSTYVMDA (SEQ ID NO:3) and a second nucleic acid sequence that encodes a light chain variable region sequence comprising the CDR1 sequence RASQDIGNYLR (SEQ ID NO:4), CDR2 sequence GATNLAA (SEQ ID NO:5), and the CDR3 sequence LHHSEYPYT (SEQ ID NO:6).
 

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