WO2000040201A2 - Method and means for reducing chemotherapeutic drug resistance in-situ within neoplasms of epithelial cell origin - Google Patents

Abstract

The present invention provides a method and composition means for reducing chemotherapeutic drug resistance exhibited in-situ by a solid mass neoplasm of epithelial cell origin. The tumor cells constituting the solid neoplasm have clinically demonstrated resistance in-situ to a single- or multiple-drug treatment regimen, and the resistant tumor cells express both the PDZK1 protein and the cMOAT protein intracellularly. The invention provides antagonistic antibody preparations which inhibit the interaction of PDZK1 and cMOAT proteins intracellularly; and thereby cause a reduction in clinical resistance to the previously administered chemotherapeutic treatment agents.

Description

METHOD AND MEANS FOR REDUCING CHEMOTHERAPEUTIC

DRUG RESISTANCE IN-SITU WITHIN NEOPLASMS OF

EPITHELIAL CELL ORIGIN

CROSS REFERENCE

This application is a Continuation-In-Part of the subject matter as a whole comprising the invention disclosed by United States Patent Application Serial No. 08/997,445 filed December 23, 1997, now pending.

RESEARCH SUPPORT

The research investigations for the present invention were supported by grants from the Beth Israel Hospital Pathology Foundation, Inc.; the Else U. Pardee Foundation and the Nell and Nancy Fund; and the Pine Mountain Benevolent Association.

FIELD OF THE INVENTION

The present invention is concerned generally with enhancing the effectiveness of chemotherapeutic drug treatments used clinically with humans and animals afflicted with solid mass cancers; and is directed in particular to methods and means for reducing drug resistance frequently demonstrated by neoplasms in- vivo to the administration of single or multiple chemotherapeutic drugs as part of a tumor treatment regimen. BACKGROUND OF THE INVENTION

Historically, the first effective drugs for treating cancer were brought to clinical trials in the 1940s. The first of these were single agent drugs such as nitrogen mustard, antifolates, corticosteroids, and the vinca alkaloids. Although initially effective, the impressive regressions of neoplastic disease created by these single agents were short-lived and the longer-term therapeutic results proved disappointing. Even when a complete remission was initially obtained, these typically lasted less than one year; and the relapse was routinely associated with a demonstrable resistance to the single drug employed for treatment [see for example, Lchnet, M. , Em\ L Cancer 32A: 912-920 (1996)].

One consequence of these early clinical findings was an ever-wider search for newer and better single agents and effective drugs, an investigative effort which continues unabated today. As a consequence, the number of single agents or drugs approved today for use as chemotherapeutic treatment substances is quite large. A representative listing of single agents is provided by Table A below.

Table A¹:

CLASS I

Asparaginase Gemcitabine

Busulfan Melphalan < 100 mg/m²

Busulfan (oral) Mercaptopurine

Chlorambucil Paclitaxel

Cyclophosphamide (oral) Thioguanine (oral)

Docetaxel Vinblastine

Floxuridine Vincristine

Fludarabine Vinorelbine

CLASS II

Bleomycin Fluorouracil < 1000 mg/m²

Busulfan 1 mg/kg (oral) Methotrexate < 100 mg/m²

Cytarabine < 500 mg/m² Thiotepa > 200mg/mm²

Etoposide Topotecan

CLASS III

Cisplatin continuous infusion <25 mg/m²/day Idarubicin

Cyckophosphamide < 600 mg/m² Ifosfamide 2000-4999 mg/m²

Cytarabine 500-1499 m/m² Irinotecan

Dacarbazine continuous infusion Methotrexate 100-249 mg/m²

Daunorubicin Mitromycin C

Doxorubicin continuous infusion <20 mg/m² Mitroxantrone

Fluorouracil >1000 mg/m² Teniposide

CLASS IV

Carboplatin 200-400 mg/m² Hexamethyl melamine Carmustine <200 mg/m² Ifosfamide >5000 mg/m² Cisplatin < 50 mg/m² Lomustine < 60mg/m² Cyclophosphamide 600-999 mg/m² Methotrexate >250 mg/m² Dacarbazine < 500 mg/m² Pentostatin Doxorubicin 21-59 mg/m² Procarbazine Table A (continued) CLASS V

Amifostine Dactinomycin Azacytidine Doxorubicin >60 mg/m²

Carmustine > 200 mg/m² Lomustine >60 mg/m² Cisplatin >50 mg/m² Mechlorethamine Cyclophosphamide > 1 g/m² Melphalan >100 mg/m² Cytarabine >1500 mg/m² Streptozocin Dacarbazine >500 mg/m² Thiotepa >100 mg/m²

See Drug Information Handbook 6th edition 1998-1999, American Pharmaceutical Association, 1998.

Subsequently, cyclic combination drug therapy was introduced in the late 1950s; and multi-drug regimens have long-since become the standard component of most effective treatment strategies for neoplastic disease. A representative listing of disseminated cancers which are deemed curable using multiple drug chemotherapy is provided by Table B below.

Table B*:

Probable

Disease Therapy Cure Rate

Adults

Intermediate- and high-grade non-Hodg ins lymphomas Combination chemotherapy 35 % -50 %

Hodgkin's disease (stage III or IV) Combination chemotherapy 50% or greater

Testicular carcinoma (stage III) Combination chemotherapy 75 % or greater followed by surgery

Gestational choriocarcinoma Methotrexate ± actinomycin D 90% Ovarian carcinoma Platinum-containing combination 10% -20 % chemotherapy

Acute myelocytic leukemia Combination chemotherapy 20 %

Children

Acute lymphocytic leukemia Combination chemotherapy 50% or greater plus cranial irradiation

Intermediate- and high-grade non-Hodgkin's lymphomas Combination chemotherapy 50 % or greater

Wilms' tumor and sarcomas Surgery, chemotherapy, 50% and radiation

Reproduced from Cancer Chemotherapy and Biotherapy: Principles and Practice. (Chabner & Lango, editors), Lippincott-Raven Publishers, 1996, page 3.

The superior results of combination or multiple-drug chemotherapies in comparison to single-agent use treatments are derived from and supported by a variety of clinical observations and considerations: (1) Initial resistance by a tumor to any known single drug or chemical agent is frequent, even with the most responsive neoplasms; and even with the most responsive tumors, the complete response rate does not typically exceed 20% of cases. (2) Initially responsive tumors rapidly acquire resistance after repeated exposure to a single drug of choice, presumably owing to a selection of some few pre-existing resistant tumor cells present within the heterogeneous tumor cell population constituting the original neoplastic mass. (3) Some anticancer drugs themselves are known to increase the rate of mutation in tumor cells initially sensitive to that given drug or agent, thereby creating the basis for an evolving drug resistance as an eventual outcome and byproduct consequence of continuing the chemotherapeutic treatment itself. (4) The use of multiple drugs or agents, each with an individual cy to toxic activity but with different mechanisms of action, would allow for independent tumor cell killing by each agent; thus a tumor cell resistant to one agent might still be sensitive to one or more other drugs if these were concurrently administered as part of the treatment regimen. (5) The route and mode of drug administration appears significant; thus while historically the most common anticancer regimens have employed intermittent bolus delivery of drugs, more recent treatments routinely prefer and use constant-infusion administrations of multiple drugs to provide a less toxic and constant exposure of different therapeutic agents to the tumor cells.

Today, despite the use of many different drugs and agents which are used routinely in chemotherapeutic treatment strategies, tumor cell resistance is and remains an ever-increasing problem for clinicians. Drug resistance, either apparent with initial multi-drug treatment or emerging at the time of relapse after an initial favorable response, almost inevitably occurs in all but a few cancer types deemed potentially curable (such as those listed within Table B). A variety of different resistance mechanisms have been put forward by research investigators seeking facts and reasons for the clinically validated drug resistance. The most widely accepted hypothesis of acquired drug resistance in tumors that are initially responsive to chemotherapy - without specifying the precise biochemical processes involved - suggests that resistance to chemotherapy by cancer cells results from random spontaneous accumulations of somatic mutations by tumor cells. See for example the detailed presentation and discussion in Cancer Chemotherapy and Biotherapy: Principles and Practice (Chabner & Lango, editors), 1996, Chapter 1; and the references cited therein. A illustrative and representative listing of some exemplary proposals and mechanisms suggested in the scientific literature for biochemical drug resistance is provided by Table C below. The reader is also directed to the following authoritative publications for more comprehensive and detailed information: Loe et al.. Eur. L Cancer 32A: 945-957 (1996); Lehnet, M., Ey_jL L Cancer 32A: 912-920 (1996); Fisher et al.. Eur, L Cancer 32A: 1082-1088 (1996); Twenhnan, P.R. and C.H.M. Versantroort, Eιn\ L Cancer 32A: 1002-1009 (1996); Ferry eLaL, Eur, Cancer 32A: 1070-1081 (1996); and the references cited within each of these publications.

Table C*:

MECHANISM OF RESISTANCE MUTATION DRUGS INVOLVED

Alteration of drug target

Dihydrofolate reductase Gene amplification, Methotrexate overexpression overexpression Thymidylate synthase Gene amplification, 5-FU overexpression

Topoisomerase II structure Point mutation, deletion VP-16, VM-26, alteration doxorubicin

Alteration of drug influx

Drug transporter proteins Point mutations?, deletions? Methotrexate?

Alteration of drug efflux or intracellular retention

Polyglytamation defect Methotrexate MDR overexpression Gene amplification, Vincristine, transcriptional dysregulation VP-16, VM-26 doxorubicin, paclitaxel, other natural products

MRP overexpression ? Probably similar to MDR

Increased drug detoxification

Glutathione, increased synthesis ? Alkylators, anthracyclines?

Glutathione-S-transferase ? Alkylators, metalators overexpression

Increased DNA repair

0°-alkyl guanine alkyl transferase Increased translation Nitrosourease, DTIC, overexpression procarbazine, temozolomide

Table C (continued):

MECHANISM OF RESISTANCE MUTATION DRUGS INVOLVED Defective adoptosis p53 inactivation Point mutations Most cytotoxic drugs bcl-2 overexpression Translocation, transcriptional Most cytotoxic drugs dysregulation

Overexpression of other Most cytotoxic drugs apoptosis inhibition genes

Reproduced from Cancer Chemotherapy and Biotherapy: Principles and Practice (Chabner & Lango, editors), Lippincott-Raven Publishers, 1996, page 9.

In sum, it is clear that the problem of tumor cell resistance to chemotherapeutic treatments using single and multiple agents and pharmacologically active drugs is and remains an increasingly difficult problem. Even while more and newer drugs are being developed and tested in varying combinations for clinical effectiveness, the risk of creating tumor resistance to these combination drug therapies grows greater. Accordingly, were a technique to be created which addresses and interrupts a specific biochemical mechanism and reaction recognized as a requisite component in tumor cell drug resistance, such a development would be recognized by clinicians and research investigators alike as an unforeseen and long-sought major achievement.

SUMMARY OF THE INVENTION

The present invention provides a method for reducing chemotherapeutic drug resistance exhibited in-situ by a solid mass neoplasm of epithelial origin, said method comprising the steps of: identifying a solid mass neoplastic cell of epithelial origin as being constituted at least in part of tumor cells clinically resistant in-situ to at least one discrete substance previously administered to the cell as a chemotherapeutic treatment agent; and administering to said neoplasm at least one antagonistic antibody preparation specific against at least one epitope presented by a protein selected from the group consisting of PDZKl protein and cMOAT protein such that intracellular binding of PDZKl protein with cMOAT protein is inhibited in-situ, and whereby clinical resistance to the previously administered chemotherapeutic treatment agent exhibited by the solid tumor cell becomes reduced.

The subject matter as a whole which is the present invention also provides a composition for reducing chemotherapeutic drug resistance exhibited in-situ by tumor cells constituting a solid mass neoplasm of epithelial origin, said composition comprising: an antagonistic antibody preparation specific against at least one epitope presented by a protein selected from the group consisting of PDZKl protein and cMOAT protein such that intracellular binding of PDZKl protein with cMOAT protein within the tumor cell is inhibited in the presence of said antibody preparation, and whereby clinical resistance to the previously administered chemotherapeutic treatment agent exhibited by the solid tumor cell becomes reduced in-situ.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more easily understood and better appreciated when taken in conjunction with the accompanying drawing, in which:

Figs. 1A-1H are photographs showing immunohistochemical studies of normal and carcinomas of human tissues and organs;

Figs. 2 A and 2B are representations of cMOAT cDNA sequences; Fig. 3 is a photograph showing SDS-PAGE analysis of different PDZKl and cMOAT protein interactions; and

Figs. 4A-4T are photographs showing cellular localization of PDZKl and cMOAT mRNAs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a method for reducing drug resistance to chemotherapeutic drugs exhibited by solid mass tumors of epithelial cell origin; and provides antagonistic antibody means for administration to living subjects whose neoplasms demonstrate single- or multi-drug resistance clinically to chemotherapeutic drug regimens. It will be recognized and appreciated that the subject matter as a whole which is the present invention concerns itself with a very sophisticated and technically difficult medical problem; and that a general familiarity and understanding of cancer in general and malignant tumors in particular is essential in order to properly understand the present invention.

It is important also to employ some basic definitions and terms properly and precisely and to relate them to a clinical context. Many clinical and medical terms regarding cancer have a more precise meaning than is commonly appreciated. For this reason, a minimal set of definitions and terminology is presented below which will be employed in the description of the invention which follows hereinafter.

Definitions and Terminology: Neoplasm: an abnormal mass of cells typically exhibiting uncontrolled and progressive growth. Neoplasms are broadly classified into two categories: (1) according to the cell type from which they originate; and (2) according to their biologic behavior, LC_., whether they are benign or malignant.

Cancer: a general term that by common usage has come to encompass all forms of malignant neoplasms.

Malignant: a concept referring to the tendency to become progressively worse and to result in death. With neoplasms, the term denotes the properties of invasiveness and metastasis.

Benign: mild, favorable or kindly; in oncology, the opposite of malignant. Benign neoplasms are usually well circumscribed and are often encapsulated; by definition, benign tumors do not invade locally and do not metastasize.

Metastasis: the process by which malignant cells are disseminated from the tumor of origin (the primary tumor) to form a new growth (the secondary tumor) at a distant site. It is the discontinuous extension of a malignant neoplasm.

In addition, in order to properly understand the experiments presented hereinafter, it is important that the user be at least familiar with the many techniques for manipulating and modifying genes and DNA fragments which have been reported and are today widespread in use and diverse in application. Merely exemplifying the many authoritative texts and published articles presently available in the literature regarding genes, DNA and RNA probes, nucleotide manipulation, and the expression of proteins from manipulated DNA are the following: Gene Probes for Bacteria (Macario and De Macario, editors), Academic Press Inc. , 1990; Genetic Analysis. Principles. Scope and Objectives by John R.S. Ficham, Blackwell Science Ltd. , 1994; Recombinant DNA Methodology II (Ray Wu, editor), Academic Press, 1995; Molecular Cloning. A Laboratory Manual (Maniatis, Fritsch, and Sambrook, editors), Cold Spring Harbor Laboratory, 1982; PCR fPolymerase Chain Reaction). (Newton and Graham, editors), Bios Scientific Publishers, 1994; and the many references individually cited within each of these publications.

The reader is also presumed to be both familiar and acquainted with the published scientific reports and the relevant patent literature regarding cMOAT, the multi-drug resistance-associated protein; with PDZKl protein; as well as with their respective functions, their attributes, and their relationship to tumors generally. However, among this very large body of information known and accumulated to date, it is often difficult, if not impossible, to focus upon unusual and critical observations which are the foundation of unforeseen developments and unexpected innovations within the field. A summary review of the scientific and evidentiary basis for the present invention will therefore serve the reader and provide the proper factual background and focus for recognizing the truly unique and unforeseen aspects of the present invention.

I. The Type Of Tumor Affected By In-Situ Inhibition Of PDZKl and cMOAT Protein Interactions It will be recognized and recalled that the specific development in-vivo of a resistance to chemotherapeutic agents is one of the major problems in the treatment of cancer; and often results in the failure of chemotherapy in treating a very large percentage of human cases. In many instances, the multi-drug resistance shown by a solid tumor mass in-vivo is believed to be dependent upon the existence, expression, and continuing interaction in-situ of the PDZKl and cMOAT proteins. Accordingly, the present invention's goal and objective is to inhibit and prevent such interactions required by a pre-existing tumor cell to survive and grow. It is the purpose of the present inhibitory methodology to reduce tumor resistance to chemotherapeutic agents generally.

With this objective and goal in mind, it is useful to first address, identify, and characterize the tumor cell target which is to be deprived of an adequate resistance to chemotherapy. For purposes of the present invention, any solid tumor mass of epithelial cell origin lying in any part of the body and in any particular tissue or cell type is suitable as the intended target for inhibition of chemotherapeutic agent resistance - presuming that both the PDZKl and cMOAT proteins are expressed intracellularly. It will be recalled that by definition a tumor is a neoplasm - an abnormal mass of cells typically exhibiting uncontrolled and progressive growth. Neoplasms are broadly classified into two categories: (1) according to the cell type from which they originate; and (2) according to their biologic behavior - whether they are benign or malignant. Accordingly, so long as the neoplasm is a solid mass of abnormal cells in which there is a distinct or discrete tumor matrix, stroma, and included and/or associated blood vasculature, that neoplasm is a proper and suitable target for inhibition of tumor drug resistance using the present methodology.

It will also be recognized that the particular state of the neoplasm or tumor - so long as it is a definable solid mass - does not influence the suitability of or use for the present invention. Thus, the tumor may be a "benign" neoplasm - that is, mild, favorable, or kindly (the opposite of malignant). Benign neoplasms are usually well circumscribed and are often encapsulated; and, by definition, do not invade locally and do not metastasize. In comparison, a "malignant" tumor is a neoplasm having the tendency to become clinically progressively worse and to result in the death of the subject. With neoplasms, the term "malignant" denotes the properties of tumor invasiveness and metastasis. In addition, the term "metastasis" is defined as the process by which malignant cells are disseminated from the tumor of origin (the primary tumor) to form a new growth (the secondary tumor) at a distant site; it is the discontinuance extension of a malignant neoplasm. Thus, it is a primary purpose and goal of the present invention to inhibit tumor drug resistance in malignant tumors of epithelial cell origin generally wherever they may be found as a discrete tumor mass.

Accordingly, the present inhibitory methodology is directed at solid tumors found clinically within the living patient in-situ; and the entire broad class of human and animal solid mass tumors is deemed suitable for such therapeutic treatment wherever the tumor may be found within the body - so long as the tumor cell is of epithelial cell origin and both the cMOAT protein and the PDZKl protein are expressed intracellularly. Equally important, and especially for purposes of malignant tumors and neoplasms, the present inhibitory methodology is suitable for use with the epithelial cell tumor regardless of what kind, type, grade, age or stage may apply to the tumor in question. Thus, many types of primary and metastatic solid tumors of epithelial cell origin can be treated in-vivo. Representative examples are breast cancer, endometrial cancer, colon cancer, lung cancer, kidney cancer, prostate cancer, and squamous cell carcinoma of the skin. For these reasons, the present method for reducing tumor multi-drug resistance is deemed to be a broadly applicable and clinically valuable therapeutic treatment.

II. The Intracellularly Expressed PDZKl Protein: The PDZKl protein is an intracellularly expressed substance whose presence, particularly in abundant quantities, already indicates an ongoing neoplastic development - and provides a part of the drug resistance mechanism for carcinomas of those particular cells and tissues.

The PDZKl protein is constituted as a 519 amino acid strand, the composition and amino acid residue sequence of which is recited by Table 1. The PDZKl protein is about 63 kD in molecular weight; and comprises four individual and distinct PDZ domains varying in size from 54 to 80 amino acids which are shown as block enclosures within the cDNA and amino acid correlation of Table 2. Also, the PDZKl protein is devoid of a SH3 binding domain; does not contain a guanylate kinase domain; but does actively interact with the membrane associated protein MAP 17 (recognized as itself being overexpressed in a number of different carcinomas). The isolation, amino acid residue composition, and overall characteristics of the PDZKl protein are described in detail by the experiments and empirical data presented within prior pending U.S. Patent Application Serial No. 08/997,445 filed December 23, 1997, the text of which is expressly incorporated by reference herein.

Table 1

MTSTFNPRECKLSKQEGQNYGFFLRIEKDTEGHLVRVVEKCSPAEKAGLQD

GDRVLRINGVFVDKEEHMQVVDLVRKSGNSVTLLVLDGDSYEKAVKTRV

DLKELGQSQKEQGLSDNILSPVMNGGVQTWTQPRLCYLVKEGGSYGFSLKT

VQGKKGVYMTDITPQGVAMRAGVLADDHLIEVNGENVEDASHEKVVEK

VKKSGSRVMFLLVDKETDKRHVEQKIQFKRETASLKLLPHQPRIVEMKKGS

NGYGFYLRAGSEQKGQIIKDIDSGSPAEEAGLKNNDLVVAVNGESVETLDH

DSVVEMIRKGGDQTSLLVVDKETDNMYRLAHFSPFLYYQSQELPNGSVKEA

PAPTPTSLEVSSPPDTTEEVDHKPKLCRLAKGENGYGFHLNAIRGLPGSFIKE

VQKGGPADLAGLEDEDVIIEVNGVNVLDEPYEKVVDRIQSSGKNVTLLVCG

KKAYDYFQAKKIPIVSSLADPLDTPPDSKEGIVVESNHDSHMAKERAHSTAS

HSSSNSEDTEM

Table 2

s c s t r s oa GOT ore

50 0 « c r t 241

t 0 249 840 r s s ore

1020

1021 CTO CCT CAT TTT TCT CCA TTT CTC TAC TAT CAA ACT CAA CAA CTO CCC AAT OGC TCT CTC 1080 330 L A H P S P r Y Y Q S Q E L P N C S V 349

1081 AAO CAC GCT CCA GCT CCT ACT CCC ACT TCT CTO GAA CTC TCA ACT CCA CCA GAT ACT ACA 1140 s s s

1200 c 389 1260 t

1320

_

1380

1440

^{V c} 0

1441 ATT OTT TCC TCC CTO OCT GAT CCA CTT GAC ACC CCT CCA GAT TCT AAA GAA GGA ATA CTG 1500 470 I V S S L A D P L D T P P D S K E G I V 489

1501 OTO GAG TCA AAC CAT GAC TCO CAC ATO OCA AAA GAA CGO CCC CAC ACT ACA GCC TCA CAT 1560 Clinical Significance

The expression and quantitative presence of the 519 amino acid residue containing PDZKl protein has major clinical value and utility in the development and occurrence of drug resistance within the neoplastic cells and tissues of epithelial cell origin where the protein is found. The presence of the PDZKl protein intracellularly, within particular tumor cell types or neoplastic tissues and/or in especially large or expressive quantitative amounts, is a reliable indicator of ongoing intracellular interactions with cMOAT protein within the cells and tissues of interest; and that the cells and tissues in question are becoming more resistant to single or multiple chemotherapeutic drug treatment while becoming more and more variable, ineffective, and dysfunctional.

Distribution In Normal Cells And Tissues of Epithelial Origin:

As evidenced by the data presented hereinafter, PDZKl protein has been found to be expressed in minimal or moderate quantities by selected normal cells and tissues; and only by some normal cells and tissues of epithelial cell origin (rather than all types generally). Other kinds of cells (of epithelial cell origin) are not able to express and produce PDZKl protein at all. Thus, mesenchymal cells and inflammatory cells, which are not of epithelial cell origin, but are medically and functionally normal in all clinical respects, have been shown not to express or produce PDZKl protein. In addition, there is considerable variety in the range of normal cells and tissues of epithelial cell origin which are able to express PDZKl protein in detectable amounts. A representative listing of such normal cells and tissues is given by Table 3 below.

Table 3: Normal Cells and Tissues

Normal Expression And Quantitative Tissue or Presence of Amount of Cell Tvpe PDZKl Protein PDZKl Protein*

Tissues (^"Normal)

Kidney Yes + +

Liver Yes + +

Pancreas Yes + +

Small Intestine Yes + +

Testis Yes +

Adrenal Cortex Yes +

Stomach Yes +

Heart No -

Brain No -

Placenta No -

Lung No -

Breast No -

Adrenal Medulla No -

Thyroid No -

Thymus No -

Colon No -

Table 3: Normal Cells and Tissues (continued)

B. Cells (Normal) Presence Quantity

Proximal tubular epithelial cells Yes + + + (kidney)

Renal parenchyma No cells (kidney, other than proximal tubular epithelial cells)

Glandular epithelial cells Yes + (stomach)

Glandular epithelial cells Yes + (small bowel)

Colonic mucosa cells No (large intestine)

+ = minimal quantity

+ + = moderate quantity

+ + + = high quantity

+ + + + = excessive quantity

Quantity above background labeling (a negative result) The listing of Table 3 thus provides several valuable criterion and standards. These include the following: (a) Among the normal cells and tissues of epithelial cell origin which were empirically tested, only about half express PDZKl protein in any detectable amount, (b) Even within a single organ, some cells comprising the organ tissues may express PDZKl protein while other cell types in the organ do not. The kidney is a illustrative example of this finding, where the proximal tubular epithelial cells express the proteins while other renal parenchyma cells do not. (c) A number of organs such as the breast, colon and lung, in the medically normal state, do not express PDZKl protein at all. (d) In those medically normal cells and tissues which consistently demonstrated expression of PDZKl protein, only moderate or minimal quantitative amounts of protein were produced; in no instance was either a large or excessive quantity of PDZKl protein produced by a medically normal cell or tissue of epithelial origin.

Distribution in Neoplastic (Malignant) Cells and Tissues of Epithelial Cell Origin

The expression and production of PDZKl protein in neoplasms of epithelial cell origin (carcinomas generally) shows a very different range and distribution among abnormal cells and tissues. A representative listing of neoplastic cells and tissues as well as their ability to express PDZKl protein is given by Table 4 below.

Table 4: Neoplasms

Neoplastic Expression And Quantitative Tissue or Presence of Amount of Cell Type PDZKl Protein PDZKl Protein*

Renal cell carcinoma 75 % of cases + + to + + + +

Infiltrating carcinomas of the breast 55 % of cases + + + to + + + +

Colonic carcinomas 25 % of cases + + + to + + + +

Lung carcinomas 25 % of cases + + + to + + + +

+ = minimal quantity

+ + = moderate quantity

+ + + = high quantity

+ + + + = excessive quantity

* Quantity above background labeling (a negative result)

The listing of Table 4 also provides valuable diagnostic criterion and standards for using the expression of PDZKl protein as a marker of increasing chemotherapeutic drug resistance. These findings include: (a) In those cells and tissues which if medically normal do not express PDZKl protein, a large number of carcinomas of such cells and tissues express and produce PDZKl protein in either large or excessive amounts. Illustrative examples are the breast, lung and colonic carcinomas, (b) In those cells and tissues which might or might not express PDZKl protein under medically normal conditions, a meaningful number of carcinomas of these cells and tissues express and produce PDZKl protein in high or excessive amounts. Examples of these are the renal cell, breast, colon, and lung carcinomas.

Inferentially and indirectly, at least one additional conclusion may be drawn which has clinical impact and predictive value: In those cells and tissues which do not express PDZKl protein under medically normal conditions, the expression and production of PDZKl protein - even in minute amounts - will serve as a reliable indicator that the cell regulatory control mechanisms are degrading and failing. Thus, it is expected that in a number of instances involving carcinomas of the adrenal, the thyroid, the thymus and the like, detectable amounts of PDZKl protein will be expressed and produced; and that such PDZKl protein expression will directly increase the resistance of such cells to chemotherapeutic drug treatments clinically.

III. The Intracellularly Expressed cMOAT Protein Human canalicular multispecific organic anion transporter or "cMOAT" protein is also known as multidrug resistance-associated Protein 2 or "MRP2" ; and is a member of the family of multidrug resistance-associated proteins (MRPs) acting intracellularly at the cell membrane to confer drug resistance in tumor cells. The MRP family of proteins and cMOAT in particular, belong to the ATP-binding cassette (ABC) or traffic ATPase superfamily of transport proteins which act to provide removal of a variety of different chemotherapeutic agents from tumor cells - thereby clinically demonstrating an acquirement of drug resistance. Human cMOAT is constituted of 1,531 amino acids; is a 190 kDa molecule; and has a carboxy (COOH) - terminal end segment which is 124 amino acid residues in size. The carboxy-terminal portion is particularly important because it is this end portion of the cMOAT molecule which directly interacts with one of the PDZ domain of the PDZKl protein at the tumor cell membrane. The amino acid sequence for the carboxy-terminal end is recited by Table 5 below. It will be recognized also that Table 5 is a partial reproduction and restatement of the published information described by Kool et al.. Cancer Research 57: 3537-3547 (1997), the entire text of which is expressly incorporated by reference herein.

Table 5: C-Terminal Sequence of cMOAT

L V L R G I T C D I G S M E K I G V V G R T G A G K S S L T N C L F R I L E A A G G Q I I I D G V D I A S I G L H D L R E K L T I I P Q D P I L F S G S L R M N L D P F N N Y S D E E I W K A L E L A H L K S F V A S L Q L G L S H E V T E A G G N L S I G Q R Q L L C L G R A L L R K S K I L V L D E A T A A V D L E T D N L I Q T T I Q N E F A H C T V I T I A H R L H T I M D S D K V M V L D N G K I I E Y G S P E E L L Q I P G P F Y F M A K E A G I E N V N S T K F

The reader is presumed to be acquainted and informed regarding the characteristics, properties, genetic origins, cDNA, and multiple drug resistance capabilities of cMOAT protein and the MRP family of transponder activities generally - all of which have been the subject of many research investigations and publications. Merely exemplifying such scientific publications are the following: Loe et al.. Eur. L Cancer 32A: 945-957 (1996); Taniguchi et al.. Cancer Research 56: 4124-4129 (1996); Cole et al.. Cancer Res. 54: 5902-5910 (1994); Kool et al.. Cancer Res. 57: 3537-3547 (1997); Paulusma et al.. Hepatology 25: 1539-1542 (1997); Koike et al.. Cancer Res. 57: 5457-5479 (1997); Keppler et al.. FASEB L 11: 509-516 (1997); S. Chaub et al.. L AJTL Soc, Nephrol. 8: 1213-1221 (1997); Paulusma et al.. Science 271 : 1126-1128 (1996); and the references cited within each of these publications, all of which are expressly incorporated by reference herein.

Distribution In Neoplastic Cells And Tissues Of Epithelial Cell Origin cMOAT protein has been found in a variety of different neoplasms of epithelial cell origin. A representative listing of tumor cells and tissues in humans is given by Table 6 below. Also, a partial listing of various drugs associated with cMOAT protein and drug resistance activity is provided by Table 7 below.

Table 6: Neoplasms

Neoplastic Presence of Associated

Tissue or cMOAT Drug

Cell Tvpe Protein* Resistance

Renal cell carcinoma Yes Yes

Infiltrating carcinomas Yes Yes of the breast

Colonic carcinomas Yes Yes

In addition, cMOAT protein is overexpressed in a large number of cell lines derived from ovarian, bladder, testicular, and squamous cell carcinomas [see for example, Kool et al.. Cancer Res. 57: 3537-3547 (1997)].

Table 7:

Resistance To Drugs

Cisplatin;

CPT-11;

SN-38;

Vincristine;

Doxorubicin;

Etoposide;

ACNU ,3-[4-amino-2-methyl-5-pyrimidynil-methyl]-l-(2- chloroethyl)- 1 -nitrosourea;

5-Flouro Uracil;

Mitomycin C

* Restatement of data extracted in part from Koike et al.. Cancer Res. 57:

5475-5479 (1997), at page 5477.

IV. Underlying Mechanism Of Chemotherapeutic Drug Resistance For

The Invention The present invention utilizes and relies upon a novel and previously unknown mechanism of direct protein interaction between cMOAT protein and PDZKl protein in-situ as the basis for overcoming single or multiple drug resistance in-situ by tumor cells of epithelial cell origin. Evidence of such direct protein interactions is provided by the experiments and empirical data described hereinafter. Such interactions between the recently isolated PDZKl protein having four PDZ binding domains and cMOAT protein have been previously unknown; in fact, no relationships or interactions between cMOAT protein and PDZKl protein have ever been proposed or envisioned before the present invention was conceived or demonstrated empirically.

As shown experimentally hereinafter, the PDZ domains of the PDZKl protein interact and bind with the carboxy-terminal amino acid residues of the cMOAT protein in-situ, locally at or near the cell membrane. The interactions between the cMOAT and PDZKl proteins is direct; no intermediaries or cofactors are involved in the binding reactions; and such direct binding interactions occur only within the cell membrane area by solid tumor cells of epithelial origin.

By their direct binding and interaction in-situ, the cMOAT and PDZKl proteins are requisite components and mediators of the active organic ion transport system acting intracellularly to deplete and remove chemotherapeutic drugs from the cytoplasmic interior of solid mass tumor cells. The cMOAT/PDZKl protein binding is believed to promote an increase in chemotherapeutic drug resistance generally for neoplasms under both clinical in-vivo conditions and under in-vitro experimental circumstances.

The methodology and means provided by the present invention for reducing single and/or multiple drug resistance within tumor cells and neoplasms of epithelial cell origin is therefore directed at preventing, inhibiting, and effectively disrupting the direct binding and protein interactions which would otherwise typically occur in solid mass tumor cells within a human or animal living subject. Such prevention, inhibition, and/or disruption is best achieved via the administration of antagonistic antibody preparations - in a present regimen of treatment, specific means for effectively reducing drug resistance by tumor cells in- situ.

An Antagonistic Antibody Preparation Effective

Against Interacting PDZKl And cMOAT Proteins The requisite manipulation is the administration to the subject of at least one antagonistic preparation effective against at least one of the interacting cMOAT and PDZKl proteins in-situ such that tumor drug resistance is reduced or inhibited in- vivo. For this purpose of explicitly antagonizing either of the respective cMOAT or PDZKl proteins in situ, the preferred means and agent is a function-blocking antibody preparation comprised of monoclonal and/or polyclonal antibodies which are specific for one or more epitopes on either of these interacting proteins.

The function-blocking antibody antagonist

The preferred function-blocking antibody antagonist will demonstrate two characteristics: It will have the capability of binding specifically to one or more epitopes present within a spatially exposed region of the cMOAT or PDZKl proteins as expressed in-vivo. In addition, the other essential characteristic of the specific function-blocking antibody is - that upon binding to the particular protein or protein part, the functional interactions between the cMOAT and PDZKl proteins will be prevented. Both properties are necessary and required.

The antigenic determinants recognized by the function-blocking antibodies are provided by the amino acid residues comprising the complete protein sequence of PDZKl or the carboxy-terminal portion of the cMOAT protein as shown by Tables 2 and 5 respectively herein. However, this specific binding capability can be demonstrated not only by a whole intact antibody, but also by F(ab')₂ fragments, as well as by Fab fragments derived from the whole antibody structure. It will be recalled that while the whole antibody molecule is a large bulky protein having two specific binding sites, the F(ab')₂ fragment represents a divalent binding fragment of the whole antibody; while the Fab binding portion is a univalent binding unit having a minimum of antibody structure. Similar smaller and genetically engineered antibody units having a specific binding capability have also been recently developed; and these entities are deemed to be equally suitable for use herein.

In addition, particular methods for preparing "humanized" antibodies have been devised. See for example, Co, M.S. and C. Queen, Nature 351 : 501-502 (1991); Winter, G. and W.J. Harris, TjPs 14: 139-142 (1993); Stephens eLaL , Immunology 85: 668-674 (1995); Kaku et al.. Eur. L Pharmacol. 279: 115-121 (1995); and the references cited within each of these publications. Humanized antibodies offer distinct therapeutic advantages; and thus are highly preferred for clinical use because they are less likely to provoke an immune response from the patient undergoing treatment.

Other methods for preparing, isolating, and purifying each of these different antibody binding sequences and units are conventionally known in the scientific literature and these techniques have been available for many years as common knowledge in this field. The user may thus choose from among all of these different structured formats - whole antibodies, antibody subunits and antibody fragments - in picking a useful antagonistic structure having a specific binding capability for an epitope in one of the spatially exposed regions.

In general therefore, the user has the option to chose whether the function- blocking antibody antagonist(s) is obtained from monoclonal, or polyclonal or broad antisera sources. Equally important, the user will decide whether the antibody or antibody fragments should be isolated and purified prior to use; whether they should be altered into humanized antibody forms; or whether the antibody antagonist can be employed as a heterogeneous mixture of different entities and varying binding affinities, only some of which will have the requisite affinity and specific binding capability for an exposed epitope on the PDZKl protein of cMOAT protein expressed in-situ. Thus, the degree of homogeneity, purity, human compatibility, affinity, and specificity of antibodies or antibody fragments and genetically engineered subunits for one or more epitopes of these proteins is left to the discretion and needs of the user. Immunogens

The entirety of the PDZKl protein or the carboxy-terminal portion of the cMOAT protein as well as different fragments thereof can serve as immunogens since antibodies obtained with such immunogens will be evaluated and selected for their specific binding and function-blocking properties, before use.

It will be noted and appreciated also that the range and variety of the intended sites for epitope binding with either of these two distinctive proteins as a whole provide a large number of potential antigenic determinants within each permissible region spatially available for use. Thus, if one chooses a peptide fragment as an immunogen, it will be recalled that a minimum of 5-7 amino acid residues (in theory) are able to be employed as a haptene in order to raise specific antibodies within a living host animal. However, longer peptide lengths of at least 10-20 residues are generally preferred. It will be noted also that the various regions in the cMOAT or PDZKl structures (shown by Tables 2 and 5) available for use as a source of antigenic determinants each provide far longer amino acid residue segments for this purpose. Thus, if an extended segment length of amino acid residues were purposely employed as the immunogen, a larger number of different antigenic determinants becomes available, given the range of residue choices. Accordingly, the number of potential epitopes becomes enormous; yet each of these epitopes is a potential specific binding site for the antibody antagonist(s).

For peptide immunogens, it is intended and envisioned that at least one peptide segment of suitable length (preferably at least 10-20 residues) be chosen as the immunogen in order to provide the antigenic determinants and the production of specific antibodies using a living host animal. Once the amino acid residue length and composition has been chosen (preferably in conformity with the desired requirement of being within a spatially exposed region) , the chosen antigenic or haptene segment must be prepared. Often, the desired amino acid segment can be synthetically prepared using conventionally known solid phase peptide synthesis methods [such as Merrifield, RB, J. Arm Chem. Soc. 85: 2149 (1963)]. Once synthesized, it is most desirable that the chosen segment be purified (such as by gel filtration) and desirably analyzed for content and purity (such as by sequence analysis and/or mass spectroscopy).

After its isolation or synthesis, the chosen peptide segment is typically coupled to a protein carrier to form the immunogen. Conventionally suitable protein carries available for this purpose are available in great variety from many diverse sources. The only requirements regarding the characteristics and properties of the carrier are: first, that the protein carrier be in fact antigenic alone or in combination with the synthesized chosen amino acid residue sequence; and second, that the carrier protein be able to present the antigenic determinants of the residue sequence such that antibodies specific against the amino acid residues are produced in a living host animal. Clearly, as the experiments described hereinafter, the preferred choice of protein carrier for immunization purposes include keyhold limpet hemocyanin (KLH), coupled by glutaraldehyde (GLDH), sulfo-m- maleimidobenzo (M-hydroxysuccinimide) ester (MBS), or bisdiazobenzidine (BDB). However, any other carrier protein compatible with the host to be immunized is also suitable for use. Examples of such other carrier proteins include bovine serum albumin, thyroglobulin, and the like.

Immunization procedure

All immunizations and immunization procedures are performed in the conventionally known manner described in the scientific literature. It is expected that under certain use conditions, adjuvants will be employed in combination with the prepared immunogens. Alternatively, the prepared immunogens may be used alone and be administered to the animal or human host in any manner which will initiate the production of specific antibodies.

In addition, the harvesting of polyclonal antiserum and the isolation of antibody containing sera or antibody producing cells follows the conventionally known techniques and processes for this purpose. Similarly, the preparation of hybridomas follows the best practices developed over recent years for the isolation of monoclonal antibodies [Marshak-Rothstein et al.. L Immunol. 122: 2491 (1979)]. Polyclonal and monoclonal antibodies

Once obtained, the polyclonal antisera and/or monoclonal antibodies and/or genetically engineered antibodies should be evaluated and verified for their ability to bind specifically with an epitope existing within a spatially exposed region of either cMOAT or PDZKl protein and for the capability to functionally block the abilities of the cMOAT and PDZKl proteins to bind to each other. If desired, cleavage with papain will produce two Fab fragments plus the Fc fragment; whereas cleavage of the antibodies with pepsin produces the divalent F(ab')₂ fragment and the Fc' fragment - all as conventionally known. It will be expressly understood, however, that regardless of whether the antibody binding portion represents polyclonal antisera, monoclonal antibodies, the F(ab')₂ fragment, Fab fragments, humanized antibodies, or other antibody species - all of these are suitable and intended for use so long as the specific function blocking capability is demonstrated after binding to at least one epitope existing within the cMOAT or PDZKl proteins and expressed in-vivo. It is therefore deemed to be expected that a wide variety of different immunoassay systems will be employed to demonstrate the specific binding and function-blocking capabilities required by the antibody antagonists of the present invention; and that the parameters of concentration, volume, temperature, carriers, and delivery systems can be varied extensively at will when choosing antibodies and/or antibody fragments and subunits. The present invention therefore presumes and incorporates by reference any conventionally known immunoassay technique, procedure, protocol, or other factor or parameter - all of which may be usefully employed for the evaluation and/or preparation of a specifically binding and functionally-blocking antibody antagonist.

Preparation of a PDZKl antibody against a PDZKl -GST fusion protein

For fusion protein production, a Bglll DNA fragment containing the complete PDZKl cDNA clone was subcloned into the BamHI site of the glutathione-S-transferase (GST)-fusion vector pGEX-3X (Amrad Corporation Ltd. , Melbourne, Australia). E. coli JM109 were transformed with the recombinant plasmids. Fusion protein production was induced with 0.5 mmol/1 isopropyl-b-D- thiogalactopyranoside (IPTG). The fusion proteins (GST and GST-PDZK1) were affinity purified using a glutathione-sepharose column (Pharmacia, Uppsala, Sweden) as previously described (Smith et al.. 1998; Kocher et al. , 1990). A polyclonal antibody against PDZKl was prepared against the GST-

PDZK1 fusion protein (Lampire, Pipersville, PA). Chicken were immunized and IgY were recovered from egg yolks. Anti-PDZKl antibodies were subsequently purified by chromatography using a GST-PDZK1 affinity column after absorption of IgY against GST-sepharose. For Western blot analysis, protein extracts were electrophoresed on SDS-PAGE, transferred to nitrocellulose and incubated with the anti-PDZKl antibody, or chicken IgY as a control, at a concentration of 3 μg/ml. The Western blot was subsequently incubated with a biotinylated anti-chicken IgG at 1/500, then incubated with Avidin-horse radish peroxidase at 1/10,000 (Sigma, St. Louis, MO). These incubations were followed by a diaminobenzidine reaction (Sigma).

For immunoperoxidase studies to demonstrate antibody specificity, tissues were collected in the operating room and fixed for 4 hours in 4% paraformaldehyde in phosphate-buffered saline, pH 7.4 at 4°C and then transferred to 30% sucrose in phosphate-buffered saline, pH 7.4, overnight at 4°C. Tissues were then frozen in OCT compound (Miles Diagnostics, Elkhart, IN) and stored in liquid nitrogen. Immunoperoxidase studies were performed on 6 μm fixed-frozen tissue sections using either the affinity-purified primary antibody against PDZKl or chicken IgY as a control at a concentration of 5 μg/ml. The sections were then incubated with a biotinylated anti-chicken IgG using a 1/200 dilution (Vector, Burlingame, CA), and subsequently treated with the Vectastain ABC reagents (Vector) and diaminobenzidine (Research Genetics, Inc., Huntsville, AL), according to the manufacturer's protocol.

Antibody against cMOAT A polyclonal antibody was prepared using the 30-mer carboxy-terminal peptide of cMOAT protein sequence (GSPEELLQIPGPFYFMAKEAGIENVN STKF). A cysteine was added to the sequence at its NH2 terminus in order to conjugate the peptide to Keyhole Limpet Haemocyanin (KLH). Rabbits were immunized and the antibody was purified by affinity chromatography using the same peptide.

For immunoperoxidase studies to demonstrate antibody specificity, tissues were collected in the operating room and fixed for 4 hours in 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, at 4°C and then transferred to 30% sucrose in phosphate-buffered saline, pH 7.4, overnight at 4°C. Tissues were then frozen in OCT compound (Miles Diagnostics, Elkhart, IN) and stored in liquid nitrogen. Immunoperoxidase studies were performed on 6 mm fixed-frozen tissue sections using either the affinity -purified primary antibody against the cMOAT carboxyl-terminal protein sequence or rabbit IgG as a control at a concentration of 5 μg/ml. The sections were then incubated with a biotinylated anti-rabbit IgG using a 1/200 dilution (Vector, Burlingame, CA), and subsequently treated with the Vectastain ABC reagents (Vector) and diaminobenzidine (Research Genetics, Inc. , Huntsville, AL), according to the manufacturer's protocol.

In solution, both antibodies inhibit the cMOAT-PDZKl interaction when used at a concentration of 50 μg/ml in the presence of PDZKl -GST fusion protein at a concentration of 40 μg/ml and cMOAT carboxy-terminal peptide immobilized on agarose beads.

VI. The In- Vivo Inhibition Of Drug Resistance The consequence in-vivo of practicing the present methodology properly and completely in all its manipulative steps will provide and produce an effective inhibition of tumor multi-drug chemotherapeutic resistance as a clinically recognizable consequence and benefit. The present invention will provide a reliable and useful procedure for reducing drug resistance by solid tumors in-vivo within the body of a human or animal subject.

Dosages. Modes of Administration, and Pharmaceutical Formulations Compositions embodying the specifically binding and functionally-blocking antagonistic antibody for the present invention can be administered in any manner which preserves the function of the antibody and delivers it to the tumor site - such as intravenous, subcutaneous or other parenteral administration. The prepared antagonistic antibody can be introduced by any means or routing.

The dosage to be administered to any patient will vary and be dependent upon the age, overall health, and weight of the human or animal recipient; the kind of concurrent treatment, if any; the frequency of concurrent treatment; and the physician's prognosis for the patient. Generally, a range doses of antagonistic antibody from 0.1 milligrams to about 10.0 milligrams per kilogram of body weight, in twice weekly or three times weekly administrations is expected to be effective to yield the desired therapeutic result. The duration of antagonistic antibody dose administration is expected to be continued so long as a favorable clinical result is obtained. It is believed that this treatment regimen will reduce and markedly inhibit tumor chemotherapeutic drug resistance in-vivo; and, in this manner, act indirectly to help control the growth of the solid tumor in-situ. However, it is unclear whether or not this inhibitory treatment method will provide for complete elimination of single- or multi-drug resistance for the tumor. For this reason especially, the treatment duration and dosage should be monitored accordingly.

In addition, since the antagonistic antibody preparation is typically to be given intravenously, subcutaneously, or other parenteral applications, the appropriate quantity of antibody will be prepared in sterile form; exist in single or multiple dose formats; and typically be dispersed in a fluid carrier such as sterile physiological saline or 5 % dextrose solutions commonly used with injectables.

The efficacy of the therapy will be assessed using standard laboratory tests (such as measurements of tumor specific antigens in patient's blood) and radiological procedures to detect change in number and size of tumor lesions.

VII. Experimental and Empirical Data

To demonstrate the merits and value of the present invention, a series of planned experiments and empirical data are presented below. It will be expressly understood, however, that the experiments described and the results provided are merely the best evidence of the subject matter as a whole which is the invention; and that the empirical data, while limited in content, is only illustrative of the scope of the invention envisioned and claimed.

Methods and Materials:

Fusion protein production

For fusion protein production, a BgHI DNA fragment containing the complete PDZKl and cDNA clone was subcloned into the BamHI site of the glutathione-S-transferase (GST)-fusion vector pGEX-3X (Amrad Corporation Ltd., Melbourne, Australia). E. coli JM109 were transformed with the recombinant plasmid. Fusion protein production was induced with 0.5 mmol/1 isopropyl-β-D- thiogalactopyranoside (IPTA). The fusion proteins (GST and GST-PDZK1) were affinity purified using a glutathione-sepharose column (Pharmacia, Uppsala, Sweden) as previously described in Kocher et al., Lab. Invest. 78: 117-125 (1998).

Specific antibody against PDZKl protein

A polyclonal antibody against PDZKl was prepared against the GST- PDZK1 fusion protein (Lampire, Pipersville, PA). Chickens were immunized and IgYs recovered from egg yolks. Anti-PDZKl antibodies were subsequently purified by affinity chromatography using a GST-PDZK1 affinity column after absorption of IgY against GST-sepharose. The affinity purified antibody has been previously characterized as described in Kocher et al.. Lab. Invest. 78: 117-125 (1998).

Immunoperoxidase studies

For immunoperoxidase studies, tissues were collected in the operating room and fixed for 4 hours in 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, at 4°C and then transferred to 30% sucrose in phosphate-buffered saline, pH 7.4, overnight at 4°C. Tissues were then frozen in OCT compound (Miles

Diagnostics, Elkhart, IN) and stored in liquid nitrogen. Immunoperoxidase studies were performed on 6 μm fixed-frozen tissue sections using either the affinity- purified primary antibody against PDZKl or chicken IgY as a control at a concentration of 5 μg/ml. The sections were then incubated with a biotinylated anti-chicken IgG using a 1/200 dilution (Vector, Burlingame, CA) and subsequently treated with the Vectastain ABC reagents (Vector) and diaminobenzidine (Research Genetics, Inc. , Huntsville, AL), according to the manufacturer's protocol.

Yeast two-hybrid system

A 2000 bp Bglll DNA fragment containing the complete PDZKl coding sequence was subcloned into the BamHI sites of pGBT9 expression vector. The orientation and sequence was verified by sequence analysis. The yeast strain HF7c was then sequentially transformed with the PDZKl -pGBT9 vector; used as a bait; and a normal human liver cDNA library in the pACT2 expression vector (Clontech, Palo Alto, CA) was prepared [previous experiments showed that PDZKl was expressed in liver]. Potentially interacting clones were selected on synthetic dropout (SD) medium containing all amino acids except tryptophan, leucine, and histidine, in the presence of 5mM 3-amino-l,2,4-triazole (3-AT). Yeast colonies expressing potential interacting proteins were further tested using an X-Gal reporter assay, according to the manufacturer's protocol. The activating domain plasmids corresponding to each positive clone were rescued on selection media lacking leucine after transformation of E. coli HB101. Plasmid DNA from each clone was purified using standard techniques and subjected to DNA sequencing using oligonucleoti.de primers (Integrated DNA Technologies, Coralville, IA) and a 373 automated DNA sequencer from Applied Biosystems (Branchburg, NJ). The Genebank was also searched for sequence homologies.

Confirmation of the interaction between CMOAT and PDZKl proteins :

In order to confirm the interaction of cMOAT and PDZKl , a biotinylated 30-mer peptide corresponding to the carboxy terminus of cMOAT was synthesized (Genemed, South San Francisco, CA), and then coupled to agarose-avidin beads (Sigma, St. Louis, MO). Briefly, 0.8 mg of peptide was incubated with 1 ml of avidin-agarose beads in PBS (pH 7.4) for two hours at room temperature. Avidin- agarose beads with or without peptides (as a control) were subsequently incubated with bovine serum albumin (0.5 mg/ml) overnight at 37°C to block non-specific binding sites and washed with PBS. The beads were then incubated with either GST or GST-PDZK1 at a concentration of 40 μg/ml for 3 hours at 37°C. After extensive washings with PBS, the agarose beads were resuspended in sample buffer and run on SDS-PAGE with appropriate size markers under reducing conditions. The protein gels were fixed and stained with Coomassie blue.

In-situ hybridization studies In-situ hybridization was performed on 6 μm fixed-frozen tissue sections using single-strand antisense or control sense ³⁵S-labeled riboprobes as previously described [Brown et al.. Cancer Res. 53: 4727-4735 (1993)]. PDZKl sense and anti-sense cRNA probes were generated with ligating an Xhol-Bglll 1 kb DNA fragment corresponding to the 5 '-end of the PDZKl cDNA clone into pGEM7Zf and linearization with Styl. cMOAT sense and anti-sense cRNA probes were generated after ligating an EcoRI-BamHI 1 kb DNA fragment corresponding to the 3 '-end of the cMOAT cDNA clone into pSP72 and linearization with BstXI.

Experimental Results:

Experiment 1: Distribution of PDZKl Protein In Normal Human

Tissues And In Carcinomas Using the prepared specific anti-PDZKl antibody, immunocytochemical studies were performed using normal human kidney, colon and breast tissue samples as well as on carcinomas derived from these organs. The results are shown by Figs. 1A-1H respectively.

As shown by the 200X magnified photographs of Fig. 1 overall, the immunoperoxidase staining using the anti-PDZKl antibody in the different tissue samples is revealed by: Fig. 1A showing normal renal cortex; Fig. IB for renal cell carcinoma; Fig. IC showing normal colonic mucosa; Fig. ID for colonic adenocarcinoma; Fig. IE showing normal breast tissue; Figs. IF and 1G for infiltrating ductal carcinomas of the breast; and Fig. 1H for a mucinous carcinoma of the breast. A careful inspection of the individual tissue samples tested shows that in normal kidney (Fig. 1A), the staining is limited to the brush border of proximal tubular epithelial cells (p), while glomeruli (g) and distal tubules (d) are not labeled. In comparison, the renal cell carcinoma shows strong staining (Fig. IB). Similarly, the intensity of staining is markedly increased in carcinomas arising from colon (Fig. ID) and breast (Figs. 1F-1H), compared to normal tissue samples (Figs. IC and IE). With breast carcinoma, in particular, the staining is limited to a subpopulation of cancer cells (Figs. lG and 1H).

Experiment 2: Isolation of cMOAT (MRP2) Using the Yeast Two- Hybrid System

A 2 Kb BglH DNA fragment including the complete coding sequence of PDZKl was subcloned into the BamHI site of pGBT9 (as described previously herein). The recombinant plasmid was used as a bait to screen a normal liver cDNA library prepared in the pACT2 vector. Yeast colonies were grown on selection media to allow growth of yeast successfully transformed with vectors encoding two interacting proteins. Such yeast colonies were subsequently tested for GAL4 activation using the X-Gal reporter assay. Using this technique, several cDNA clones potentially encoding for proteins interacting with PDZKl were selected for further study and submitted for DNA sequencing. The results are illustrated by Figs. 2 A and 2B respectively.

Fig. 2 A shows that two cMOAT cDNA clones were obtained from the yeast two hybrid systems screening using PDZKl protein as a bait. Both cDNA clones contain a portion of the carboxy terminal translated sequence of cMOAT (ORF) as well as part of the 3 '-untranslated region (3'UT). Fig. 2B shows the synthetic peptide sequence, corresponding to a portion of the carboxy terminus of cMOAT, which was used for the biochemical interaction between cMOAT and PDZKl proteins. The putative PDZ domain-binding site is shown by italicized letters. Overall therefore, the empirical sequence analysis revealed that two cDNA clones corresponded to the carboxy terminus of cMOAT, the multi-drug resistance- associated protein (Fig. 2A).

Experiment 3: Determination of the Interaction of PDZKl with cMOAT Using a Carboxy-Terminal Peptide Sequence analysis of the protein interacting with PDZ-domains as well as screening with random peptides uncovered a consensus sequence for the PDZ domain binding site: Ser/Thr-X-Val-COOH, in which X represents any amino acid and Val can be substituted with other hydrophobic amino acid. Review of the carboxy-terminal sequence of cMOAT revealed a potential PDZ domain interaction site, with a threonine in position -2 and a hydrophobic amino acid (phenylalanine) in the last position (shown by Fig. 2B).

Using a 30-mer biotinylated peptide corresponding to the carboxy-terminus of cMOAT (bound to agarose-avidin beads) and a GST-fusion protein containing the complete PDZKl sequence, this experiment confirmed the occurrence of an interaction between the GST-PDZK1 fusion protein and the cMOAT carboxy terminal peptide, while the GST protein alone did not interact with the cMOAT peptide. This result is shown by the evidence of Fig. 3. Fig. 3 shows the SDS-PAGE assay demonstrating the interaction between the GST-PDZK1 fusion protein and the carboxy-terminal peptide of cMOAT. GST-PDZK1 fusion proteins bound to agarose-avidin beads coated with the cMOAT biotinylated peptide (lane a) while GST peptide alone did not (lane b). Neither of the GST-PDZK1 nor GST proteins were able to bind to agarose-avidin beads not coated with the cMOAT peptide (lanes c-d). Samples of both PDZKl and GST-PDZK1 fusion proteins have been loaded on the gel to indicate the respective position of both these proteins (lanes e-f). Experiment 4: Colocalization of PDZKl and cMOAT by In-Situ

Hybridization To determine the cellular localization of PDZKl and cMOAT mRNAs, in- situ hybridization studies were performed. Using antisense RNA probes derived from PDZKl and cMOAT cDNA clones, a number of human adult tissue samples were evaluated including kidney, liver, colon and breast, as well as carcinomas arising from these organs. The results are shown by Figs 4A-4T respectively. Overall, Fig. 4 (magnification XI 00) includes the following: the in-situ hybridization of normal kidney is shown by Figs. 4A-4D; of normal liver by Figs. 4F-4H; of renal cell carcinoma by Figs. 4I-4L; of colonic adenocarcinoma by Figs. 4M-4P; and of infiltrating breast carcinoma by Figs. 4Q-4T. All assays used either the antisense PDZKl cDNA probe (left two columns) or the anti-sense cMOAT cRNA probe (right two columns). Each pair of photographs (left two columns and right two columns) represents the same microscopic field photographed alternatively in bright and dark fields.

In normal kidney (Figs. 4A-4D), the labeling with both cRNA probes is limited to proximal tubules while the glomeruli (G) are not labeled. In comparison, liver hepatocytes labeled with both cRNA probes have similar intensities (Figs. 4E-4H). Also the renal cell carcinoma (Figs. 4I-4L) and the colonic adenocarcinoma (Figs. 4M-4P) labeled with both cRNA probes show comparable intensities, while the infiltrating carcinoma of the breast is strongly labeled with PDZKl probe (Figs. 4Q-4R) and minimally labeled with cMOAT probe (Figs. 4S-4T).

The empirical data demonstrates that in normal kidney, PDZKl was highly expressed in proximal tubular epithelial cells, while it was either not expressed or expressed at low levels in the remainder of the renal cortex (Figs. 4A and 4B). cMOAT showed a similar distribution; however, the level of expression was significantly lower than that of PDZKl (Figs. 4C and 4D). In normal liver PDZKl mRNAs (Figs. 4E and 4F) and cMOAT (Figs. 4G and 4H) mRNAs were both expressed at moderate levels by hepatocytes.

The expression of PDZKl and cMOAT mRNAs was further studied in twelve carcinomas from renal, colonic and breast origin. A wide range of results were observed. Both PDZKl and cMOAT mRNAs were expressed at high levels in 17% of tumors studied (Figs. 4I-4P) while 58% of the tumors studied expressed significant levels of PDZKl mRNA and only low levels or no significant amounts of cMOAT mRNA (Figs. 4Q-4T). cMOAT mRNA was never expressed in significant amounts within tumors which did not express PDZKl mRNA.

In all cases studied, PDZKl and cMOAT expressions were confined to cells of epithelial origin; mesenchymal and inflammatory cells were not detectably labeled. Control hybridization using a sense of cRNA probe were negative on all tissue studied.

Conclusions: cMOAT, the canalicular multispecific organic anion transporter [also known as MRP2, the multidrug resistance-associated protein] is a member of the ATP binding cassette (ABC) transporter family. In normal tissues, it is highly expressed in the liver, where it is associated with the canalicular cytoplasmic membrane of hepatocytes. In addition, it is also associated with the apical membrane of proximal tubular epithelial cells of the renal cortex and is found in the exact same localization as MAP17 and PDZKl proteins. Moreover, the main function of cMOAT known to date is the excretion of anionic conjugates with glutathione, glucuronate or sulfate moieties into the bile. cMOAT is not expressed in Dubin- Johnson syndrome, an hereditary deficiency in the secretion of amphophilic anionic conjugates into the bile associated with hyperbilirubinermia and rat congenital jaundice. In addition, cMOAT is overexpressed in a number of cultured cell lines which have developed resistance to cisplatin. The empirical results presented herein show protein interacting with

PDZKl; and the data reveals the essential binding area as the carboxy-terminal sequence of cMOAT. The data also demonstrated that PDZ domains of PDZKl protein interact with a consensus sequence localized at the carboxy terminus; and that the three last amino acids of cMOAT in particular provide such a consensus sequence - including a threonine in position (-2) and a phenylalanine (hydrophobic amino acid) in the last position. The direct interaction between PDZKl and cMOAT proteins is further confirmed using a synthetic peptide corresponding to the carboxy terminus of cMOAT and a GST-PDZK1 fusion protein - using the evidence of Fig. 3. Furthermore, in-situ hybridization studies using PDZKl and cMOAT cRNA probes on the same tissue samples confirms the expression of both mRNAs in the same normal and tumor cell populations (see Fig. 4). The finding that cMOAT interacts with a PDZ protein provides a mode of overcoming multidrug resistance genes expressed by tumor cells. PDZKl and cMOAT proteins do in fact form functional clusters at the membrane/cytoplasm interface - thus promoting resistance against a variety of chemotherapeutic agents. Specific antibodies directed to either or both of these directly interacting proteins in-situ will thus serve to interfere with and reduce single and multiple drug resistance in tumor cells which express PDZKl and cMOAT proteins intracellularly.

The present invention is not to be restricted in form nor limited in scope except by the claims appended hereto.

Claims

What I claim is:

1. A method for reducing chemotherapeutic drug resistance exhibited in-situ by a solid mass neoplasm of epithelial origin, said method comprising the steps of: identifying a solid mass neoplastic cell of epithelial origin as being constituted at least in part of tumor cells clinically resistant in-situ to at least one discrete substance previously administered to the cell as a chemotherapeutic treatment agent; and administering to said neoplasm at least one antagonistic antibody preparation specific against at least one epitope presented by a protein selected from the group consisting of PDZKl protein and cMOAT protein such that intracellular binding of PDZKl protein with cMOAT protein is inhibited in-situ, and whereby clinical resistance to the previously administered chemotherapeutic treatment agent exhibited by the solid tumor cell becomes reduced.

2. The method as recited in claim 1 wherein said antagonistic antibody preparation is specific for at least one epitope within the PDZ domains of PDZKl protein.

3. The method as recited in claim 1 wherein said antagonistic antibody preparation is specific for at least one epitope within the carboxy-terminal portion of cMOAT protein.

4. The method as recited in claim 1 wherein said antagonistic antibody preparation comprises at least some binding fragments of a polyclonal antiserum.

5. The method as recited in claim 1 wherein said antagonistic antibody preparation comprises at least one binding fragment of a monoclonal antibody.

6. The method as recited in claim 1 wherein said antagonistic antibody preparation comprises an admixture of different antibodies in which a first portion is specific for PDZKl protein and a second portion is specific for cMOAT protein.

7. A composition for reducing chemotherapeutic drug resistance exhibited in- situ by tumor cells constituting a solid mass neoplasm of epithelial origin, said composition comprising: an antagonistic antibody preparation specific against at least one epitope presented by a protein selected from the group consisting of PDZKl protein and cMOAT protein such that intracellular binding of PDZKl protein with cMOAT protein within the tumor cell is inhibited in the presence of said antibody preparation, and whereby clinical resistance to the previously administered chemotherapeutic treatment agent exhibited by the solid tumor cell becomes reduced in-situ.