CA2113578A1 - Trifunctional compounds having specificity for multi-drug resistant cells - Google Patents

Abstract

The present invention relates to trifunctional compounds having specific reactivity with P-glycoproteins associated with multi-drug resistant (MDR) tumor cells. The compounds can optionally be specific for diagnostic and/or therapeutic agents as well and are thus contained in pharmaceutical compositions. Methods for detecting and treating patients with tumors expressing P-glycoproteins associated with MDR cells are also provided.

Description

WO 93/1)210~ 2 1 ~ 3 ~ 7 ~ PCI /lJS92/06043 ,` :~
` ~ 1 TRIFUNCTIONAL COMPOUNDS HAVING SPECIFICITY FOR
MULTI-DRUG RESISTANT CELLS ::

FTELD OF THE I2~VENTION

The present invention generally relates to multi-drug re~istance exhibited by many tumor cells.
~ore specifically, the present invention relates to trifunctional compounds having specificity for multi-drug resistance (MDR) cells that overexpress P-glycoproteins and to their use for diagnosis and therapy.
.
.
BACKGROIIND OF THE INVENTION

The ability of certain tumor cells to acquire multi-drug resistance has been a significant barrier to the effective use of chemotherapy in the treatment of ~`
such tumors and other neoplastic disorders.~ Multi-drug resistance is the phenomenon by which certain cancer cells~selected~for resistance to one drug can simultaneously exhibit resistance to structurally and functionally unrelated drugs to which they have not been previously~exposed.

Cells exhibiting the multi-drug resistance phenotype show reduced intracellular accumiulation of such ;~
drugs~compared~with the corresponding drug-sensiti~e cellsi~ A common characteristic of many MDR cells is the overexpression of cell surface P-glycoproteins that are absent or expre$s~ed,in ~ow levels in drug-s,ensitive and normal cells. One function of the P-glycoproteins is to act as a drug efflux pump by which intracellular toxins are actively pumped out of the cell. It is believèd ',;
-certain regions of the P-glycoprotein bind to the energy-carrying compound, ATP, which provides the energy to pump the non-related toxins out of the cell.

.

W O 93/0210~ P ~ ~US92/06043 At least two P-glycoprotein genes have been identified in humans and three in mice, suggesting that a family of such glycoproteins may exist. One human P-glycoprotein has an apparent molecular weight of about 180 Kd, while the other has an apparent molecular weight of about 170 Kd. Nonoclonal antibodies specifically reactive with these P-glycoproteins are known and have been described, for example, in Hamada & ~suruo, PNAS
(U.S.A.) 83:77~5-7789 (1986) and in European Patent Application No. 87309939.4, published on May 18, 1988.
These monoclonal antibodies are commonly referred to as -~
anti-MDR antibodies.

Bispecific anti-MDR antibodies have been reported in van Duk et al., Int. J. Cancer 44:738-743 (1989). 'These bispecific antibodies have one specificity ~
for the 170 Kd P-glycoprotein and a second specificity ~-for~CD3 to induce lysis of MDR tumor oells.

.
Polyclonal and monoclonal anti-MDR antibodies are~;lLmiting since they'can only bind to the site of action.~'~If~diagnostic or therapeutic agents are also ';
d-s;ired,; they must be chemically coupled to such antibodies. Bispecific antibody molecules are also limlted. For example, ~uch~bispecific antibodies have ~ ~ .
the~inherent limitation of effectively being monospecific once~b;ound to the antigen binding site in that they can target~only a single~agent to such binding site. In addition, such bisp-cific antibodies are limited in their ability to recognize at most two different antigen bindi~ng!sites.il~ ! ! ~ ~ i ~ ` ~ ' ' ~ . ~
It would be advantageous to use compounds that are capable of being more than monospecific at the site ~'~; of action or have the~ability to recognize more than two different P-glycoproteins. For example, one trifunctional compound having three arms each specific ,, WO93/0210~ PCT/US92/06043 . .

for different P-glycoproteins or different P-glycoprotein binding sites can be used in place of three monoclonal ~-antibodies. ~lternatively, a trifunctional compound 7 having one arm specific to a P-glycoprotein, a second arm specific for a diagnostic agent and a third arm s~ecific -for a therapeutic agent would be useful to diagnose and treat a patient without administering àdditional antibodies that would be necessary if monospecific or bispecific antibodies were used. `~

Thus, a need exists for compounds that have more than two specificities. The present invention satisfies this need and provides related advantages as ~;`
well. `~

SUHUARY OF THE INVENTION

The present invention relates to trifunctional . ~
compounds of the following Formula:

X

~ .
wherein L is a cross-linking agent, X1, X2 and X3 are antibody Fab'-like moieties, and at least one of the ~- Fab'-like moieties has specific reactivity for a P-glycoprotein associated with a MDR cell. The Fab'-like moieties can also be specific for diagnostic and therapeutic agents. The pre~ent invention also provide pharmaceutical ¢ompositions containing the,`trifunctional ; compounds and pharmaceutically acceptable carriers.
;~ :
The trifunctional compounds can have arms that recognize up to three different P-glycoproteins or three different antigenic binding sites on the same P- `
glycoprotein. Alternatively, such compounds can have one :; ' ` ~;
, :

W093/0210~ PCT/US92/06043~-~: .

arm specific for a P-glycoprotein and two arms specific for one or two diagnostic agents or therapeutic agents.
A particularly useful compound has one arm specific for a P-glycoprotein, a second arm specific for a diagnostic agent and a third arm specific for a therapeutic agent.
The desired trifunctional configuration will depend on its intended use.
, Particularly useful diagnostia agents are complexes of chelating agents and an imaging agent, such as a radionuclide or a paramagnetic metal ion.
Therapeutic agents include cytotoxic agents and complexes of chelating agents and radionuclides.

Also provided are methods for inhibiting the activity of P-glycoproteins in conferring multi-drug resistance to certain tumor cells. Such methods include ~;~
ad~inistering the trifunctional compounds of the present invention.~ Chemosensitizers can optionally be administered~with the trifunctional compounds.

The present invention further provides for the -~
detection of MDR cells. The detection of such cells can be accomplished by in vitro assay of samples or in vivo maging of a patient~ In the ln vlvo imaging methods, P~the trifunctional compounds of the present invention are administered either `sLmultaneously with or prior to the ``
admin~stration of a~diagnostic agent.

In another aspect, the present invention also provides methods foritreating a patient having à disorder associated with MDR cells. The trifunctional compounds of the invention can also be similarly administered either simultaneously with or prior to the administration of a therapeutic agent.

The present invention further relates to '; ~ `
. ~

WO ~3/0210~ 3 ~ rç1 ~ PCr/US92/06043 methods for the diagnosis and treatment of a patient having or suspected of having MDR cells by administering a trifunctional compound having specificities for a diagnostic agent and a therapeutic agent.

Finally, kits useful for performing the methods of the present invention are also provided. The kits contain the trifunctional compounds of the present ;.
invention and any desired ancillary reagents.

BRIEF ~ESCRI~ION OF THE DRAWINGS
`:
Figure l shows the relative activities of -intact LS2H241 antibody, bifunctional F(ab)'2 and c:
trifuncitonal F(ab)'3.

Figure 2 shows the binding of LS2~241 and trif~nctional (LS2H)2/xCHA to CEM membranes.

Figure 3 shows the effect of LS2H241 and trifunctional (LS2H)2/xCHA in modulating actinomycin D
~; uptake in multi-drug resistant cells.

Figure 4 shows the results of (LS2H)2/xCHA
biodistribution studies in nude mlce bearing 3ROtMDR1 xenographs. :

DETAILED ~ESCRI~TION OF THE INVENTION

The present;in*ention generally relates to ! `
-~ trifunctional compounds having at least one specificity ~r,'', ~ for an antigenic determinant on a P-glycoprotein expressed by multi-drug resistant cells of certain ;~ tumors~. Such tumors include, for example, ovarian, colon, renal and breast carcinomas,.leukemia, bladder ~ carcinoma, lymphoma, myeloma, adrenal carcinoma, ,",~
,.,~,:
~-, .

WO93/0210~ PCT/US92/06043 teratocarcinoma, sarcoma, seminoma and the like. Since the various P-glycoproteins are not tissue specific, the compounds of the present invention are likewise not tissue specific. Thus, the compounds have applicability in the management of a broad spectrum of tumors.
, The trifunctional compounds of the present -~
invention have the following general Formula I: `
-~;

¦ (I) ;;
,~
L is a cross linking agent. X1, X2 and X3 can be antibody fragments or antibody-like fragments, particularly Fab'-like moieties, having a combination of various antigenic specificities. As used herein, the term ~Fab'-like ~
molety,~ when referring to X" X2 or X3 ~ iS used nterohangeably with the tenm "arm." The term "specificity" refers to the specific binding reactivity of a compound with a particular antigenic determinant.
: :: ` :
The term "trifunctional," as used herein, refers~to~the three func~tional arms or valencies of a compound-~ Thus~ a~trifunctional compound can also be referred~to as a "trivalent" compound.

A trifunctional compound can be monospecific, bispecific or~trispecific. Used in this sense, the term ~; "specific" refers to the number of different antigenic ~ - determl~ants orlepitdpes~a particular compound aan bind.
- For example, a bispecific, trifunctional compound has one ~ arm~æpecific for an antigenic determinant, while the -~ ~ other two arms are specific for a different antigenic determinant. The arm~ of a triæpecific, trifunctional compound each have specificitieæ for different antigenic determlnants relative to the other two arms, i.e., three , ,~: :~ , ,, , ~

2 1 :~ 3 3~ ~ PCT/US92/06043 unique determinants. The antigenic determinants recognized by the bispecific and trispecific compounds of the present invention can be from the same antigen or different antigens. The arms of a monospecific, trifunctional compound are specifically reactive wi~th the same antigenic determinant.

The term "Fab'-like moiety," also referred to herein as an ~Fab'-like fragment,~' includes Fab or longer ~;
Fab~ fragment of an antibody, as described in the U.S. ;~
Patent Application Serial No. 07/491,406, filed on March 9, 1990 IAttorney Docket No. H-7912), by Ahlem and Huang, which is incorporated herein by reference. As is known ;-to those skilled in the art, a Fab~ fragment includes a portion of the constant Fc region that is not included in a Fab fragment. The Fab and Fab' fragments include the Fv fragment, which is the variable region of an antibody that determines the antigen binding specificity. The Fab'-like fragment can be obtained directly from an antibody~by digesting the antibody into the desired fragments or indirectly by chemical synthesis of a ~;
po~ypeptide having the Am;no acid sequence of a Fab or Fab~ fragment from such an antibody. The Fab'-like fragment can be a single chain antibody derived from the binding region of an antibody as described in U.S. Patent No~. 4,946~778, issued August 7, 1990 to Ladner et al., which is incorporated herein by reference.
Alter~ati~ely, the fragments can be obtained indirectly ;
from an antibody by recombinant engineering techniques ; well known in the art.

The Fab'-like moietie~ can also be obtained from chimeric antibodies`/ particularly those having a .;
human constant region ~Fc) and a non-human variable region (Fv), such as a murine Fv region. The hum2n constant region minimlzes the risk of inducing an immune ~ response when the compounds of the present invention :~

WO93/0210~ PCT/~S92/06043 having a variable region from a non-human species are administered to a human~ For example, the potential immunogenicity of murine antibodies in humans, referred ;~
to as HAMA (human anti-murine antibody), can result in severe adverse reactions with repeated administration of murine antibodies. Those skilled in the art will appreciate that desirable compounds can be selected for non-human mammals ~ased on sim;lar considerations for specie differences.

As used herein, "mammal" is used interchangeably with the term "patient" and means any mammal capable of expressing a P-glycoprotein associated with multi-drug resistant cells, including, for example, dogs mice, hamsters, humans, and the like.

~- The trifunctional compounds of the present ~~
invèntion have~at least one~ arm directly or indirectly derived from an~antibody~having specificity for a P-glycoprotein associated with MDR cells. Such antibodies are generally referred to as anti-MDR antibodies. Anti-MDR~-antibodies from which the trifunctional compounds are derived~can be polyclonal, monoclonal, chemically synthesized or recombinantly produced. Anti-MDR
- ~ ~ antibodies~are known in the art and include, for example, MRK16 and MRK17 antibodies described in ~amada & Tsuruo, PNAS~ (U.S.A.~ 83:7785-7789 llg86~ and C219 described in U~.S. Patent~No. 4,837,309, issued June 6, 1989 to Ling et al. ~The antibodies listed in Table 1 can also be used, particularly the monoclonal antibody produced by the - hybridomà;designated asiLS2H241 AT~C DEPOSI~ NO.: ~B
10814. The antibodies were generated following the procedure of Example I.

,, ".~

.,, i, WO93/0210~ PCT/US92/06043 ... .

Table 1 Drug Resistance Related Monoclonal Antibodies ELISA Ratiosn ~:

Antibody Isotype MC-IXC/VCR SH-SY5Y/VCR P-glycoprotein~ -HYB-241 IgGl 40 12 HYB-612 IgGl 17 19 +
HYB-034 IgGl 27 13 +
HYB-057 IgGl 7 11 +
HY~-237 IgGl 97 6 +
~Y~-374 IgGl 7 11 + ~.
~Y~-162 IgG2b 41 ~ 3 +
HYB-~243 M 50 8 +
Y~-19~5~ M ~ 1 5 HYB-065 M 55 : 1 - ~.

,, , ~
Antibody~binding ratios, resistant sensiti~e Antibo1y reacts with the human MDRI transfectant cell line, ~.
: BROtMDRI: and not the parental nontransfected cell line by : ELISA

~ The anti-MDR antibodies recognize epitopes on `
:~ . different P-gly~op~oteins having molecular~weights in the range of about 170-180 kilodaltons (Xd). The existence :~`
of a family of MDR-associated P-glycoproteins haæ been reported~in Meyers et al., "Characterization of ~`
: Monoclonal Antibodies Recognizing a 180 Xd P- -:.
Glycoprotein: Differential Expression of the 180 kDa and ~ 170 kDa P-Glycoproteins in Multidrug-resistant Human .~

u~Iu~ PfCT/US92/06043 f ,"
Tumor Cells," Cancer Res. 49:3209-3214 (1989). For example, antibody LS2H241 recognizes an epitope on a P- `
glycoprotein having an apparent molecular weight of about 180 Kd. Antibody C219 recognizes an epitope on a P~
glycoprotein having an apparent molecular weight of about -~
170 Kd.

~` One desired function of the anti-MDR Fab'-like moieties is to target the trifunctional compound to cells exhibiting the MDR phenotype for diagnostic or therapeutic purposes. Another function of the anti-MDR -, Fab~-like moiety is to inhibit or block the P-~-~ glycoprotein drug efflux pump activity in conjunction -~, ~ with the administration of MDR drugs in arder to ,~
facilitate the intracellular accumulation of such drugs and consequent cellular death.

The trifunctional~compounds of the present inve ~ ion can~also have Fab'-like moieties directed to antigens, haptens or epitopes (collectively;referred to a~s~ antlgéns~ other than the MDR-associated P- , ;
; ,glycoproteins. ~For example, such antigens can optionally be diagnostic agents for the in yitro detection of P-glycoproteins~ or ln vlvo imaging of tumors expressing s~uGh~P-glycoproteins.~ The~antigens can optionally be therdpeutic agents for the treatment of a patient having a disorder, such as cancer or other neoplastic disorders, dssociated with~MDR~cells.

The term "diagnostic agent" generally means any agent capable of being detected either with or without ;
the aid'of instrumentation. When used for ln vivo ~ ~-~ detection, a diagnostic agent is also referred to herein "~ as an imaging agent. A detectable~marker can be directly or indirectly attached to a diagnostic agent. Useful diagnostic agents are described U.S. Patent Application Serial No. 07/491,406, filed March 9, 1990, by Ahlem and WO93/0210~ 21~ i 7 ~ PCT/US92/06043 1~' , Huang, which is incorporated herein by reference. Such agents include, for example, enzyme detection systemæ, radionuclides or paramagnetic metal ions. Conventional diagnostic agents for the in vitro detection of target antigens can also be used in the present invention and are well known to those skilled in the art.

Imaging agents useful in the present invention permit the imaging of a patient's organ or tissue for the in vivo detection of tumors expressing P-glycoproteins.
The in vitro or ln vivo detection of MDR cells and tumors aid in the diagnosis of a disorder or disease associated with the o~erexpression of such P-glycoproteins.
Although the imaging agent can be a radionuclide or paramagnetic metal ion attached directly to an antigen recognized by an arm of a trifunctional compound, preferably the imaging agent is a physiologically . ~
compatible chelate complex that has been formed between ~
eithèr~a~chelating agent and a radionuclide or a ~`
chelating agent and a paramagnetic metal ion. For exampl~e,~radionuclides permit the imaging of tissue and/or~organs by gamma scintillation photometry, positron emisgion~tomography (PET), single photon emission computed tomography lSPECT)~and gamma camera whole body ima ~ g,~while paramagnetic metal ions permit viæualization by magnetic resonance imaging (MRI). Such imaging techniques are well known to those skilled in the art.

The term "physiologically compatible chelate coMplex,?' as used herein, means a complex between a ! `
chelating agent and a radionuclide or paramagnetic metal ion that does not permit physiologically incompatible amounts of the radionuclide or ion to dissociate from the comp}ex in vivo.

Suitable complexes and chelating agents are WO93/0210~ f~ PCT/US92/06043 identified in U.S. Patent Application Serial No.
0~/491,406, filed on March 9, 1990 and incorporated herein by reference. Methods for preparing bifunctional derivatives of chelating agents are described in Meares et al., U.S. Patent No. 4,678,667, issued July 7, ~987, incorporated herein by reference, and in Moi et al., J.
Am. Chem. Soc., 110:6266 (1988). The chelating agents-described therein form physiologically compatible chelate `
complexes with a variety of metal ions. Other suitable chelating agents are disclosed in Gries et a}., U.S.
Patent No. 4,647,447, which is incorporated herein by ;
reference. A particularly useful chelating agent is DBX, the use of which is described in more detail in the examples herein. DBX ha~ the following chemical structure: ~
(~02CCH2) 2N~ : ' N(CH2cO2H)2 H02C ~ )~N ~ ~ ¦ (II) ~ H N ~ NCH2COiH

N ICH2C02H) 2 ''`
DBX i5 prepared by the reaction of the In3+complex of (~p-aminobenzyl)EDTA and (p-am nobenzyl)DTPA
;with~disuccinimidyl carbonate. To ~ynthe~ize DBX, the indium complex~of ~p-~ ~nobenzyl)EDTA and (p-aminobenzyl)DTPA~are reacted with disuccinlmidyl carbonate in~an agueous~solution. Reaction of the aromatic amine groups of (p-am;nobenzyl)EDTA and ~p-aminobenzyl)DTPA with the disuccinimidyl carbonate yields DBX, which contain~!~a ui~ea linkage between the benzyl ! `
DTPA and In-benzyl EDTA moieties. The crude DBX reaction -;~ mixture is purified by liguid chromatography using a C18 -revers~ed phase ~PLC column, followed by anion exchange on a SephadeY~;DEAE column (Pharmacia, Piscataway, N.J.).
-Sephadex i8~ a register trademark of Pharmacia. Other methods of preparing DBX can be readily determined by W093/0210~ tj~ ~V PCT/US92/06043 those skilled in the art.

Radionuclides that are suitable as imaging agents include gamma-emitting radionuclides, for example, In, 67Ga, 99~rC ~ 5lCr~ l97Hg~ 203Hg 169yb 05Sr 8~S d particularly ~l'In. The~e gamma-emitting radionuclides are chelatable in ionic form. The preparation of chelated radionuclides that are suitable for binding by Fab' fragments is taught by Nicoletti et al, U.S~ Patent ;~
No. 4,659,839, which is incorporated herein by reference.

Paramagnetic metal ions that are suitable for use as imaging agents in MRI include, for example, the ~`
lanthanide elements of atomic number 57-70, or the transition metals of atomic numbers 21-29, 42 or 44.
Gries et al. in U.S. Patent No. 4,647,447, incorporated herein by reference, describe the use of chelated paramagnetic metal ions for imaging.

One or two arms of the trifunctional compounds of the~pres~ent invention can have specificity for a therapeutic agent~ A "therapeutic agent," as used herein, refers to any agent administered to a patient to treat~a disease, condition or other disorder associàted with~MDR~cells, such as tumor cells that overexpress a cell surface P-glycoprotein.

The trifunctional compounds of the present invention can have direct or indirect specificity for a therapeutic agent. Direct specificity means a functional arm is s~ecificallylrea~tive with the therapeutic agent.
Indirect specificity, for example, means a functional arm specific for a substrate conjugated to a therapeutic agent. Thus, for an enzyme ~ubstrate, an enzyme or active fragment thereof is required to activate the therapeutic agent by releasing it from the substrate. -More details of the direct and indirect specificities are ~

;

.

WO93/~2105 ~ ~I J~ ~ PCT/US92/06043 . ,....... ,;

provided in U.S. Patent Application Serial No. `~
07/491,406, filed March 9, 1990, which is incorporated herein by reference.

Suitable therapeutic agents of the present invention can be cytotoxic agents that are useful in the treatment of neoplasms. Such cytotoxic agents or drugs include, for example, the vinca alkaloids, the daunomycin family, the mitomycins, the bleomycins and colchicines.
Drugs associated with MDR are particularly useful and are referred to herein as MDR drugs. Such drugs include, for instance, doxorubicin, daunorubicin, mitom~cin C, etoposide, vinblastine, vincristine, colchicine, dihydroxyanthracenedione, mitoxanthrone, mithramycin, ~
actinomycin D, puromycin, gramicidin D, ethidium bromide, ~-taxol, tenoposide, cytochalasin B, l-dehydrotoastosterone and the like.

Alternatively, such therapeutic agents can be chelate~complexes that are formed between chelating agents and radionuclides, for example, ~-emitters that emit ~- particles of sufficient energy and frequency so as to provide a beneficial effect in the treatment of such disorders. Suita~le ~-emitters include s'Cu, l86Rh, ~-Rh , l89Rh , l53Sm, lllIn (AUger), and particularly 90Y . The . ~ ~
emltters are chelatable when in ionic form, typically when~in the +1 to +4 oxidation state. Chelating agents u eful~for such complexes are described in U.S. Patent Application Serial No. 07/491,406, and in Meares et al., U.S. Patent No. 4,678,667, issued July 7, 1987, both of which are incorporated her`ein by reference, and in Moi! et al., J. Am. Chem. Soc., 110:6266 (1988).

Suitable cross linking agents are also descri~ed in U.S. Patent Application Serial No.
07/491,406, filed March 9, l990, which is incorporated herein by reference. Particularly useful cross linking '' .

~ 1 ~ , ~J '' WO93/0210~ PCT/US92/06~43 agent~ for the trifunctional compounds of the present invention are the tris-maleimides described in U.S. ;
Patent Application Serial No. 07/491,386, filed March 9, l990, which is incorporated herein by reference.
~ .
Methods for preparing trifunctional compounds are described in ~.S. Patent Application Serial No.
07/49l,406, filed March 9, l990, the methods of which are incorporated herein by reference. Other methods of preparing such compounds are described in the examples herein.

The trifunctional compounds of the present invention can be used for a variety of purposes, including use in the diagnoætic and therapeutic methods of the present invention. The compounds can also be used to purify MDR-associated P-glycoproteins. For example, if a mixture of different P-glycoproteins is desired, the .
trifunational antibodies would be particularly useful to isolate~suc~h proteins from a sample. Those skilled in ;
the art can readily identify other potential uses for the trifunctional compounds.

The present invention further provides pharmaceutical compositions comprising a trifunctional compound of Formula I and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known nd include, for example, aqueous solutions such as bicarbonate buffers, phosphate buffers, Ringer's solution and physiological saline supp}emented with 5% dextrose or human ~erum albumin, if'desired.

The pharmaceutical compositions, useful for imaging and therapy, can be prepared by methods known in the art, including the simple mixing of the reagents.
Those skilled in the art will know that the choice of ;;
pharmaceutical carrier and the appropriate preparation of ,`:

W093/02l0~ 2 1 ~ 357 ' PCT/US92/06043 the composition will depend on the intended u~e and mode of administration.

The present invention also provides methods of inhibiting the ability of the P-glycoprotein molec~le to pump drugs out of the cell across the membrane. The methods involve contacting an MDR cell with a trifunctional compound of Formula I. The trifunctional compound can be monospecific, bispecific or trispecific to different epitopes or different P-glycoproteins.
Trispecific, trifunctional compounds are particularly ~;
useful over known monospecific and bispecific antibodies since more potential binding sites can be blocked with one compound compared with a monospezific or bispecific antibody. Although the exact mechanism is not known, it is believed that the binding of the LS2H241 compound at the cell surface may induce a ccnformational change in the P-glycoprotein molecule that interferes with the ability of the molecule to pump drug out of the cell across the membrane. These methods can therefore be used for therapeutic purposes to enhance the intracellular accumulation of MDR drugs.

The methods further include contacting the cells with a chemosensitizer, such as verapamil, either prior to, sLmultaneously with or subsequent to contacting such cells wi*h the trifunctional compound.
Chemosensitizers are compounds that compete with NDR
drugs as substrates for binding to the ATP dependent efflux pump, thus rendering multi-drug resistant tumor cells sensitive to drugs lthat would otherwise be !
ineffective. Such chemosensitizers or modulators include calcium channel blockers as described in Tsuruo et al., ancer Res. 41:1967-1972 ~1981); calmodulin inhibitors as described in Ganapathi & Grabowski, Cancer Res. 43:3696-3699 (1983); antiarrythmics as described in Tsuruo et al., Cancer Res. 44:4403-4407 (1984) and Chauffert et ' WO93/02105 ~ 3 ~ PCT/US92/~6043 . .

al., Br. J. Cancer 56:199-222 (1987); antimalarials as described in Zamora & Beck, Biochem. Pharm. 35:4303-4407 (1986); other lysoosmotropic agents as described in ;
Zamora et al., Mol Pharmacol. 33:454-462 ~1988); steroids ~ r as described in Yan et al., J~ Biol. Chem. 264:78~-788 (1989); antiestrogens as described in Ramu et al., Cancer Res. 44:4392-4395 (1984); and cyclic peptide antibiotics as described in Slater et al., J. Clin. Invest. 77:1405-1408 (1986). ~owever, many chemosensitizers, such as verapamil, are highly toxic. The use of the trifunctional compounds allows the use of a less toxic dose of such chemosensitizers to obtain similar results and thus reduce the toxic side effects.

As used herein, the term "contacting encompasses any means by which two substances come into close physical proximity. For example, the trifunctional compound comes into contact with a P-glycoprotein when it comes into physical proximity sufficiently close for the ~;
compound to bind to an antigenic determinant on the glycoprotein. For the in vitro methods of the present invention, such contact can be accomplished, for example, by adding the compound to a sample or dipping the sample into a solution containing the compound. For the ln vivo ~`
methods, such contact can be accomplished by administering the compound to a patient.

As used in the methods of the present invention, the trifunctional compounds can be admini~tered to a mammal by any means known to those skilled ln the ar,t, ! including parenteral injection or !
topical application. Injection can be done intravascularly, intraperitoneally, subcutaneously or intramuscularly. For parenteral administration, the compounds can be administered in admixture with a suitable pharmaceutically acceptable carrier. ` `
~ ~ `

:~ ' WO93/0210~ PCT/US92/06043--i , . .,;
1~ ' .
The dosage regimen for diagnosing and treating MDR-associated disorders depends on a variety of factors, including the type of disorder, age, weight, sex and ;
medical condition of the patient, the severity of the condition, the route of administration and the type of diagnostic or therapeutic agent used. A skilled physician or veterinarian can readily determine and prescribe the effective amount of the compound or pharmaceutical composition required to diagnose or treat the patient. Conventionally, one skilled in the art would employ relatively low doses initially and subsequently increase the dose until a maximum response is obtained. Because the trifunctional compound of the present invention localizes the diagnostic or therapeutic agent, much lower doses are believed to be effective than with conventional methods.

Methods for detecting the presence or absence of a multi-drug resistant cell are also provided by the present invention. Such methods include (a) contacting a sample containing or suspected of containing a multi-drug resistant cell with a detectable trifunctional compound of Formula I, said trifunctional compound having at least one Fàb'-like moiety specific for a diagnostic agent; and (b~) detecting the binding of the compound to the sample, whereby binding indicates the presence of a multi-drug resistant cell in the sample. In these methods, at least one of the Fab'-like moieties of the trifunctional antibody is specific for a diagnostic agent and another is specific for a P-glycoprotein associated with a MDR cell.

The trifunctional compound can have any combination of specificities described previously.
Particularly useful compounds have one specificity for a diagnostic agent and the remaining two arms are specific for two different epitopes of the same P-glycoprotein, or ;
preferably, different P-glycoproteins. Thus, the --W093/02105 ~ PCT/US92tO6043 19 '~:
trifunctional compound of the present invention is particularly useful for assaying samples that contain a mixture of P-glycoproteins.

Assays useful in the in vitro methods include any immunoassay known to those skilled in the art in which antibody binding is determinable. Such Lmmunoassays include, ~LISA, agglutination assays, flow cytometry, serum diagnostic assays, immunohistochemical assays, including immunoperoxidase staining of tissue ;~
sections and others described, for example, in R. Aloisi, PrinciPles of ImmunolooY and Immunodiaqnostics. 152-176 and 221-222 (1988), which is incorporated herein by reference.

In a further aspect, the present invention relates to methods for detecting tumors expressing P-glycoproteins in a mam~al having or suspected of having such tumors~ The methods include (a) administering the ~- trifunctional compound of Formula I, wherein at least one of~ the~Fab-like moieties has specific reacti~ity with a diagnostic agent; (b) administering a diagnostically effective amount of the diagnostic agent; and (c) ~detecting the binding of the trifunctional compound to the mamm~l, whereby binding indicates the presence of a tumor~expressing P-glycoprotein. The methods can be used to diagnose or monitor a patient or to determine the ; appropriate therapy. Such determinations depend on the extent of binding. For example, a diagnosis can be made by those skilled in the art by comparing the extent of binding ln a pati'ent or~'patient sample with the extent of binding in a control, such as a mammal free of the `
-~ disorder. Thus, an elevated amount of ~inding compared to such a control can be diagnostic of the disorder.
~.
For the ln vivo detection methods, the trifunctional compounds have one arm specific for a P-W093/0210~ 2 1 ~ 3 !~ ~ 8 PCT/US92/06043 ~.. ,. .

glycoprotein associated with a MDR cell and a second armspecific for an imaging agent. Suitable imaging agents have been identified a~ove. The third specificity can be against the siame or different P-glycoprotein, or against the same or different imaging agent. For monitor~ng, the third specificity is preferably a different Lmaging agent.

The trifunctional compound and the imaging agent can be administered to the patient simultaneously or sequentially. For sequential administration, the imaqing agent can be administered after the trifunctional compound is given sufficient time to localize to the P-glycoprotein sites, preferably abo~t 24 hours. One skilled in the art can readily determine the appropriate time period.

The present invention further relates to methods for treating a mammal having a disorder ~; ~ associated with multi-drug resistant cells. These methods~invol~e (a) administering a therapeutically effective amount of a trifunctional compound of Formula , , ~ .
wherein at~least one of said Fab'-like moieties has speciic~reactivity with a therapeutic agent; and (b~
administering a therapeutically effective amount of the therapeutic agent to the mammal. Suitable therapeutic agents have been identified above.
.: ~ .
The trifunctional compounds of the therapeutic methods can have any combination of specificities as long ,as ~ne sjpe~ificity i8 against a MDR-associated P~
glycoprotein and a second specificity is against a therapeutic agent. The third specificity can be against either the same or a different P-glycoprotein as~oiciated with MDR or against the same or different therapeutic ` agent.
,,,.~,~ ,~
,; ~ , : ' WO93/0210~ 3~ PCT/US92/06043 ;.
21 '' The methods herein advantageously provide sLmultaneous inhibition of P-glycoprotein activity and delivery or focusing of therapeutic agents to the site in order to kill the MDR cells. Similar to the in ivo detection methods, the trifunctional compound and~
therapeutic agent can be a~m;niætered simultaneously or ~'sequentially.

Methods for diagnosing and treating a mammal having or suspected of having a disorder associated with multi-drug resistance are advantageously provided by the ' present invention. Such methods include (a) - administering a trifunctional compound of Formula I to ; the = l, wherein one Fab'-like moiety has specific reactivity with a P-glycoprotein, a second Fab'-like moiety has specific reactivity with a diagnostic agent ~;
; and a third Fab'-like moiety has specific reactivity with ' a therapeutic a~ent; ~b) administering a diagnostically ef~ ective;amount of the diagnostic agent to the mammal;
(c)';~detectin~ the binding of the trifunctional compound to~the;~'~mammal, wherein an elevated amount of binding of the compound is diagnostic of the disorder; and (d) if `''warranted, administering a therapeutically effective amount of the therapeutic agent to the mammal. Because these methods use one trifunctional compound to diagnose ant,~if~needed, treat a patient, the risk of HAMA is signifieantly reduced.~
", ~
,- '. ; ' : ~ :::
Finally, kits useful for performing the methods of the present invention are also provided. The kits containla trifunctlonal ¢ompound of the pre~ent invention and any necessary or desired ancillary reagent. Such ancillary reagents can be diagnostic or therapeutic agents, signal detection systems, buffers, stabilizers, pharmaceutically acceptable carriers and the like.

The following examples are intended to .,, ~: , , ," ~

~, :

WO93/0210~ PCT/US92/06043 ~ .., ~

illustrate, but not limit, the present invention.

EXAMPLE I
Production of MDR Antibodies .~ , Monoclonal antibodies were produced either against the multi-drug resistant human neuroepithelioma cell line MC-IXC/VCR or the multi-drug resistant human neuroblastoma cell line SH-SYSY/VCR. These cell lines were provided by Dr. June Beidler (Sloan-Kettering Cancer Center, New York) and are described in Scotto et al., Science 232:751-755 (1986).

To produ~e the LS2H241 hybridoma, balb/c mice were immunized with 5 x 10~ SH-SY5Y/VCR cells intraperitoneally. Three weeks later, the mice received a second intraperitoneal injection of 5 x 106 of the same GellS~ Four~days prior to fusion, the mice received a final~intravenous boost of 5 x Io6 cells.

Cell fusion was carried out according to the procedure of Xohler and Milstein, Nature 256~:495-497 `~
(197~51~as modified by Gerhard, Monoclonal Antibodies, R. `~
Kennett~et~al., 370-371 (Plenum Press, 1980). Briefly, 0~8~splenocytes were fused with 2.5 x 107 P3-X63-Ag8.653 (ATCC~yCRL~1580), a mouse~myeloma cell line, in 1.0 ml of 35% polyethylene qlycoI (PEG 1500) in AP-MEM medium.
Following the~fusion, the cells were cultured in medium "~
supplemented~with;hypoxanthine, ~;nopterin and thymidine at 370C in a humidified 5% C02 incubator.

Initial screening of hybridoma cultures was done by indirect enzyme-linked immunoabsorbent assay (ELISA) using a horseradish peroxidase conjugate of sheep anti-mouse Ig (Cappel, Malvern, Pennsylvania). Drug-sensitive and~drug-resistant neuroblastoma cell lines, ~ which grow as monolayers, were placed in microtiter ,;

WO93/0210~ v PCT/US92/06043 plates and allowed to grow overnight. Cells growing in suspension were addzd to microtiter plates that had been coated with 0.1% poly-L-lysine according to the procedure ~-~
of Cobbold, J. Immuno. Methods 44:125-133 (1981).

The following day, cells were fixed with 0.125%
glutaraldehyde. Following removal of the fixative and blocking with 10% bovine serum albumin, 40 yl of hybridoma supernatant was added and allowed to incubate for one hour at 37C. Plates were washed with PBS and incubated with peroxida~e-conjugated sheep anti-mouse immunoglobulin (Ig). After five washes with PBS, positive clones were identified by addition of 100 ~1 of a solution containing 1 mg/ml o-phenylenediamlne, 0.1%
hydrogen peroxide, 50 mM citrate and 100 mM sodium phosphate buffer (pH 5.0). The plates were read at 490 nm on an EIA Autoreader (Bio-Tek Instruments, Winooski, Vermont).

~^`?~ Ra*ios were determined by dividing the signal -~ obtained on the drug-resistant lines by the signals obtained on the corresponding drug-sensitive lines.
Hybridomas that demonstrated a 6-fold or greater ; ~ reactivity with the resistant cell lines relative to the drug~-sensitive cell lines were screened again in the same manner. ~Antibodies that continued to demonstrate a 5-fold or greater reactivity were selected for further characterization.
"'~' ~ `
The other MDR antibodies were generated followin~ the same general procedure. Two fusions ! ' produced 1,139 hybridomas, of which fifteen clones consistently demonstrated greater reactivity with drug-resistant cells relative to the corresponding drug-enQitive cells. These clones are identified in , .~
Rittmann-Grauer et al., "Monoclonal Antibodies To Target Multidrug-Resistant Cells," AACR Proceedinqs 20:394 ~ .

W093/0210; `2 ~ 5 7 ~ PCT/US92/06043 ~1987). The hybridoma producing monoclonal antibody LS2H241 ~also referred to as ~YB 241) ATCC No. H~ 10814 was selected for synthesizing a trifunctional compound of the present invention.

EXAMPLE II
Synthesis of LS2H FLab)'? Fraaments F(ab) ~2 fragments of monoclonal antibody LS2H241 were prepared according to the procedures of Rousseaux et al., J. Immunol. Methods 64:141-146 (1983), which is incorporated herein by reference. Thirty-three ml of LS2H241 monoclonal antibody at 5.9 mg/ml was dîalyzed in 2 L of a 0.1 M sodium formate and 0.1 M NaCl solution ~pH
2.8) overnight at 4C. The dialysis buffer was changed to~0.1 M NaOAc and 0.1 M NaCl (pH 4.5~. ~he LS2~241 :~ antibody was~ again dialyzed overnight at 4C. The :an~tibody~solution was~digested with 3% pepsin (5.7 mg) at -:-3:7C~for~110~:mlnutes. The~digestion was then terminated .~.
wi~h~ ml 2M tri~-Cl (pH 8.0~ and 100 ~1 10 M NaO~ with a final pH of about 8Ø The terminated digest was then dialyzed in:1 ~ borate buffered solution (B8S: 50 mM
sodium~borate, 50 mM NaCl, pH 8.2) for 48 hours at 4C.

EXAMPLE III
S~nthesis:of xCHA F(ab) ~? Fraqments The antibody designated herein as "xC~A" is a mouse human chimeric antibody having speciflcity for the ~- ,In-EDT~ ,chelate.complexl. ~he xC~ antihody was prepared ~-`
by essentially the same method used for the preparation of chLmeric CEM antibody as described in U.S. Patent Application Serial No. 07/274,106, by M.J. Johnson, filed .~, .
on November 17, 1988, incorporated herein by reference.
::The ~onoclonal antibody from which the murine variable , ~ .
~ region of xC~A was derived is described in U.S. Patent : : ~

WO93/0210; ~ PCT/US92/06043 ... .

No. 4,722,892, issued to Meares et al., incorporated herein by reference. ~
,;
The preparation of xCHA F(ab)' ragments is described in U.S. Patent Application Serial No. -~07/491,406, filed March 9, 1990, incorporated herein by reference. Briefly, xC~A antibody solution having a concentration of 5-15mg/ml were dialyzed overnight in an '~
acetate buffered saline solution (pH 4.1) overnight at ''-' 4C. A concentrated pepsin solution (2% of the antibody ' mass) was added to the dialyzed solution and incubated ~
for about 4-24 hours at 37C. The reaction was ~`;
termlnated~by addLng 50-100; mM tris buffer and adjusting `' the~pH to about 8 with 10 N NaOH. The terminated digests were dialyzed against BBS (p~ 8.2). The F(ab~ ~2 fragments were purified by either ~PLC or gel filtration on a~ ~Sephadex G-150 column (Pharmacia, Piscataway, N.J.). '' EX~MPLE IV
8ynthesis of Trifunctional ComPound LS2H(2)/xCHA

A~trifunctiona~l compound of Formula I was synthesiz:ed~using the F(ab) ~2 fragments of Examples II and ,;in~;which elements X1 and X2 were derived from the LS2~241 fragments and X3 was derived from the xC~A
fr ~ nt~. ;The synthesis of the trifunctio~nal compound 'was~general~ly~in accordance with the procedures set forth in~`U~.S.~Patent Application~Serial No. 07/491,406, filed Marob~9,~ 1990,~ incorporated herein by reference.

First,,6Ç~ml,of a solution of the LS2H241 F(ab) ~2 fragment (4.5 mg/ml) in BBS was incubated with 14 a-s M~DTPA at 37 C for 15 minutes. Then, 280 ~1 of 0.5 M cysteine in a citrate-DTPA buffer (50 mM ammonium citrate, 0.1 M NaCl and 0.5 mM
diethylenetriaminepentaacetic acid (DTPA) solution, p~
- . - :
~ 6.3)~were added to the antibody fragment mixture and . ~:.
~, .

~VO 93/02105 PCI /US92/06043 .
2113 r~ r~ ~ ~

incubated for an additional 10 minutes at 37C.

After reduction, excess cysteine was removed by gel filtration on a 90 ml column of P-6 DG (BioRad Laboratories, Richmond, California) in citrate-DTP~
buffer. The protein fraction was collected by monitoring absorbance at 280 nm. The volume of the protein fraction (Fab', molecular weight 50 Kd) was 11 ml, the absorbance '~
at A280 was 2.97, which is equivalent to a protein concentration of 2.1 mg/ml. The reactive sulfhydryl content of the Fab' product was determined with 5,5'~
dithiobis-(2-nitrobenzoic acid) (DTNB) in BBS (pH 8.2). -'~
The absorbance was measured at 412 nm using a molar extinction coefficient of 13,600. The absorbance at 412 nm was 1.8, which is equivalent to 132 ~M sulfylhydryl.
The sulfhydryl to Fab' ratio was determlned to be 3.2. ;

' To prepare the xCHA fragment, 1.4 ml of xCHA -~
F(ab)~'2 was;incubated with 3 yl of 0.5 M DTPA for 15 minutes at 37-C, followed by the addition of 2.8 yl of 0.5 M dithiothreitol ( DTT ) . The resulting solution was incubated for an additional 10 minutes at 37C. Excess DTT was removed on a 15 ml (1.5 x 28 cm) P-6 DG column in ';
citrate-DTPA buffer. The Fab' fraction was collected by monitoring at 280 nm. The total volume colle ted was 4.4 ml. The absorbance at A280 was 3.07, which is equivalent to 2.2 mg/ml. Free sulfhydryl concentration was 180 yM
as determined with DTNB as described above. The ., ~
~ sulfhyd:ryl to Fab' ratio was 4.1.
, .
' T~he LS2H ~ab';ifraction was derivatized with a;
30 fold molar excess of N,N'-bis~3-male;midapropionyl)-2-hydroxy-1,3-propanediamine (BMP), in which 5.4 mg BMP
were dissolved in 400 yl of DMF/water (50:50) fol'lowed by the addition of 10.8 ml of the LS2H Fab' fraction with stirring. The derivatization was allowed to proceed at room temperature for 10 minutes. Excess BMP was remove~

:-WO93/0210~ S 7~ ` PcT/us92/o6o43 . . . .

on a 240 ml P-6 DG column in citrate-DTPA buffer. The Fab'-BMP fraction was collected by monitoring at an absorbance of A280 nm. The total volume collected was 16.1 ml, with an a~sorbance of 1.82, which is equivalent to 1.3 mg/ml (26 yM). The active maleimides, as ~ `~
determined by cysteine back titration, were present at a concentration of 22 yM, which represents an average of 0.85 maleimides/Fab'.

A 15.5 ml (20 mg~ sample of the LS2H Fab'/BMP
~olution was added with stirring to 3.5 ml (7.7 mg) of ;-the xC~ Fab' solution. The reaction was incubated at room temperature for 1 hour and terminated by the addition of 20 yl of 1 M NEM in DMF. `
`.~
The Fab'3 trifunctional and Fab'2 bifunctional products were~purified on a 2.5 x lO0 cm G-150 SF column in BB~S (pH~8.2). Column fractions were analyzed by gel ~
filtration ~PLC and SDS-PAGE. Fractions 3g-43, ;;
aorre~ponding;to the 150 Kd product, LS2H(2)/xCHA, were pooled~ The~volume was 22.5 ml, with an absorbance at A28~0;~of .469,~which is equivalent to 0.33 mg/ml. The ;~
Fab'3 trifunctional product (7.4 total mg) was recovered after purification.

The Fab'3 trifunctional compound contained two LS2~241 Fab' fragments and one xCHA Fab' fragment. The bifunctional Fab'2 compound contained one LS2H241 Fab' fragment and one xCHA Fab' fragment.

, I ;,EXAMPLE V
In Vitro Studies A. ComDarison of Activity ~.
The activities of Fab'3, Fab'2 and intact ',~
monoclonal antibody LS2~241 were compared by ELISA to .

W~93/0210~ PCT/US92/06043 determine if the preparation of the bifunctional and trifunctional compounds resulted in any loss of activity.
The antibody compounds were tested against the CCRF-CEM
cell line, which is drug-sensitive, and CEM~VLBloo, the corresponding drug-resistant cell line. Both cell~lines were obtained from William Beck, et al., Cancer Res.
39:2070-2076 (1979). -~

To perform the membrane ELISA, 5 yg of sucroYe purified CCRE-CEM membranes and CEM/VLBloo membranes were aliquoted into wells of flat bottom microtiter plates (Dynatech, Alexandria, Virginia). The plates were incubated at 37C in an oven overnight. The plates were -washed three tLmes with cold tap water and then incubated for 3Q minutes at room temperature with 4% bovine serum albumin in phosphate-buffered saline. The buffer was removed and 50 ~1 of one of the antibodies (intact, Fab' 2 or Fab'3) was added to the wells and incubated at room temperature for one hour. Five concentrations of ~``
antibody were~assayed (1 ng/ml, 10 ng/ml, 100 ng/ml, 1 g/ml~and 10 ~g/ml). The plates were washed six times with tap water prior to the addition of peroxidase conjugated~goat anti-mouse kappa and lamda immunoglobulins (Southern Biotechnology, Birmingham, Alabama) at a 1:500 dilution. The plates were incubated for one hour at room temperature and then washed five time~ with tap water. Color was developed after the `addition of 100 ~1 of 1 mg/ml o-phenylenediamine, 0.03%
hydrogen peroxide in 0.1 M citrate phosphate buffer (pH
5.0). The plates were incubated in the dark with shaking ~for 20 minutes.i The reaction was quenched by adding 50 ~1 of 4 N ~2SO~ to each well. The wells were then read on ~-~ an ELISA reader at an absorbance of 49Q nm.

As 3hown in Figure 1, the LS2~241-containing compounds bound to the multi-drug resistant CEM/VIBloo membranes, which are known to express P-glycoprotein. By .

.

W093/0210~ 3 v ~ 3 PCT/US92/06043 .... ,............................................................................ ~,~,~
29 !-~
comparison, as shown in Figure 2, the LS2~24l-containing compounds bound minimally to the drug sensitive CCRF-CEM
membranes, which are known to contain very low levels of ;
P-glycoprotein. Thus, the ability of the trifunctional compound to bind P-glycoprotein does not appear t~ be -significantly affected by the chemical synthesis of the product.

B. Modulation of P-alycoprotein To determine the effect of cytotoxic drugs in the presence and absence of intact LS2~24l or Fab' 3 , trifunctional antibody, MC-IXC/VCR drug-resistant cells were plated out into 2 ml wells at lO6 cells/well in the absence of vincristine. Cells were incubated overnight and allowed to attach to the wells. The next day, the media was removed and replaced with l ml of fresh media containing 50 yg/ml of one of the antibodies. Control wells~reoeived fresh mèdia with PBS or a control antibody, GDJ352, which does not bind to P-glycoprotein.
Plates were incubated for 30 minutes at 37C, followed by removal of media and replacement with media containing 50~g/ml of intact or Fab'3 antibody plus 3~-actinomycin D
at 0O3 yCi/ml. Control wells were replaced with fresh media with the same amount of 3H-actinomycin D but without Fab' 3 - . The cells were incubated in the presence of the drug for 4~hours at 37C in 5% C2-' The supernatants were remov~ed from the wells and the cells were carefully and rapidly wa~hed by serial dipping in 3 beakers of cold PBS. The cells were then released from the wells by mild trypsinization!~250 ~L~of trypsin) and the, entire ! `
suspension`was transferred to a scintillation vial and counted.

The results of the study are shown in Figure 3.
Preincubation of the drug-resistant cells with the Fab' 3 trifunctional compound prior to incubation with ~113'j7~ -actinomycin D resulted in an approximately 4 fold -increase in intracellular drug concentration over the ~-control cells. Similarly, preincubation of the cells with intact LS2H241 resulted in an approximately 5 fold "~
increase in intracellular drug concentration over ~he -control cells. The data demonstrate that the trivalent compound binds to the drug resistant cell line and blocks `-the ATP-dependent efflux of drug from the cells.

EXAMPLE VI
Synthesis of the Chelatinq Aqent DBX

The chelating agent, DBX, was prepared by the reaction of disuccinimidyl carbonate with the In3' complex of p-amlnobenzylethylene-diaminetetraacetic acid, -~
referred to herein as (p-amlnobenzyl)EDTA, and p-aminobenzyldiethylenetriamine-pentaacetic acid, referred to herein as (p-aminobenzyl)DPTA.

A. ~ ~ nthesls~of~the In3~ complex of (p-aminobenzyl)EDTA

The following reagents were used to prepare the complex: (p-aminobenzyl)EDTA, indium metal, at least 99.99~% pure, 6 M Ultrex HCl (J.T. Baker Chemical Co., P~hillisberg, New Jersey), distilled water and 10 M NaOH.
First 344~mg of indium metal (foil or pellets) were weighed~oot in an acid-washed 100 mL polypropylene volumetria flask.~ After 20 ml of 6 M Ultrex HCl was added~,~ the f~lask was lightly stoppered and placed in a 60C water bath overnight to hasten dissolution. After the indium~ was disso~vedS the flaskl~was cooled to;room !
temperature, followed by the addition of distilled water to volume. The molar concentration of In3' was calculated as approximately 30 mM using the mass determlned in the initial weighing step and 114.82 as the atomlc weight for indium.
.'`.

WO93/0210~ 2 1 1 3 lj 7 o PCT/US92/06043 ~

3l A 50 mM solution of (p-aminobenzyl)EDTA in distilled water was prepared. The concentration of the solution was determined by measuring the absorbance of a solution at 240 mn and using ~ = l0,800Q M~l cm~l. PBS (l0 mM Na2HPO4, 150 mM NaCl, p~ 7.2) was used as the di~luent.

The total moles of (p-aminobenzyl)EDTA was calculated. An aliquot of the indium chloride solution was added to the (p-aminobenzyl)EDTA solution such that the indium was 10% in exce~s over the chelate. The resulting mixture was stirred and the pH adjusted to about 8 by adding l0 M NaO~. The uncomplexed indium was removed by filtering the solution through a 0.2 ~m Acrodisc filter.

The resulting mixture was analyzed by reversed phase HPLC with isocratic elution using 8% v/v solvent B
and 92% v/v solvent A and compared with a sample of (p-amlnobenzyl)EDTA. A~single peak with a retention time greatèr~than that of ~p-aminobenzyl)EDTA indicated that all of the (~p-aminobenzyl)EDTA was complexed with indium.
The concentration of the indium complex was measured spectophotometrically as described above and used to prepare DBX.

B. ~Synthesis of ~p-Nitrobenzyl)EDTA

(S)-p-nitrobenzyl EDTA was prepared and converted to (S)-4-isothiocyanatobenzyl EDTA ("ITCBE") as described in Meares et al., U.S. Patent No. 4,622,420, and Meares, Anal. ~iochem. l42:68-75 (1984). The lyophilized ITCBE was resuspended in 0.3 M Ultrex ~CL to a final concentration of approximately 50 mM ("Stock ITCBE solution"). This solution was stored at -70C.

C. Synthesis of DBX

WO93/0210~ PCT/US92~06043 .

Indium p-aminobenzyl-EDTA (In-ABE) (50 mM, 250 ;~
~1~ was mixed with p-aminobenzyl-DTPA (137 mM, 100 ~
and the solution diluted with 0.5M NaHCO3, pH 9 (3S0 ~1).
Excess N,N-disuccinimidyl carbonate was added and the reaction vortexed periodically. Three products were evident in the HPLC analysi~ (3.2, 3.7, and 4.2 min~ in a 1:2:1 ratio, consistent w~th urea formation. The reaction mixture was diluted to 50 mh with water and applied to an A-25 column (12 mL). The column was eluted with a gradient (200 mL) of 100 mM to lM ammonium formate, pH 8. Fractions were combined based on HPLC
analysi~ and the fraction containing the 3.7 min peak analyzed. HPLC analysis showed that this compound bound both indium (4.2 mln) and yttrium (4.3 and 4.4 min). W -analysis showed the compound had an absorbance maximum at 260 nm. Incorporation of l'lIndium (as explained abo~e) was >95% and CHA-255 binding of the lllIndium (III) was 94%. The 13C NMR spectrum contained a signal at ~ 156.5, consistent with a urea carbon, as well as signals ,~
corresponding to Indium benzyl EDTA and benzyl DTPA. The proton NMR spectrum was consistent with the assigned structure.

NMR (13C, D2O, p~=4.5) ~: 177.2, 176.7, 176.6, 176.3, 171.2, 171.1, 155.8, 138.3, 138.0, 132.2, 131.1, 130.7 (CH), 121.7 (C~), 63.4 ~CH), 62.0 (C~), 60.6 (CH2), 58.7 (CH2), 58.2 (CH2), 57.7 (CH2), 55.8 (CH2), 55.0 (C~2), 54.3 (CH2), 54.0 (CH2), 52.4 (CH2), 49.5 (CH2), 33.0 (CH2), 32.5 (CH2); (proton NMR, D20, pH=4.5) ~: 7.25 (d, 2H, J =
7.8 Hz), 7.23 (d, 2H, J = 7.8 Hz), 7.15 (d, 2H, J = 7.8 Hz), 7.09 (d, 2E, J = 7.l8 Hz), 2.20` - 3.80 (32 EXAMPLE VII
Synthesi~ of 'l~In-DBX

Indium chloride (In-lll) was diluted to 2.5 mCi/ml with 0.05 M HCl. Buffered indium chloride (BIC) WO93/0210~ 211 3 j 7~ PCT/~S92/06043 was prepared by mixing 60 ~l of the diluted indium chloride and 40 yl of 0.22 M glycine (pH 8.2).

The indium-lll labeled DBX was prepared by adding 160 ~l (400 ~Ci) of BIC at 2.5 mCi/ml to l~0 yl of DBX at 2,000 pmol/ml. The mixture was incu~ated for 30 minutes at room temperature. An equivalent ~olume (320 yl) of a solution containing 150 mM ascorbate, 50 mM `~
tris, 0.1 mM ~TPA (pH 7.4) was added to quench the reaction. Samples (1 ~l) of the reaction were spotted on the origin of silica gel plates and allowed to dry completely. Buffered indium chloride radioisotope (0.5 yl) was also spotted on the plate as a control. The plate was placed in a thin layer chromatography (TLC3 chamber that had been equilibrated with 10% ammonium acetate (aq):methanol (l:l) solution. Following chromatography, the plates were dried and cut from the - bottom to slightly above the origin and from the top to ~ : "
the solvent front. The strips were transferred to gamma counting tubes and the amount of radioactivity of each strip was determined. Counts in the solvent front represent ~llIndium-labeled DBX. Counts remaining at or near~the origin represent BIC. Routinely, 98~ of the counts are incorporated into the DBX.

EXAMPLE VIII
In Vivo Biodistribution Studies Tumors were established by ~ubcutaneously injecting 1 x 105 BROtMDRl cells from tissue culture into nude;miqe. BROtMDRl cells`were obtained from Dr. Piet Borst (Netherlands Cancer Institute, Amsterdam). The BROtMDRl cell line was developed from the human melanoma cell line BRO following transfection of the human liver mdrl gene. When the tumors had reached 0.6-0.8 grzms in ~ .
size~ 20 yg of Fa~'3 trifunctional compound, also referred to herein as (LS2~)2xC~A, was injected into the tail vein ':~
~::
' W093~02105 ~z 113 r~ 7 ~ PCT/US92/06043~

of the mice. Twenty-four hours later, lO ~Ci of -~
1llIndium-labeled DBX was injected into the tail vein of each mouse.

At 4, 24, 48 and 120 hours after injection of the radiolabeled hapten, the animals were sacrificed and the biodistribution of the radioactivity was determined.
Tumors, visceral organs, blood, bone, skin, muscle, urine and feces were weighed and counted in a gamma counter for activity. The amount of activity remaining in the tail was subtracted from the injected dose to calculate the percentage of the injected dose per gram of tissue~

The results of the biodistribution study are summarized in Figure 4. The trivalent antibody (LS2H)zxC~A targeted the human drug resistant tumor xenograft and bound minimally to all the normal tissues that;were evaluated. By 25 hours 13% dose/gram was localized~in~the tumor and remained at the tumor by 48 hours~(l4% dose~/gram~, while all the normal tissues, with the exception of kidney, had levels under 5%~ doselgram.
The kidney contained 7% dose/gram, which reflects the rapid clearance of the labeled DBX. The rapid clearance ~;
of DBX~is also s~hown in the high counts in urine. Thus, the results Qf these studies indicate that the trifunctional compounds of the present invention have diagnostic and therapeutic utility to detect and treat , multi-drug resistant tumors, including colon, renal, breast and ovarian carcinomas.
, .
The foregoingidescription of the invention is exemplary for purposes of illustration and explanation.
It will be apparent to those skilled in the art that changes and modifications will be possible without departing from the spirit and scope of the invention. It l`
: ~ 1 ;~ ~ i8 tended that the following claims be interpreted to embrace all such changes and modifications.

Claims (54)

We claim:

1. A trifunctional compound having the formula:
wherein L is a cross-linking agent;
wherein X1, X2 and X3 are Fab'-like moieties;
and, wherein at least one of said Fab'-like moieties has specific reactivity with a P-glycoprotein associated with a multi-drug resistant cell.

2. The trifunctional compound of claim 1, wherein said P-glycoprotein has an apparent molecular weight in the range of about 170-180 Kd.

3. The trifunctional compound of claim 1, wherein at least one of said Fab'-like moieties is derived from monoclonal antibody LS2H241.

4. The trifunctional compound of claim 1, wherein said trifunctional compound is a bi-specific compound.

5. The trifunctional compound of claim 4, wherein one Fab'-like moiety has specific reactivity with said P-glycoprotein, a second Fab'-like moiety has specific reactivity with a diagnostic agent, and the third Fab'-like moiety has specific reactivity with said P-glycoprotein or said diagnostic agent.

6. The trifunctional compound of claim 5, wherein said diagnostic agent is a complex of a chelating agent and an imaging agent.

7. The trifunctional compound of claim 6, wherein said complex is a physiologically compatible chelate complex.

8. The trifunctional compound of claim 6, wherein said imaging agent is a radionuclide or a paramagnetic metal ion.

9. The trifunctional compound of claim 8, wherein said radionuclide is selected from the group consisting of 113mIn, 67Ga, 99mTC, 51Cr, 197Hg, 203Hg, 169Yb, 85Sr, 87Sr and 111In.

10. The trifunctional compound of claim 8, wherein said paramagnetic metal ion is selected from the group consisting of lanthanide elements of atomic number 57-70 and transition metals of atomic number 21-29, 42 or 44.

11. The trifunctional compound of claim 7, wherein said chelating agent is DBX.

12. The trifunctional compound of claim 4, wherein one Fab'-like moiety has specific reactivity with said P-glycoprotein, a second Fab'-like moiety has specific reactivity with a therapeutic agent and the third Fab'-like moiety has specific reactivity with said P-glycoprotein or said therapeutic agent.

13. The trifunctional compound of claim 12, wherein said therapeutic agent is a cytotoxic agent or a complex of a chelating agent and a radionuclide.

14. The trifunctional compound of claim 13, wherein said cytotoxic agent is selected from the group consisting of doxorubicin, daunorubicin, mitomycin C, etoposide, vinblastine, vincristine, colchicine, dihydroxyanthracenedione, mitoxanthrone, mithramycin, actinomycin D, puromycin, gramicidin D, ethidium bromide, taxol, tenoposide, cytochalasin B, 1-dehydrotoastosterone.

15. The trifunctional compound of claim 13, wherein said radionuclide is selected from the group consisting of 67Cu, 186Rh, 188Rh, 189Rh, 153Sm, 111In (A?ger) and 90Y.

16. The trifunctional compound of claim 13, wherein said chelating agent is DBX.

17. The trifunctional compound of claim 1, wherein said trifunctional compound is tri-specific.

18. The trifunctional compound of claim 17, wherein one Fab'-like moiety has specific reactivity with said P-glycoprotein, a second Fab'-like moiety has specific reactivity with a diagnostic agent and the third Fab'-like moiety has specific reactivity with a therapeutic agent.

19. The trifunctional compound of claim 18, wherein said diagnostic agent is a complex of a chelating agent and an imaging agent.

20. The trifunctional compound of claim 19, wherein said chelating agent is DBX.

21. The trifunctional compound of claim 10, wherein said imaging agent is a radionuclide or a paramagnetic metal ion.

22. The trifunctional compound of claim 18, wherein said therapeutic agent is a cytotoxic agent or a complex of a chelating agent and an imaging agent.

23. The trifunctional compound of claim 22, wherein said complex is a physiologically compatible chelate complex.

24. The trifunctional compound of claim 23, wherein said chelating agent is DBX and said imaging agent is a radionuclide or a paramagnetic metal ion.

25. The trifunctional compound of claim 1, wherein said cross-linking agent is a tris-maleimide.

26. The trifunctional compound of claim 25, wherein said tris-maleimide is N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine.

27. A composition comprising the trifunctional compound of claim 1 and a pharmaceutically acceptable carrier.

28. The composition of claim 27, wherein at least one of said Fab'-like moieties is derived from monoclonal antibody LS2H241.

29. The composition of claim 28, wherein said trifunctional compound is bi-specific.

30. The composition of claim 29, wherein one Fab'-like moiety has specific reactivity with said P-glycoprotein, a second Fab'-like moiety has specific reactivity for a diagnostic agent and the third Fab'-like moiety has specific reactivity with said P-glycoprotein or said diagnostic agent.

31. The composition of claim 29, wherein one Fab'-like moiety has specific reactivity with said P-glycoprotein, a second Fab'-like moiety has specific reactivity with a therapeutic agent and the third Fab'-like moiety has specific reactivity with said P-glycoprotein or said therapeutic agent.

32. The composition of claim 27, wherein said trifunctional compound is tri-specific.

33. The trifunctional compound of claim 32, wherein one Fab'-like moiety has specific reactivity with said P-glycoprotein, a second Fab'-like moiety has specific reactivity with a diagnostic agent and the third Fab'-like moiety has specific reactivity with a therapeutic agent.

34. A method for inhibiting the activity of a P-glycoprotein associated with a multi-drug resistant cell, said method comprising contacting said multi-drug resistant cell with a trifunctional compound of claim 1.

35. The method of claim 34, wherein said compound has one Fab'-like moiety having specific reactivity with a first P-glycoprotein binding site and a second Fab'-like moiety having specific reactivity with a second P-glycoprotein binding site.

36. The method of claim 34, further comprising the step of contacting said multi-drug resistant cell with a chemosensitizer.

37. The method of claim 34, further comprising the step of contacting said multi-drug resistant cell with a MDR drug.

38. The method of claim 34, wherein contacting said multi-drug resistant cell with a trifunctional compound of claim 1 is accomplished by administering said trifunctional compound to a mammal having a disorder associated with said multi-drug resistant cell.

39. A method for detecting a multi-drug resistant cell, comprising:
(a) contacting a sample containing or suspected of containing said multi-drug resistant cell with a detectable trifunctional compound of claim 1, said trifunctional compound having at least one Fab'-like moiety specific for a diagnostic agent; and (b) detecting the binding of said trifunctional compound to the sample, wherein binding indicates the presence of said multi-drug resistant cell in said sample.

40. A method for detecting tumors expressing P-glycoprotein in a mammal, comprising:
(a) administering a trifunctional compound of claim 1 to said mammal, wherein at least one of said Fab'-like moieties has specific reactivity with a diagnostic agent;
(b) administering a diagnostically effective amount of said diagnostic agent to said mammal; and (c) detecting the binding of said tri-functional compound to said mammal, wherein binding of said compound to said mammal indicates the presence of said tumor in said mammal.

41. The method of claim 40, wherein at least one of said Fab'-like moieties is derived from monoclonal antibody LS2H241.

42. The method of claim 40, wherein said diagnostic agent is a complex of a chelating agent and an imaging agent.

43. The method of claim 40, wherein said trifunctional compound and said diagnostic agent are administered simultaneously.

44. The method of claim 40, wherein said trifunctional compound and said diagnostic agent are administered sequentially.

45. A method for treating a mammal having a disorder associated with multi-drug resistance, said method comprising:
(a) administering a therapeutically effective amount of a trifunctional compound of claim 1 to said mammal, wherein one of said Fab'-like moieties has specific reactivity with a therapeutic agent; and (b) administering a therapeutically effective amount of said therapeutic agent to said mammal.

46. The method of claim 45, further comprising the step of administering a MDR drug to said mammal.

47. The method of claim 45, wherein at least one of said Fab'-like moieties is derived from monoclonal antibody LS2H241.

48. The method of claim 45, wherein said therapeutic agent is a cytotoxic agent or a complex of a chelating agent and a radionuclide.

49. The method of claim 45, wherein said trifunctional compound and said therapeutic agent are administered simultaneously.

50. The method of claim 45, wherein said trifunctional compound and said therapeutic agent are administered sequentially.

51. A method for diagnosing and treating a mammal having or suspected of having a disorder associated with multi-drug resistance, said method comprising:
(a) administering a trifunctional compound of claim 1 to said mammal, wherein a second Fab'-like moiety has specific reactivity with a diagnostic agent and the third Fab'-like moiety has specific reactivity with a therapeutic agent;
(b) administering a diagnostically effective amount of said diagnostic agent;
(c) detecting the binding of said tri-functional compound to said mammal, wherein binding of said compound to said mammal is diagnostic of said disorder; and (d) if warranted, administering a therapeutically effective amount of said therapeutic agent to said mammal.

52. The method of claim 51, wherein said diagnostic agent is a chelating agent complexed to an imaging agent.

53. The method of claim 51, wherein said therapeutic agent is a cytotoxic agent or a complex of a chelating agent and a radionuclide.

54. A kit comprising the trifunctional compound of claim 1 and an ancillary reagent.