CA1306414C

CA1306414C - Affinity enhancement system

Info

Publication number: CA1306414C
Application number: CA000547184A
Authority: CA
Inventors: Jacques Barbet; Michel Delaage; Jean-Marc Le Doussal
Original assignee: Immunotech Partners SA
Current assignee: Immunotech SAS
Priority date: 1986-09-19
Filing date: 1987-09-17
Publication date: 1992-08-18
Anticipated expiration: 2009-08-18
Also published as: AU613318B2; AU7865687A; GR3004914T3; KR900005622B1; DE3778281D1; JP2612454B2; ATE74769T1; EP0263046B1; ES2032468T3; FR2604092A1; FR2604092B1; EP0263046A1; JPS63159327A; KR880004312A; US5256395A

Abstract

ABSTRACT
AFFINITY ENHANCEMENT IMMUNOLOGICAL REAGENTS FOR DETECTION AND KILLING
OF SPECIFIC TARGET CELLS

Immunological reagents, consisting of a) antibody or fragment conjugates having both an anti-cell specificity and an anti-hapten specificity, and b) synthetic tracers containing at least two hapten sites and at least one site suitable to attach radioactive isotopes, paramagnetic ions, drugs or toxins, are provided. These reagents are capable of binding to target cells in a specific way, and the tracer localizes preferentially on the membrane of antigen-bearing cells, even in the presence of excess antibody conjugate (affinity enhancement). These reagents are usefully employed, either in vitro or in vivo, to detect tumors, metastases, or other tissue injuries, when the synthetic tracer carries radioactive or paramagnetic compounds, and to kill target cells when the synthetic tracer carries radioactive compounds or drugs or toxins.

Description

AFFINITY ENHANCEMENT IMMUNOLOGICAL REAGENTS FOR DETECTION AND
KILLING OF SPECIFIC TARGET CELLS

FIELD OF THE D~NTION

The present invention relates generally to specific immunological reagents including monoclonal antibodies that recognize human target cells. In one aspect, this invention relates to the use of radioactive or paramagnetic compounds in association with antibodies for immunodiagnostic purposes. In another as~ect, it relat~s to the use of radioactive materials, or drugs, or toxins, or enzymes in asso-ciation with antibodies for irnmunotherapy of cellular disorders, particularly cancer.

BACKGR~UND OF THE ~NTION

The use of antibodies labeled with radioactive iodine isotopes has been proE~osed to detect tumor-associated antigens. For instance, in the US Patent 3,927,193 labeled goat anti-l1uman carcino-15 embryonic antigen antibodies have been injected to Syrian hamstersinoculated with human carcinoma and shown to locali~e preferentially in the tumor. It was thus suggested that labeled antibodies might be used to visualize tumors after injection to patients using detectors available in the art. Such diagnostic applications are cG~monly referred 20 to as immunodiagnostic. As early as 1956, Beierwaltes and coworkers cured a patient with advanced malignant melanoma by injecting large amounts of 131I-labeled gamma-globulins from a rabbit immuni~ed with the patients~ own tumor cells. Further use of directly labeled polyclonal antibodies has not met with equal success. For convellience, this, and 25 other rela-ted applications of antibodies, will bereferredto as immuno-therapy.
Since the discovery of monoclonal antibodies (Kohler and Milstein), monoclonal antibodies capable of specific binding to cells of a particular -type, or, in a less specific way, to cells of a few 30 different types, have been obtained in many laboratories and industries.
Such monoclonal antibcdies are most attractive because they are hcmo-geneous and potentially more specific than polyclonal antibodies extracted from antisera. They have been widely used to identify cells in tissue sections and various biological samples, and to diagnose 3~

cancer and metastases in vitro (Gat.ter et al.). An obvious application of these reagents was to label them with a suitable radioactive isotope and inject them in animals or human in order to visualize in vivo specific cell subsets (e.gO tumors or metastases) using existing devices such as the gamma camera. Another application was to inject large quantities of monoclonal antibodies labeled with radioactive isotopes capable of killing the cells (e.g. malignant cells) to which the anti-body became bound.
Isotopes generally used in radioimmunoscintigraphy are .
131 iodine and 123 iodine (covalently coupled to tyrosines of the anti-body, Hunter and Greenwood) ; 111 indium, 9 ~ c and other metals (attached directly of by means of suitable chelating agents covalently coupled to the antibody, Hnatowich et al.). For radioimmunotherapy, high linear energy transfer (L~r) isotopes are usually preferred (e.g. 131I, At, Bi).
The state of the art and the major limitations of radio-immunoscintigraphy and radioimmunotherapy have been discussed by Bradwell et al. The essential parameters in these techniques are the fraction of the injected dose specifically localized at the site(s) where target cells are present and the uptake ratio (i.e. the ratio of the concentration of specifically bound antibody tQ that of the radioactivity present in surrounding normdl tissues~. These parameters are related in a non-trivial way. Usually the fraction of injected dose localized in the tumor is not much better than 0.1 %, and contrast not better than 2 to 3. These figures translate in the common observa-tion that tumors (or other tissue injuries) smdller than 1 to 2 cm in diameter cannot be detected, and that radioimmunotherapy has met with little success so far. Non specific uptake by non-target organs such as the liver, kidneys or bone-marrow is another major limitation of the technique, especially for radioimmunotherapy, where irradiation of the kone marrow often causes the dose-limiting toxicity.
Recently, the use of low molecular weight tracers, such as indium chelates, associated with dual specificity antibody conjugates combining antibodies (or fragments) to the target cells with antibodies (or fragments) to the indium chelate, has been proposed (Reardan et al.).
Increased uptake ratios and faster localization of the tracer are expected, since the radioactivity would be associated to low molecular weight structures capable of fast distribution through the body tissues and of rapid clearance. If the radioactive isotope has a rapid radio-active decay, such as 123I of 99mTc, images recorded sooner after injection will be obtained with higher activities remaininy than with the conventional techniques. Simllarly, fast localization and rapid clearance of excess radioactive isotopes, or drugs or toxins would reduce damage to normal cells and tissues in immunotherapy.
However, the tracer may be effectively trapped by excess circulating dual specificity conjugate, and its specific localization and its clearance would be impaired. This is a major limitation of the proposed two-step technique in immwlodiagnostic and immunotherapy. To take advantage of the theoretical potential of the method, excess dual specificity conjugate should be removed from the circulation prior to injection of the tracer (Goodwin). This would involve cumbersome in vivo manipulations, which have not been substantiated yet. Thus further improvements of the method are still required.
C*her useful techniques for the diagnostic of cancer and tissue injuries which do not necessarily involve the use of antibodies are known to the art. In addition to the techniques derived from X-ray radiography, of which an elaborated version is the computer assisted tomography (CAT scanning), sophisticated detectors have been developped to nitor the magnetic resonance properties of living organisms, and particularly to produce images of organs or whole bodies in a technique called Magnetic Resonance Imaging IMRI). The association of the exquisite spatial resolution of MRI and the specificity of imrnunological reagents such as the rnonoclonal antibodies has been conternplated (Unger et al., Curtet et al.). It has been proposed to label monoclonal antibodies to specific cellular antigens with chemical groups capable of enhancing the relaxation of the protons contained in body tissues and fluids, and particularly to use pararnagnetic rnetal ions such as Fe, Mn, or Gd.

r r _ However, the techniqle is far rom achieving clinically useful resultsr particularly because the concentrations of relaxation agents that must be deposited at the target sites are very high. Theoretically, the problem would be solved by conjugating several thousand relaxation agents per antibody molecule, but this has not been possible yet without compromising the ability of the antibody to recognize the antigen. m us, in this area also, substantial advances must occur.
In an entirely different domain of the prior art, a few natural multivalent ligands are recognized to bind more tightly to mul-tivalent receptors than the corresponding monovalent ligands (e.g.
binding of IgE and IgM to cell membrane receptors, binding of agregated IgG to the polymeric Fc receptor, or c1q binding to immune complexes).
Similarly, synthetic mu~tivalent ligands for receptors such as the DNA
have been described (Le Pecq et al.) with increased affinity as compared to the monovalent ligand. However, no useful app]ication of this kncwledge has been proposed :in the fields of in vivo immunodiagnostic or immunotherapy.

SUMMARY OF THE INVENTION
The present invention is based upon an entirely new approach using low molecular weight tracers with definite tropism towards cell-bound, as opposed to excess free, dual specificity conjugate. qhus tracers possessing such a troptsm have been designed by taking advantage of -the fact that multiple simultaneous bm ding to receptors distributed a-t the external side of the membrane of target cells may be much stronger than monovalent binding to the same receptors in solution.
a) It is an object of the present invention tha-t reagents, referre~to as dual specificity conjugates, may be provided to associate to target cells (defined by their expression of a given membrane ~ltigen) receptors for soluble ligands (haptens).
b) It is another object of the present invention that binding of the dual specificity conjugates to antigens expressed at the surface of the target cells result in a distribution of receptors for the soluble ligands within tne surface of the cell membrane that may !~

5.

behave as a poten-tial multivalent reeeptor for the soluble ligands.
c) It is a further object of the present invention to provide low molecular weight molecules, referredto as affinity e~nhancem2nt probes, suitable for radioisotopic labeling, or earrying paramagnetic compounds, or drugs or toxins.
d) It is still a further object of the present invent:ion tha-t the affinity enhancement pro~e ccmprises at least tw~ hapten groups and one or several effeetor groups. me said effector groups are either suitable for radio-labeling, or eomprise one or several paramagnetie eompounds, or drugs, or toxins. A general strueture of the affinity enhancement probes may be sehematized as follows, where X refers to any suitable hapten~ Y to any suitable effeetor group, the lines referringto any sequence of atoms or chemical groups linked by covalent che~ical bonds and forming a single stable moleeule :

~o~ ~roup IJapten ~

For some particular application, the hapten and the effeetor group may be one and a single entity. In that ease, the affinit~ enhaneement probe may be constituted of two, or more, of this hapten/effector group linked together through eovalent ehemieal konds.
e) It is still a further objeet of the present invention that the affinity enhaneement probes, as deseribed in b) are eapable of binding s~eeifieally to the target eells, properly treated with dual speeificity conjugate as described in a).
f) It is an other ob]eet of the present invention that multivalent complexes eomprising two or more molecules of dual specificity conjugates and at least one moleeule of the affinity .. . ~

3&i~ lf~
6.

æl~ncement -probe may become bound to the specific antigens expressæd at the surface of the target cells. Such multivalænt complæ~es m~y be usefully schæ~ati~ed as follows, wherein X, Y and the solid lines have ~he meanings described in d), and the dual specificity conjugate is represented as an F(ab')2, recognizing a cell membrane target antigen, couplæd to an Fab' recognizing the hapten X :

~f~ln1~y enh~nce~en~ pro.~e ~u.~ e~ifl~lty ~on~,u~ate I~n. r~
T~rge~ 2nti~en ~ell g) It is still another object of the present invention that the affinity ælihancement probe is made with two different, non cross-reactive, haptæns (X and X'). In that case, two different dual specificity conjugates, one with specificity to one cell~llar antigel-and to hapten Xr a second with specificity to an other cellular antigen and haptæn X' are used. As a result the affinity enhancæment probe will react preferentially with cells expressing both cellular antigens, as opposed to those æ~pressing only one or the other.

~,~fini~y enh~n.~e~en~ p~ oke ~ '.~u~l spe~ifi~l~y ~onju~.?te T~get ~n~igen Cell ~ s~3~

h) It is still another object of the present invention that specific binding of the affinity enhancement probe is not precluded by the presence of excess dual specificity conjugate present in the surrounding medium.
i) It is still another object of the present invention that these reagents may be used to detect or kill specific cells in animals and human for immunodiagnosis, magnetic resonance imaging, and immuno-therapy.
I'hese and other objects of the present invention are provided by dual specificity conjugate associating receptors for cell membrane antigens and soluble ligands, particularly by antibodies or fragments recognizing cell membrane antigen(s) covalently coupled to antibodies or fragments recognizing a soluble hapte~ (dual specificity con~ugate), and by synthetic molecules, suitahle for radio-isotopic labeling or carrying paramagnetic compounds, or drugs, or toxins,-presenting two, or more, hapten groups (affinity enhancement probe). Preferred embodi-ments of the present invention are monoclonal antibodies and their fragments as the receptors for cell-surface antigens or as hapten-specific receptors. Other preferred embodiments of the present invention are F(ab')2 fragments as the cell-specific antibody and Fab' fragments as the hapten specific receptor. Other preferred embodiments of the present invention are dinitrophenyl (DNP) as the hapten, and tyrosines or chelating agents as the group suitable for radioactive labelingO
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Monoclonal antibodies with specificity to tumor-specific or tum~r-associated antigens, or to cell-surface antigens of specific organs, or to serw~ proteins, or to muscular proteins, or to any other normal or pathologic constituents of the human body are useful in preparing the dual specificity conjugate. Monoclonal antibodies against t~mor-associated antigens of melanoma, or T-cell lymphoma, or breast, or Lung, or colorectal cancer, or clotting antigens such as fibrin, or intracellular antigens such as villin or myosin, or viral or microbial antigens are preferred. r~e specilici~y and binding affinity of each such monoclonal antibody with respect to its target antigen may be about that of monoclonal antibodies conventionally used for the binding 3~

assay being performed. High affinity monoclonal antibodies (dissociation constant smaller t.~n 10 M) are pre~erred.
Monoclonal antibodies to a wide varie~y of small molecular weight haptens of natural or synthetic origin are known or may be produced by techniques known to the previous art. Preferred monoclonal antibodies recognize specifically dinitrophenyl, or succinyl-histamine, or metal chelates. ~ost preferred anti-hapten monoclonal antibcdies exhibit an affinity toward the monomeric hapten that does not allow -the circulating dual specificity conjugate to trap the affinity enhancement probe before it binds to target associated dual specificity conjugate. Typically, most preferred dissociation constant of each such antibody is choosen beetween 10 9 and 10 7 M.
~ he monoclonal antibodies may be produced from tissue culture supernatants ox from animal implanted tumors, and purified in large quantities according to well-established techniques. It is advantageous for the present invention to hydrolyze them b~y limited enzymatic digestion, under defined conditions, to yield fragments of various molecular sizes, of which some retain the ability to bind the antigen. ~lost preferred fragments are F(ab')2 fragments of about 100,000 Da, or Fab of Fab' fragments of about 50,000 Da which can be prepared and purified using techniques known to previous art.
Monoclonal antibodies, or their fxagments, may be reacted with a variety of heterobifunctional reagents capable of cross-linking the cell-reactive anti~cdy to the hapten reactive anti~cdy. For example, succinimidyl 4-(N-maleilNdomethyl)cyclohexane-1-carboxylate (SMCC) or succinimidyl 3~(2-pyridyldithio)propionate (SPDP) are suitable to attach to one an-tibody or fragment a thiol-reactive group.
The other antibody or fragment may be derivatized in such a way as to attach a thiol group. A preferred embodiment is to generate a thiol group by partia:L reduction of the antibody, or, most preferably, of its F (ab')2 fragment. Mixtures of the two in appropriate molar ratios and incuba-tion conditions xesult in the formation of conjugate which has the requisite properties for the present inventionO
Particularly, it exhibits good stabili~y in various media, including J~

~la3~ 1 Ll:l aqueous buffers, tissue culture media and body fluids under reasonable conditions of pH and temperature. In addition, it is able to bind the hapten and the cells for which it has specificity. Alternatively, dual specificity antibodies may be produced by somatic fusion of hybridoma cells producing antibodies of two different specificities, as claimed in the U.S. patent 4,474,893 and in the International patent W0 83/03679.
Any hapten to which monoclonal antibodies are available or may be produced may be suitable for the present invention. Those haptens which are not present in human tissues ar preferred. Are also preferred haptens which are not degraded too rapidly after in vivo administration and those which do not present too high a toxicity to animals or h~n. Tw~ or more haptens and at least one ffector group may be linked together in a single molecule by any chemical or enzymatic procedure. Those procedures that result in a uniquely defined chemical st~ucture are preferred. Structures in which the distance between t~o hapten groups may be larger than 25 A are also preferred. An example of suitable chemical structure is provided by peptides of small molecular weight whose side chains and terminal amino and/or carboxylate residues are substituted by the haptens and -the effector groups. Peptides which contain one or several D-amino acids are preferred. Preferred chemical structure is one which allows radiolabeling with a radioisotope suitable for radioimmunoimaging or radioimmunotherapy. ~lost preferred structures are those in which a phenol or phenyl group is present. In that case, labeling may be performed with radioactive isotopes of the halogens such as, for le 18F 76Br 77Br 123I 125I, 131I, 211 At. In that case too, preferred radioactive isotopes are I for diagnosis and 131I or 211At for therapy. Other most preferred structures are those in which one or several chelating group have been introduced. In -that case, a radioactive metal cation may be used as a label, such as, for 57C 67Ga 68Ga 67Cu 90Y, 97Ru, 99mTc, In, In, 203pb~ 212Bi. In tha-t case too~ preferred radioactive isotopes are 111ln or 99~c for diagnosis and 9 Y or 1 Bi for therapyO

.

~ 3~
10.
In another a.spect of the invention, a stable paramagnetic ion, such as Gd, Fe, Mn, may be used. Another suitable structure is one which carries one or several paramagnetic compounds such as, for instance, paramagnetlc ions (e.g. Gd, Fe, or other heavy metals), or S stable free radicals ~e.g. derivatives of the nitroxide radical).
Superparamagnetic complexes such as those produced by precipitation of magnetite in the presence of dextran are also suitable (Ohgushi et al.)~
Alternatively, the radiolabeled or paramagnetic moiety of the tracer may serve as a hapten, as, for instance, when a chelating agent is used to bind 111In, 99mTc, or a stable paramagnetic metal such as Gd, or any other metal isotope. In that case, two or more chelated metals should be included into the tracer molecule.
In all cases, the radicactive or stable isotope may be introduced after the synthesis of the non-labeled tracer has ~een ccmpleted, as in the case of tyrosine radioiodination or radioisotopic metal chelation, or before.
Still another sui~able structure is one which associates in the same m~lecule two or more haptens and one or several molecules of a cytotoxic drug or toxin. Preferred cytotoxic drug æe methotrexate, a deriva-ti~e of the antitumor Vinca alkaloids, or of the platinum complexes, or of the anthracycline. A suitable -toxin may be a plant or bacterial toxin or its separated toxic A chain, such as diphtheria toxin, ricin, abrin, gelonin, or poke-weed antiviral protein.
. .
\\

... ..

3~
11 .
A preferred hapte3l is the dinitrophenyl gro~. A suitable affinity enhancement probe is represented by the following chemical structure :
~ ~ 2 NO
?I~
~ NO2 (,CH2)4 O N ~ -?~H-tCH2)~-CH-NH-C C~l2-N-(CH212 N (C~2)2 , ~ "
COO~ C~2 C~2 C~2 COO~
COO~ COOH COOH

15 Another suitable affinity enhancement pro~e is represented by the following chemical StrlCtU~e ~H

20 N02 ~ lH2 O2N ~ NH-tcH2)4-c-NH-c~-c-NH-c~-cooH

O O ~CH2)4 N2 ~H-c-icH2~4`N~ ~ -N2 A preferred procedure for the use of the reagents of the present invention is intravenous injection of a suitable dose of dual specificity conjugate, together with, or followed after a time delay of a few minutes -to several hours by, injection of the radioactive affinity enhancement probe at a dose adjusted to allow detection of the target cells by the imaging device, or killing of the target cells.
The imaging device may be either a detector of radioactivity available ~"~
t in the art, or a magnetic resonance imaging apparatus also available in the art. In immunotherapy applications of the present invention, the cytotoxic effector group, ~lich may be a radioactive nucleide, a drug or a toxin, will locali7e onto the target cells and exert its action with or without being internali~ed by the said target cells.
The preferred time delay between injection of the dual specificity conjugate and of the affinity enhancement probe may be adjusted to allow localization of the dual specificity conjugate at the target site and partial clearan oe of the excess. One of the major advantage of the present invention over techniques according to previous art, is that clearance of the excess dual specificity conjugate is not required prior injection of the tracer. Accordingly, this time delay may be very short. For radioimmunodiagnostic applica-tions, imaging may be performed a few hours after injection of the affinity enhancement probe, at a time when optimum localization has been achieved. This time may be selected according to the pharmacc-kinetic properties of the affinity enhancement probe, the radioactive decay of the isotopes and the rate at which the affinity enhancement probe is able to localize at the specific targe~ sites. As mentioned ab~ve, the affinity enhancement system is particularly useful ~hen the radioactive isotope has fast radioactive decay.
Preparation of several embodiments of the present invention will be more particularly described hereafter.

SYNTHESIS OF THREE DUAL SPECIFICIIY CONJUG~TES
(COMPOUNDS 1 TO 3) Pu ication of _onoclonal antibodies I~e anti-CALLA ant~tody, a mouse monoclonal IgG1, clone ALB1, the anti-CD5 antibody, a mouse monoclonal IgG2a, clone BL1a, the anti-Lyb8.2 antibody, a mouse mon w lonal IgG1, clone CY34, and the anti-2,4 dinitrophenyl (DNP) antibody, a mouse monoclonal IgG2a, clone U7-27, are purified from ascites fluid by affinity chromatography on protein A-Sepharose (Pharmacia).
* Trade Mark 13.
Preparation of F (ab'~2 fragments q~he anti-CALLA antibody (clone PIB1, 2 to 5 mg/ml) is dialyzed onceagainst 10 mM formate buffer pH 2.8, then onceagainst 50 mM acetate buffer pH 4.2. Pepsin IcrYstallized x2, Calbiochem), 5 % w/w, is then added and allowed to react for 2 h at 37C (Lamoyi and Nisonoff). The mixture is fractionated by ion-exchange chromato-graphy on a Mono S column (Pha~macia), equilibrated with 50 mM
acetate buffer pH 4.5. Elution is obtained with a linear gradient of NaCl (0 to 0.6 M). The F(ab')2 fragment of the anti-Lyb8.2 antibody (clone CY34) is prepared according to the same technique. The anti-CD5 (clone BL1a) and anti-DNP (clone U7.27~ antibodies are dialyzed against 10 ~M acetate buffer pH 3.8 and 5 % (w/w) pepsin is added. Digestion is performed over 5 h at 37C and stopped by raising the pH to 8 with 2M Tris-HCL buffer pH 8.5. The F(ab')2 fra~ments are separated from intact IgG and Fc fragments by affinity chromatography on protein A-Sepharose.
Fab' fragment of the anti-DNP antibody The F(abl)2 fragme,nt of the anti-DNP antibody is dialyzed against 0.1 M phosphate buffer pH 6.0 and cysteamine is added to a final ooncentration of 10 mM. After 1 h at 37C, the resulting Fab' frag~ent is purified by gel filtration on a TSK 3000 SW HPLC colum ~LKB) equilibrate,d with 0.1 M phosphate buffer pH 6.0 supplemented with 5 mM EDTA.

Derivatization with ~SMCC
... ...
To the F(ab')2 fragments (2 to 5 mg/ml) is added a 10 fold molar e,xcess of SMCC (10 mg/ml in dimethyl-form~ide) and the mixture is incubated for 1.5 h at 30C. After centrifugation (2 min, 10,000 g), the mixture is made free of excess SMCC by gel filtration on a PD10 column (Pharmacia) equilibrated with 0.l M
phosphate buffer pH 6Ø

14.
Preparation of the anti-CD5-anti-DNP dual specificity conjugate (campound 'I) The anti-DNP Fab' and the SMCC-derivatized anti-CD5 F(ab')2 æe mixed in a 2 to 1 molar ratio. The mixture is concentrated by ultrafiltration under positive pressure and allowed to react at 4C
for 24 h. rme dual specificity conjugate is separated from unreacted F(ab')2 and Fab' by gel filtration on a TSK 3000 SW column in 0.1 M
phosphate buffer pH 6Ø The fraction corresponding to an apparent molecular weight around 150,000 Da is collected and stored at -20C
(oompound 1). Conjugates between the F(ab')2 fragments of the anti-CALLA or the anti-Lyb8.2 antikcdies and the Fab' fragment of the anti-DNP antibody have keen prepared according to the same technique (compound 2 and 3 respectively).

SYNTHESIS OF CHEL~TING D~ERIC AND MON0MERIC PROB~S
(COMæOUNDS 4 AND 5) Synthesis of bis-(N-C~(2,4-dinitrophenyl)-L-Lysyl) diethylenetriamine-pentaacetic acid (com To a solution of DNP-Lysine (1.2 mmol in 10 ml of a 1:1 mixture of 100 mM borate pH 8.5 and dimethylformamide) are added 0.6 mmol of DTPA cyclic anhydride. The mixture is allowed to react overnight at room temperature, and evaporated to dryness under reduced pressure. The residue is redissolved in 10 ml of water and excess HCl (1~) is added. The precipitate is redissolved in water by addition of NaOH (1M) and further purified by ion exchange chrcmatography on an FPLC mono Q (Pharmacia) column. Compound 4 has an rF of 0.15 by thin layer chromatography (TLC) on silica gel in n-butanol:acetic acid:water (4:1:1). Some (N-~-t2,4-dinitrophenyl)-L,Lysyl)-diethylene-triamine-pentaacetic acid (compound 5) is formed as a by-product (rF = 0.05). Both products have ~een further purified by HPLC on a C18 reverse phase column using a 1:1 mixture of 0,05 % trifluoroacetic acid in water and methanol. Compound 4 and 5 ha~e been shown to be able to chelate 111In by TLC on silica gel in methanol:10 % ammonium acetate (1:1).

J.
15.

SYNI~ESIS OF RADIOIODINAI~D DIMERIC ~ND ~ONOMERIC PROBES
(COMæOUND 6 AND 7) Synthesis of N-hydroxysuccinLmlde 2,4-dinitro~phen~l-aminocaproate 2,4-dinitrophenyl-aminocaproic acid (300 mg) is dissolved in 6 ml of dioxane and N-hydro~ysuccinimide, 120 mg in 4 ml of ethyl acetate, is added. N,N'-dicyclohexyl-carbodiimide, 200 mg in 2 ml of dioxane, is added, and the mixture is allowed to react for 3 h at room temparture. The precipitate which forms during the reaction is filtered off and the solution is evaporated to dryness under reduced pressure. The yellow residue is crystallized in boiling absolute ethanol. The yellow needles of the N-hydroxysuccinimide ester are collected and dried (Rf = 0.59 by TLC in chloroform-ethyl acetake, 1:1 on silica gel3.
Synthesis of N~-(2,4-dinitrophenyl-aminocaproyl)-I~tyrosyl-N-~-(2,4-dinitrophenxl-aminocaproyl)-L,lysine (ccmpound 6~
L,tyrosyl-J.-lysine (10 mg) is dissolved in 1 ml of 50 mM Hepes buffer pH 8.0, a three fold molar excess of N-hydroxy-succinimide 2,4-dinitrophenyl-aminocaproate in 2 ml of dioxane is added, and the mlxture is allowed to react for 16 h at room temperature. After lyophilization, the mixture is redissolved in 1 ml of water and acidified with 1 N HCl. The precipitate is crystallized in boiling absolute ethanol (Rf = 0.80 in n-b~ltanol/acetic acid/water 4/1/1). Further purification of compound 6 is obtain~d by HPLC on a C18 ultrosphere ODS column with 60 ~ methanol, 40 ~ trif1uoroacetic acid (0.05 % in water).
Iodination o~ compound 6 :
Compound 6 (2 nmol) is dissolved in 100 ~l of 50 mM
phosphate 150 ~M NaCl buffer pH 7.3 supplemen-ted with 20 ~ e-thanol ~ 3~

16.

and transferred into a small plastic tube containing 10 ~g of Iodogen. Na131I (1 mCi) is added and the reac-tio~ is cont mued for 30 m~. at ro~m temperature~ The monoiodo-derivative of compound 6 is purified by HPLC on C18 column with 70 % methanol, 30 % trifluoroacetic acid (0,05 % in water~.
Synthesis of N ~-(2,4-dinitrophenyl-amlnocaproyl)-L-tyrosyl-glycine (compound 7) L-tyrosyl-glycine (10 mg) is dissolved in 1 ~1 of 50 mM Hepes buffer pH 8.0, a 1.5 fold molar excess of N-hydroxy-succinimide 2,4-dinitrophenyl-a~inocaproate in 2 ml of dioxane is added, and the mixture is allowed to react for 16 h at roan temperature. After lyophilization, the mixture is redissolved in 1 ml of water and acidified with 1 N HCl. The precipitate 15 (compound 7, Rf = 0.72 in n-butanol/acetic acid/water 4/1/13 i5 purified by HPLC on a C18 ultrosphere ODS column with 60 % methanol, 40 % trifluoroacetic acid (0.05 % in water).
Iodination of compound 7 Ccmpound 7 is radiolabeled under the same conditions as compound 6. Purification of the monoiodo derivative is perfo i by HPLC using the same column but with 50 ~ methanol, 50 % trifluoro-acetic acid (0.05 ~ in water).
E~MPLE
A) - SPECIFIC BINDING OF THE AFFINITY EN~A~CEMENr PROBE TO TARGET
CELLS IN VITRO rN THE P~ESENCE OF EXCESS DUAL
SPECIFICITY CONJUGATE
Experimental conditions In an Eppendorf plas-tic tube are incubated 100 ~l of a cell suspension at 3.10 cells/ml of a human ~-cell line (HPBALL) or of a human B ly~hona line (Namalwa) in phospha-te buffered saline supple~ented with 0.1 % bovine serum alb~nin, 0,02 % azide and 50 n~ deoxycJlucose, 100 ~l of a solutlon of dua ? ~
'~: ' ` ' `' `

~7 specificity conjugate, anti-CD5 ~compound 1) or anti-CALL~ ~compound 2), at 0.5 ~g/ml in the same buffer and 150 ~l of radioiodinated compound or compound 7. After 3 h at 4C or 37C with agitation, 100 ~l of the rèsulting cell suspension are transferred into triplicate 0.4 ml plastlc tubes containing 200 ~l of a 1.2:1 ~ixture of dibutyl-phthalate and ethyl-hexyl-phthalate. After 30 sec. centrifu~ation at 10,000 g, 50 ~l of the supernatant are collected and counted, and the bottom of the tube, containing the cell pellet, is cut and counted.
Results are then expressed as bound/free percentages.
Results The experimental settings allo~ cross-controlled experiments, since HPBALL cells express the T-cell mar~er CD5, but not the tumor-associated antigen CALLA, whereas Namalwa cells express CALLA, but not CD5.

8Oun~/free (%) Dimer Monomer Cell Temperature I1~62+6 SB I 1+7 2+7 S3 HP~LL 4-C I 72 16 56 I 38 3 35 ~1+,2-~37-C I 67 15 52 } 12 3 9 Namalwa 4cC I 2 23 21 I 2 6 4 ~1-,2-~)37~C I 4 21 17 I 1 5 ~I
~SB = Specific ~lnding) B) SPECIFIC BINDING OF THE AFFINITY ~HA~ ENT PROBE TO TAROET
CELLS IN VIT~O AFTER WASHING THE EXCESS DUAL
SPECIFICITY CONJUGATE
Experimental conditions.
The experimental conditions are similar to that described above but the dual specificit~ conjugate is incubated first for 1 h, then the cells æe washed 3 times and the tracer is added. The cells are then pelleted after another 2 h incubation.

Results und/free (X) Dimer Monomer Cell Temperature I 1+6 2+6 SB I ~7 2~7 SB
---------I----_________ I---------___ ~___I
HPBALL 37-C I 45 9 36 I 11' 3 7 Namalwa 4~C I 15 145 130 I B 50 42 -,2+j 37 C I 4 11 7 I 2 4 2 ~SB = Speclflc Bindlng) EX~ME~LE S
Compound 4 and the monomeric analogue, compound 5, (0.2 nmol) have been labeled with 0.2 mCi of 111 In chloride by incubation at room tem~erature in 0.1 M citrate buffer for 30 m m.
The mixture is then diluted into phosphate buffered saline and used without further manipulation. After incubation of freshly isolated BALB/c mouse spleen cells (10 cells/ml) at 37C for 2 h in the presence of anti-Lyb8.2-anti-DNP conjugate ~ccmpound 3) at 3.10 M, binding of the labeled cGmpounds 4 and 5 was monitored as described in example 4. Vnder these conditions, 26 % (bound/free) of labeled comFound 4 became bound to the mouse spleen cells (of which about 70 ~ are Lyb8.2 positive), as opposed to only 6 % (bound/free) of the moncmeric tracer (ccmpound 5). In the absence of conjugate, the non-specific binding of the labeled tracers was about 0.2 %.
Examples 4 and 5 demonstrate that :
1) The tracers (affinity enhancement probes), as defined in the present invention (compounds 4 and 6), become bound to the target cells at either 4C and 37C, provided that the cell have been preincubated with the specific dual specificity conjugate (in the experiments presented cibove : anti-CD5 for HPBALL cells, anti-CALLA
for Namalwa cells anti-Lyb8.2 for BALB/c mouse spleen cellsl 2) The presence of excess specific dual specificity conjugate does not prevent specific binding of the affinity enhancement probes (compounds 4 and 6).

iO~

3) Tracers (compounds 5 and 7) prepared with the same hapten DNP and radiolabeled to the same specific activity, but presenting a single hapten group exhi'bit much less specific binding to -the target cells in these assays, especially at 37C.

4) Compara'ble results are obtained using radio-iodinated tracers (co~pounds 6 and 7) and tracers derived from a chelating agent with a bound radioactive metal, 111In, ~compounds ~ and 5~.

The eff'ect described above cannot'be taken as a particular 'behavior of the experimental system selected as an example. On the contrary, the target cells express low amounts of tæget antigen at their membrane (2.104 CALLA antigens for Namalwa cells, 4.105 CD5 antigens Eor HP~LL cells, and 3.104 Lyb8.2 antigens for BALB/c mouse spleen cells). Most tumor or normal cells that would be selected as targets for in vivo diagnostic or therapeutic applications will express at least similar amounts of target antigen. The excellent results obtained with the affinity enhancement probes demonstrate that the affinity enhancement system will be extremely advantageous in such applications.
While certain specific ~mbodlm~nts have been disclosed in the foregoing description, it will be understood that various modifications within the scope of the invention may occur to those skilled in the art. merefore, it is intended that adoptions and modifications should and are intended to be comprehended within the scope of th~ appended claims.

.

.

f ~, . . .
.~. .. ;, I

20.
REFEæENCES CITED
Patent documents Hansen H.J., Prinus F.J., Localization of tumors by radiolakelled antibodies (1975), US patent n 3,927,193.
Adams T.H., David, G.S., Halpern S.E., Anticorps noclonaux marg~lés par un radionucl~ide et leur application ~ la visualisation d'une tumeur (1982), French Patent No. 2,515,046.
Gansow O.A., Strand M., Metal chelate conjugated monoclonal 10 antibcdies (1982), US Patent No. 4,472,509.
Gansow O~A., Strand M., Use of metal chelate conjugated antibodies (1982), US Patent n 4,454,106.
.~eares C.F., David, G.S., Monoclonal antibodies against 15 metal chelates (1984), International Patent No. WO 86/01407.
Reading C.L., Recombinant Monoclonal Antibodies (1984), US Patent No. 4,474,893.
Martinis J., Bartholomew R~M., Davud G.S.~ Adams T.H., Fincke J.M., Antibodies having dual specificities, their preparation 20 and use therefor ~1983), International Patent N~ WO 83/03679.
Other publications Kohler G., Milstein C., Continuous cultures of fused cells secreting antikodies of pre-defined specificity (1975)l Nature, 25 256, 496-497.
Gatter K.C., Alcock C., Heryet A., Pulgord K.A., Heyderman E., Taylor-Papadimitriou J., Stein H., Mason D.Y., The diEferential diagnosis oE routinely processed anaplastic tumors using monoclonal antibodies (1984), Amer. J. Clin. Pathol., 82, 33-43 Bradwell A.R., Fairweather D.S., Dykes P.W., Keeling A., Vaughn A., Taylor J~ Limiting factors in localization oE tumours with radiolabeled antibodies (1985), Immunol. Today, 6, 163-170~

Reardan D.T., Meares C.F., Goodwin D.A., ~ rigue M.
David G.S., S-tone M.R., Leung L.P., Bartholomew R.M., Frerich J.M., Antibcdies against metal chelates (1985), Nature, 316, 265-268.
Hunter W.N., Greenwcod F.C., Preparation of io~ine131 s labelled growth hormone of high specific activity (1962), Nature, 94, 495-496.
Hnatowich D.J., Layne W.W., Chids R.L., Lanteigne D., Davis M.A., Radioactive labelling of antibcdy : a simple and efficient method (1983), Science, 220, 613-615.
Goodwin D.A., Antibody-hapten complexes for in~aging (1986), Nato Advanced Study Institute "Radio-labeled monoclonal antibodies for imaging and therapy", p. 9.
La~.oyi E., Nisonoff A., Preparation of F(ab')2 fragments 15 from IgG of various subclasses (1983), J. Immunol. Methods, 56, 235-243.
Unger, E.C., et al. Magnetic resonance imaging using gadolinium labeled monoclonal antibody 11985), Invest. Radiol., 20, 693-700.
Curtet, C., et al. Selective modification of NMR relaxation time in human colorectal carcinoma by using gadolinium~diethylene-triamlnepentaacetic acid conjugated with noclonal antibody 19-3.
(1986) Proc. Natl. Acad. Sci. USA, 83, 4277-4281.
Le Pecq J.B., Le Bret M., Barbet J., Rcques B.P. DNA poly-intercalating drugs: DN~ binding of diacridine derivatives (1975), Proc.
Natl. ~cad. Sci. USA, 72, 2915-2919.
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Claims

1. Immunological reagents, comprising :
a) A monoclonal antibody or fragment with binding affinity for a desired antigen, conjugated to a monoclonal antibody or fragment with binding affinity for a desired hapten ;
b) A synthetic molecule constituted by - At least two haptens, which all have binding affinity for the above conjugate ;
- At least one site suitable for radiolabeling, or for labeling with a stable paramagnetic metal, or to covalently couple a drug or a toxin ;
- A chemical structure, which covalently links said haptens and said site; it being understood that the chemical structure cannot be a polymer.

2. Immunological reagents, comprising :
a) A mixture of two different conjugates, where each conjugate, as in claim 1a, has binding affinities to a desired antigen and to a desired hapten ;
b) A synthetic molecule constituted by :
- two different haptens, where each hapten has binding affinity to one of the two conjugates of said mixture ;
- at least one site suitable for radiolabeling, or for labeling with a stable paramagnetic metal, or to covalently couple, a drug or a toxin ;
- a chemical structure, which covalently links these functional groups.

3. Immunological reagents according to claim 1, wherein the monoclonal antibodies are mouse antibodies.

4. Immunological reagents according to claim 2, wherein the monoclonal antibodies are mouse antibodies.

5. Immunological reagents according to claim 3, wherein the monoclonal antibodies are of IgG class.

23.

6. Immunological reagents according to claim 4, wherein the monoclonal antibodies are of IgG class.

7. Immunological reagents according to claim 5, wherein the fragments are Fab'2 or Fab' or Fab.

8. Immunological reagents according to claim 6, wherein the fragments are Fab'2 or Fab' or Fab.

9. Immunological reagents according to any one of claims l, 3, 5 or 7, wherein the hapten is a derivative of 2,4-dinitrophenol.

10. Immunological reagents according to any one of claims 2, 4, 6 or 8, wherein one of the hapten is a derivative of 2,4-dinitrophenol.

11. Immunological reagents according to any one of claims l, 3, 5 or 7, wherein the dissociation constant of the binding interaction beetween the conjugate and the hapten is in the range from 10-9 to 10-7 Min physiological conditions.

12. Immunological reagents according to any one of claims 2, 4, 6 or 8, wherein the dissociation constant of the binding interaction between each conjugate and its specific hapten is in the range from 10-9 to 10-7 M in physiological conditions.

13. Immunological reagents according to claim 1, wherein the synthetic molecule contains a chelating agent suitable for labelling with metal cations.

14. Immunological reagents according to any one of claims 2, 4, 6 or 8, wherein the synthetic molecule contains a chelating agent suitable for labelling with metal cations.

24.

15. Immunological reagents according to claim 13, wherein the synthetic molecule is represented by the structure :

16. Immunological reagents according to claim 2, 4, 6 or 8, wherein the synthetic molecule contains a phenol group suitable for labeling with halogens.

17. Immunological reagents according to claim 1, 3, 5 or 7, wherein the synthetic molecule contains a phenol group suitable for labeling with halogens.

18. Immunological reagents according to claim 2, wherein the linking chemical structure is a peptide.

19. Immunological reagents according to claim 1, wherein the linking chemical structure is a peptide.

20. Immunological reagents according to claim 19, wherein the synthetic molecule is represented by the structure :

25.

21. Immunological reagents according to claim 19, wherein the peptide contains one or several D-amino acid.

22. Immunological reagents according to claim 18, wherein the peptide contains one or several D-amino acid.

23. Immunological reagents according to claim 1, 3, 5 or 7, wherein the labeled sites are the haptens so that the conjugate of claim 1a) has more binding affinity for the labeled synthetic molecule than for the unlabeled one.

24. Immunological reagents according to claim 2, 4, 6 or 8, wherein one labeled site is one of the two haptens so that one conjugate in the mixture of claim 2a) has more binding affinity for the labeled hapten than for the unlabeled one.

25. Immunological reagents accord m g to claim 1, wherein the desired antigen is a tumor-associated antigen.

26. Immunological reagents according to claim 1, wherein the desired antigen is a cell-associated antigen.

27. Immunological reagents according to claim 1, wherein the desired antigen is a tissue-associated antigen.

28. Immunological reagents according to claim 2, wherein one desired antigen is a tumor-associated antigen and the other one a tumor or a cell or a tissue-associated antigen.

29. Immunological reagents according to claim 25 or 28, wherein the tumor is melanoma or lymphoma, or breast or lung or colorectal tumor .

30. Immunological reagents according to claim 26 or 28, wherein the cell-associated antigen is myosin or villin.

31. Immunological reagents according to claim 27 or 28, wherein the tissue-associated antigen is fibrin.

32. Immunological reagents according to claim 1, for the manufacture of a diagnostic agent for targeting desired cells in vivo.

33. Immunological reagents according to claim 32, wherein the synthetic molecule is labeled with a suitable radioisotope whose radio-activity is detected.

34. Immunological reagents according to claim 33, wherein the radioisotope is 18F, 76Br, 77Br, 123I, 125I, 131I, 211At, 57Co, 67Ga, 68Ga, 67Cu, 90Y, 97Ru, 99mTc, 111In, 113mIn, 203Pb or 212Bi.

35. Immunological reagents according to claim 1, characterized in that, the synthetic molecule is labelled with paramagnetic metal ions.

36. Immunological reagents according to claim 35, wherein paramagnetic metal is Gd, Mn or Fe.

37. Immunological reagents according to claim 33, wherein the isotope is 131I, 211At, 90Y or 212Bi.

38. Immunological reagents according to claim 1, characterized by coupling one or several molecules of a cytotoxic drug, or one or several molecules of a bacterial or plant toxin, to the synthetic molecule.

39. Immunological reagents according to claim 38 wherein the drug is methotrexate, or a derivative of the anti-tumor Vinca alkaloids, or platinum complexes, or anthra-cyclines, or a combination of any of them.

40. Immunological reagents according to claim 39, wherein the toxin is ricin, or abrin, or gelonin, or poke-weed antiviral protein, or diphtheria toxin, or a com-bination of any of them.