AU677006B2 - Process for preparing macrocyclic chelating agents and formation of chelates and conjugates thereof - Google Patents

Description

11 zno /'13

PCI'

ANNOUNCEMENr OF THE LATER PUBLICATION OFINTERNATOAL SWACH REPORTS I NTERNATIONAL APPLICATION PUBISHIED UNDER THE P3IATE3NT COOPERATION TREATY (I'CT.

(51) International Patent Classification A3: (Ui) Interntional Publication Number: WO 93/20852 A61K 49/02 (43) International Publication Date: 28 October 1993 (28,10,93) (21) International Application Number:- (22) lIternational Filing Date: Priority data: 07/868,078 13 April I PCT/US93/03483 13 Apri1 1993 (13.04,93) 992 (13.04.92) (71) Applicant: THE DOW CHEMICAL COMPANY [US/ US]; 2030 Dow Center, Abbott Road, Midland, Ml 48640 (US).

(72) Inventors: CHENG, Roberta, C. 3873 Old Pine Trail, Midland, MI 48642 FORDYCE, William, A,.

4511 Concord Street, Midland, MI 48642 KRU- PER, William, Jr. ;230 B~arden Road, Sanford, MI 48657 LITWINSKI, George, R, 1911 Ardmore, Midland, Ml 48642 (US).

(74)Agent.: ULMER, Duane, The Dow Chemical Corn.

pany, Patent [)apartment, P.O. B~ox 1967, Midland, MI 48641-19-,' (US).

(81) Designated States., AU, CA, JP, NO, European patent (AT, BE1, CHi, DI:, DK, E-S, FR, 013, Gil, 113, IT, LU, MC, NL. PT, SE).

aniendiiu'as (88) D~ate of publiclltton of the interniatlinl se.arch report: 25 Novem~ber 1993 (25.11.93) (54)Title: MACROCYCLIC CHELATING AGENTS, CHELATES AND CONJUGATES THEREOF (57) Abstract The present invention is directed to an improved process for preparing macrocyclic chelating agents and for conjugating thc macrocyclic chelating agents to biological molecules, I~Rsllr~llll~-~- 31~ 11111 1 il.-l WO 93/20852 PCT/US93/03483 PROCESS FOR PREPARING MACROCYCLIC CHELATING AGENTS AND FORMATION OF CHELATES AND CONJUGATES THi-REOF The p:sent invention relates to a process for preparing isothiocyanato functionalized macrocyclic chelating agents and to a process for conjugating the macrocyclic chelating agents to biological molecules.

Metal ions may be attached to biological molecules by means of bifunctional chelating agents. Such chelating agents are compounds which contain a metal-binding moiety which forms a chelate with metal ions and a second functional group, which is chemically reactive in nature and is capable of forming a covalent bond with biological molecules. The rea tive functionality is usually ine of the various known useful chemically reactive groups such as bromoacetyl group, a diazonium ion, an isothiocyanate or a carboxylic acid derivative, the reactive functionalities being capable of binding to an amino acid of a protein the lysine moiety of an antibody). The biological molecules usually recognize distinctive external or internal cell markers and, thus, act as target directing groups for the metal ion.

When the bifunctional chelating agent is covalently attached to an antibody having specificity for cancer or tumor cell epitopes or antigens, radionuclide chelates of such antibody/chelating agent conjugates are useful in diagnostic and/or therapeutic applications as a means of conveying the radionuclide to a cancer or tumor cell. See, for example, Meares et al., Anal. Biochem., 2, 68-78 (1984); and Krejcarek et al., Biochem. and Biophys.

Res. Comm., 77 581-585 (1977).

Isothiocyanato functionalized derivatives of ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) are reported in the literature and are being used to conjugate radioactive isotopes to antibodies. For example see Gansow et al., Inorg. Chem., 252772-2781 (1986); Meares et al., Anal. Biochem., 142 68-78(1984); and U.S.

Patent 4,454,106.

When using short-lived radionuclides, it is desirable to chelate the radionuclide to the target dci acting group as close as possible to the time of injection into the patient to provide maximum specific radioactivity and minimum degradation of the radioimmunoglobulin. When using chelating agents such as EDTA and DTPA, the rapid sequestration of metal ions by these chelating agents allows the preparation of the radionuclide/antibody/chelating agent conjugates (referred to herein as conjugates) to be prepared by activating the chelating agent, reacting the chelating agent with an antibody and then chelating the radionuclide to form a complex followed by purification of the complex.

However, although chelating agents such as EDTA and DTPA rapidly chelate the radionuclide, they suffer from the disadvantage that such binding is kinetically labile. In addition, the use of labile radionuclides for antibody labeling in this manner allows substitutionally labile trace metals (which may not be radioactive) to be incorporated into the ~-a WO 93/20852 I'Cr/US93/03483 chelate. Competition for such non-active trace metals diminishes the biological efficacy of the antibody/chelate complex since a lower quantity of radionuclide is delivered to the target site.

A disadvantage associated with chelating agents such as ED1M and DTPA is the premature release of the chelated radioactive isotope. To lower the rate of metal release in vivo, bifunctional chelating agents, based on r.macrocyclic ligands, such as DOTA (1,4,7,10-tetraazacyclododecane-N,N',",N"'-tetraacetic acid), have been used. See, for example, Mirzadeh et al., Bioconjugate Chem., 1, 59-65 (1990); and Deshoande et ol., J. Nucl.

Med., 31, 473-479 (1990).

A disadvantage in the use of macrocyclic chelating agents is the relatively slow chelation rates which take place at room temperature, see for example, Mirzadeh et al,, Bioconjugate Chem., 1, 59-65 (1990). To reduce the radiolysis which occurs with prolonged chelation times, the medium in which the chelation takes place is often heated to increase the rate of chelation. As the isothiocyanate functionality is heat sensitive, U.S. Patent 5,006,643 discloses the preparation of isocyanato functionalized macrocycles containing primary or secondary amines by first reacting the chelating agent with rhodium and then activating the chelate. U.S. Patent 4,885,363 discloses chelating gadolinium to a tetraazamacrocycle containing tertiary amines prior to activating with an isothiocyanate functional group. Using this procedure for preparation of metal chelate-proteiil conjugates to be used in radioimmunotherapy or radiodiagnostics requires the formation of the chelate, activation of the chelate and then conjugation of the activated chelate to an antibody in a short period of time to avoid a substantial loss in activity.

Another disadvantage in the use of macrocyclic chelating agents is that during their synthesis and purification, the reagents and materials used must be free of extraneous r:'ptal ions. Metal :on contaminants present during these steps may become tightly bound to the macrocycles and not easily displaced during chelation of the desired metal ion. Metal ion contamination during synthesis and purification will reduce the purity of the product obtained during chelatio, of the desired metal ion as well as reduce the purity of product obtained in subsequent steps in producing the conjugate.

Consequently, it would be advantageous to have a process to prepare purified macrocyc.ic chelating agents which are substantially pure of contaminating metal ions.

It would also be advantageous to have a process to prepare the isothiocyanate activated chelating agent in an aqueous environment with an increased yield and higher purity than can oe prepared when the reaction is performed in an organic solvent/water environment.

It would be desirable to provide a chelating agent which will react rapid'y with a adionuclide at room temperature forming a chelate which does not readily dissociate, allowing the chelating agent to be activated to an isothiocyanate prior to forming the chelate.

Activating the chelating agent prior to chelation of the metal ion allows the bifunctional M1~9~12s1~88slltl UI~ ~XY IP- -P WO 93/20852 PCT/US93/03483 chelating agent to be prepared in an activated form in advance of when it is needed to produce a conjugate.

It would be further advantageous to have a process which allows rapid conjugation of the chelate with an antibody to reduce radiolysis and antibody aggregate formation. Rapid formation of the conjugate with a reduction in undesired reactions would result in a more complete conjugation reaction and result in easier product purification. Als', having a process wherein the yield and purity from each step is increased is particularly advantageous in preparing compounds and compositions which will be used in pnarmaceutical applications where the pharmaceutical composition must meet the purity criteria set-forth by the U. S. Food and Drug Administration.

The present invention provides a chromatographic process for prevention of extraneous metal ion incorporation in a macrocyclic chelating agent during purification. The process is advantageous over existing procedures in that multigram quantities of macrocyclic chelating agent free of divalent cations can be produced and purified in a single unit operation.

The invention also provides a process for preparing isothiocyanate compounds which comprises reacting thiophosgene with a polyaza chelating agent in an aqueous environment in the absence of an organic solvent to form an isothiocyanato derivatized polyaza chelating agent, wherein the polyaza chelating agent is of the formula

Q

R

3 X Q 1 2

N-Q

C (CH2)- C- (CH2)r n m Y H

N

114 Q

Q

(I)

wherein: each Q is independently hydrogen, (CHR5)pCO 2 R or (CHR5)pPO 3

H

2 Q1 is hydrogen or (CHR5),CO 2

R;

each R independently is hydrogen, benzyl or C1-C 4 alkyl; with the proviso that at least two of the sum of Q and Q1 must be other than hydrogen; each RS independently is hydrogen, C 1

-C

4 alkyl or -(C 1

-C

2 alkyl)phenyl; X and Y are each independently hydrogen or may be taken with an adjacent X and Y to form an additional carbon-carbon bond; n isO or 1; m is an integer from 0 to 10 inclusive; I -as~l laR WO 93/20852 PCT/US93/03483 p 1 or 2; r Oor 1; w or 1; with the proviso that n is only 1 when X and/or Y form an additional carbon-carbon bond and the sum of r and w is 0 or 1; R2 and R 4 are independently hydrogen, or amino; R3 C 1

-C

4 alkoxy, -OCH 2

CO

2 H, hydroxy and hydrogen; with the proviso that R2 and R 4 cannot both be hydrogen but one of R2 and R 4 must be hydrogen.

The present invention also describes a process for preparing a conjugate which comprises reacting an isothiocyanato activated chelate with an antibody at between 25°C and wherein the chelate is a chelating agent complexed with a metal ion, the chelating agent is as described in Formula I and the metal ion is is35m, 166Ho, 175yb, 1 77 Lu, 159Gd, 1 4 0La, 142pr, 1 49 Pm, 90Y or 111In.

The current invention also provides an improved process for formation of a chelate-antibody conjugate wherein the improvement comprises contacting the chelate with an antibody at 25C to about 40 0

C.

The present invention provides an improved process to prepare polyaza metal conjugates. In particular, the present invention is a process for preparing conjugates having at least the steps of: purifying a polyaza chelating agent, the polyaza chelating agent being as defined in Formula I; contacting the purified polyaza chelating agent with thiophosgene to form an isothiocyanato activated polyaza chelating agent and recovery of the isothiocyanato activated polyaza chelating agent; adding a metal ioh to the isothiocyanto activated polyaza chelating agent to form an isothiocyanato activated polyaza chelate; and contacting the isothiocyanato activated polyaza chelate with a biological molecule to form a conjugate; the improvement which comprises: in step purifying the polyaza chelating agent by chromatography on a silica gel column, wherein the silica gel has been acid washed prior to loading the polyaza chelating agent onto the column; in step contacting the purified polyaza chelating agent with thiophosgene in an aqueous environment in the absence of an organic solvent at a pH from 1 to 5; and in step contacting the isothiocyanato activated polyaza metal chelate with a biological molecule at between 25 0 C to 40 0

C.

II~ u r~l IWIIIP~PB~PI WO 93/20852 PCr/US93/03483 As used herein, the terms "chelating agent" or "ligand" mean a compound capable of chelating or sequestering a metal ion. The term "chelate" means a chelating agent which has sequestered a metal ion.

The term "bifunctional chelating agent" refers to compounds that have a moiety capable of chelating a metal ion and a linker/spacer moiety covalently bonded to the chelating moiety that is capable of being activated or functionalized to serve as a means to covalently attach to a biological molecule.

The term "biological molecule" refers to any protein, antibody, antibody fragment, hormone, antigen or hapten which functions to recognize a specific biological target site. Such a biological molecule, when attached to a functionalized chelate, serves to carry the attached metal ion to specific targeted tissues. Preferably, the biological material is an antibody or antibody fragment.

As used herein, "antibody" refers to any polyclonal, monoclonal, chimeric antibody, heteroantibody, or recombinant or derivative thereof, preferably a monoclonal antibody. As used herein the term "antibody fragment" includes Fab fragments and F(ab') 2 fragments and any portion of an antibody, including recombinants and derivatives thereof, having specificity toward a desired epitope or epitopes. The antibody fragments may be produced by conventional enzymatic methods or by genetic or protein engineering techniques, such as the production of single chain antibodies, The terms "activated" or "activating" in relation to a chelating agent mears the chelating agent has been modified with a functional group which is capable of forming a covalent bond with a biological molecule.

The term "conjugate" as used herein refers to a complex of a biological material attached to a bifunctional chelating agent or bifunctional chelate. The term "antibody/chelate conjugate" refers to an antibody which is covalently attached to a bifunctional chelate, the bifunctional chelating agent having a chelated metal icn).

A general overall procedure giving the steps for preparation of conjugates used in the present invention is given in the following Scheme 1. Steps and of the process scheme may be reversed. When sequestering metal ions which are radionuclides with short half lives, it is preferred that the activation occurs before formation of a chelate to avoid radiolysis which would occur during the subsequent activating step and any subsequent purification.

~IIL PqCI IP L~I~J I~ti*dlDlli~PIIIII~-- II- I I WO 93/20852 PCT/US93/03483 Scheme 1 Synthesis of a Chelating Agent Purification of the Chelating Agent on Silica Gel Step (a) Activation of the Chelating Agent Step (b) Chelation of a Metal Ion by the Activated Chelating Agent to Form a Chelate Step (c) Conjugation of the Chelate to a Biological Material to Form a Conjugate Step (d) Purification of the Conjugate -C I WO 93/20852 PCT/US93/03483 The present invention provides a process for reducing extraneous metal ions, specifically calcium ions during the purification of macrocyclic chelating agents by flash chromatography using silica gel. It has been found that commercially available silica gel used to purify the macrocyclic chelating agents unexpectedly contains a source of undesired metal ions, particularly divalent cations such as calcium, which become tightly bound to the chelating agent during purification. The washing of the silica gel with a strong mineral acid, such as hydrochloric, nitric, sulfuric, perchloric or hydrobromic, prior to use substantially reduces the amount of undesired metal ions bound to the chelating agent during the purification process.

Preferably, the mineral acid used to wash the silica gel is hydrochloric acid. Procedures for washing silica gel with a strong acid to remove impurities are known in the art, see, for example, Ralph K. Iler, The Chemistry of Silica, John Wiley Sons (1979).

The elimination of undesired metal ions from the macrocyclic chelating agents during the initial purification results in several advantages in the subsequent steps for forming a conjugate. The elimination of undesired bound metal ions from the macrocyclic chelating agents allows rapid cheiation of the desired metal ions at room temperature as opposed to heating or long reaction times required to displace the calcium. The ability to perform the chelation step at room temperature, rather than at temperatures which would destroy the functionalizig group, allows the activation of the macrocyclic bifunctional chelating agent prior to chelation of the rretal ion. The ability to functionalize the macrocyclic chelating agent prior to chelation of the meta! ion is particularly advantageous as the activated chelating agent can be synthesized, stored and Then sequestration of the metal ion performed shortly before conjugation to the biological material. This is particularly advantageous when the metal ion is a radionuclide with a half lif of 10 days or less, as a significant amount of radiolysis can occur during the time necessary for functionalizing the chelate, conjugation, and purification of the products from each step. Preclusion of undesired metal ions is particularly important in insuring rapid uptake of radioactive metals of the lanthanide (111) series.

The ability to rapidly chelate the radionuclide after activation of the chelating agent also allows for simpler and more efficient purification procedures to be used in preparation of the conjugate.

The present process for purifying bifunctional chelating agents can be used for many classes of ligands including any tri to hexa -(carboxylated), -(phosphonomethylated), or -(phosphinomethylated) polyazamacrocycle where the polyazamacrocycle has 3 to 6 nitrogens in the ring and the total number of atoms in the ring is 9 to 24 atoms. Of particular interest are aminocarboxylic acid chelating agents, aminophosphonic chelating agents and polyaza chelating agents containing secondary and tertiary amines. Preferred polyazamacrocyclic chelating agents are of the formula WO 93/20852 PCT/US93/03483

Q

R

3 R2 N Q 1 N

H

2 C (CH, (CH2)r N-Q Sn

N

R4

Q

Q

(I)

wherein: each Q is independently hydrogen, (CHR5)pCO 2 R or (CHR5)pPO 3

H

2 Q1 is hydrogen or (CHR5)wCO 2

R;

each R independently is hydrogen, benzyl or C 1

-C

4 alkyl; with the proviso that that at least two of the sum of Q and Q1 must be other than hydrogen; each Rs independently is hydrogen, C 1

-C

4 alkyl or-(C 1

-C

2 alkyl)phenyl; X and Y are each independently hydrogen or may be taken with an adjacent X and Y to form an additional carbon-carbon bond; nisOor 1; m is an integer from Oto 10 inclusive; p 1 or 2; r Oor 1; w or 1; with the proviso that n is only 1 when X and/or Y form an additional carbon-carbon bond, and the sum of r and w is 0 or 1;

R

2 and R4 are independently hydrogen, nitro, amino or carboxyl;

R

3 ii C 1

-C

4 alkoxy, -OCH 2

CO

2 H, hydroxy or hydrogen; with the proviso that R 2 and R4 cannot both be hydrogen but one of R 2 and R4 must be hydrogen.

Preferred amines of the polyaza chelating agents of Formula I are tertiary amines, preferably where r is 0 and each Q is (CHRS)CO 2

R.

Procedures for synthesizing polyaza macrocycles are well known in the art.

Examples of preferred chelating agents are given in Table I and are named as follows: I A is 1-(4-aminophe .yi-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; I B is 1-[2-(4-aminophenyl)ethyl] -1,4,7,10-tetraazacyclodode(.ane- -4,7,10-triacetic acid; O aasa WO 93/20852 WO 9320852PCT/US93/03483 I C is 1-[2-(4-aminophenyl)ethyl-1 1 0-tetraazacyclododecane- 1,4,7,1 0-tetraacetic acid; I D is 1 -(5-ami no-2-methoxybenzyl)- 1,4,7,1 0-tetraazacycl ododecane- -1,4,7,10-tetraacetic acid; 1 E is 1 -(5-amino-2-hydroxybenzy)- 1,4,7,1 0-tetraazacyclododecane- -4,7,10-triacetic acid; and F is 1-[2-(4-aminophenyl)ethyll -1,4,7,1 0-tetraazacyclododecane- 1 -(R,S,-aceti c-4,7, 1 O-tri methyl acetic) acid, the preparation of which is given in European Patent Publication No. 0420942, published April 10, 1991.

The preparation of IA-IE is given in European Patent Publication No. 0353450, published February 7, 1990, and European Patent Publication No. 0420942, published April 1991.

3S WO 93/20852 WO 9320852PCr/,US93/03483 TABLE I CO 2H

(-COOH

E111' N COOH O

NJ

LCOCH

/-COCH

'N

COOH

N

N

\-COOH

rcll~~nanrrrram*~~l-r-n~--rr~~~~l F rr- WO 93/20852 PCf/US93/03483 TABLE I CONT'D

H

2

N

COOH

COOH

N COOH

OCH

3

\--COOH

COOH

H, N N O OH

-COOH

w v

-COOH

CH

3 )OH

COOH

IH

N

Methods for the carboxylation of an amine of a ligand to give amine derivatives containing a carboxyalkyl group are well known, as are the methods which give alkyl phosphonic and hydroxyalkyl substituents on the amine nitrogens. [See, for example, U.S.

Patents 3,726,912 and 3,398,198.] Aminophosphonic acid derivatives of ligands can be prepared by a number of known synthetic techniques. Of particular importance is the reaction of a compound containing at least one reactive amine hydrogen with a carbonyl compound (aldehyde or ketone) and phosphorous acid or derivative thereof. (See the procedure of Moeoritzer and Irani, J. Org. Chem., 31 1603 (1966).]

I

-lliBP IIUCI r WO 93/20852 PCT/US93/03483 The polyaza chelating agents of the present invention purified by flash chromatography on acid washed silica gel can be activated with any of the known functional groups capable of forming a covalent bond with a biological molecule. Examples of such functional groups are isothiocyanate, bromoacetamide, maleimide, imidoester, thiophthalamide, diazonium and carboxyl. The use of polyaza chelating agents which are essentially free of undesired metal ions are particularly important when using functionalizing groups which are pH and/or thermally unstable, such as isothiocyanate. The activating functional groups are located at positions R2 or R 4 of Formula I on the macrocyclic chelating agent.

It has been unexpectedly found that when reacting the polyaza chelating agent with thiophosgene to activate the chelating agent with an isothiocyanate group, conducting the reaction in an aqueous environment, preferably in a dilute acid, with vigorous mixing in he absence of an organic solvent results in an activated chelating agent of greater purity than that obtained in the presence of an organic solvent. This is in contrast to the known procedures of using a pH of 8.5 and/or adding the thiophosgene in the presence of an organic solvent. See, for example, Brechbiel et al., Inorg. Chem., 25 2772-2781 (1986); Keana and Mann, J. Org.

Chem., 55, 2868-2871 (1990); Westerberg et al., J. Med. Chem., 32, 236-243 (1989); and Kline et al., Bioconjugate Chem., 2, 26-31 (1991). The reaction of the thiophosgene with the chelating agent is at a pH from 1 to 7. Preferably, the pH is from 1 to 5. More preferably, the pHisfrom 1 to3.

The reaction between the chelating agent and thiophosgene is preferably carried out in an aqueous environment having an acid concentration of from about 0.005 to about normal preferably 0.01 to 0.2 N. The acid can be any mineral acid, preferably hydrochloric acid. The reaction between the chelating agent and thiophosgene in a dilute acid with vigorous mixing and excess thiophosgene is very fast and usually complete in less than minutes at room temperature (15°C to 25 0 Higher or lower temperatures can be used 0°C to 50"C) but room temperature is preferred.

The amount of excess thiophosgene added to the mixture depends on the concentration of the polyaza chelating agent. The lower the concentration of chelating agent, the larger the excess of thiophosgene to insure the rapid and complete conversion of amine to isothiocyanate. For example, if the concentration of chelating agent is 10-3 M, the ratio of thiophosgene to chelating agent is 5-20:1. If the concentration of chelating agent is 10-8 M, the ratio of thiophosgene to chelating agent is several thousand times larger 10s:1). The excess thiophosgene is removed by conventional techniques such as evaporation, chromatography or extraction.- Rapid mixing of thiophosgene with the aqueous solution may be accomplished using conventional equipment known to those in the art which is capable of producing sufficient shear to oroduce dispersion of the thiophosgene within the aqueous solution.

1 P~ 1F- l IPr~ C IP 9 W lllls~ 111111~1~ 1IBLslRIIII Cllr~ WO 93/20852 PCT/US93/03483 Illustrative of such mixing means is the use of a Waring blender for large scale preparations and with a Mixxor" type mixer available from Alltech Inc., for smaller scale preparations.

By conducting the reaction of the chelating agent with thiophosgene as described above, the obtained purity of the isothiocyanato activated chelating agent is about 9U to about 95 percent as measured by high performance liquid chromatography (HPLC), After the chelating agent has been activated, the activated chelating agent is placed in contact with a metal ion to form the chelating agent/metal ion chelate. Although any metal ion, whether a radioactive metal ion or not, can be used which is sequestered by the chelating agent, the chelates formed should have reasonable stability such that the metal complex is not readily disassociated. Radionuclides are preferred because of the use of the resulting products in a radiopharmaceutical drug for therapy and/or diagnosis. Especially preferred radioactive isotopes are those of samarium (Sm-153), holmium (Ho-166), ytterbium (Yb-175), lutetium (Lu-177), gadolinium (Gd-159), lanthanum (La-140), praseodymium (Pr-142), promethium (Pm-149). yttrium (Y-90) and indium (In-111).

Radionuclides can be produced in several ways. In a nuclear reactor a nuclide is bombarded with neutrons to obtain a radionuclide, e.g., Sm-152 neutron Sm-153 gamma Another method of obtaining radionuclides is to bombard nuclides with particles produced by a linear accelerator or a cyclotron. Yet another way is to isolate the radionuclide from a mixture of fission products. The method of obtaining the nuclides employed in the present invention is not critical thereto.

The radionuclides can be complexed with the bifunctional chelating agent by adding the bifunctional chelating agent to a solution of the radionuclide. Chelates form readily upon mixing in an aqueous solution at a pH of 1 to 10. Preferably, the reaction is carried out in a medium having a pH of 1 to 7 and more preferably 5 to 7. Ambient temperatures of about 10°C to 40°C can be readily employed for metal ion chelation. The amount of metal ion employed may be from trace amounts to an amount in excess of equimolar with the chelate.

Preferably, the formation of the chelate occurs at room temperature, between 15°C to Chelation proceeds rapidly and yields of about 90 percent and greater as measured by high performance liquid chromatography are obtained when using a chelating agent which has been initially purified on substantially calcium free silica gel and activated with thiophosgene prior to chelation of the desired metal ion.

The ability to rapidly form chelates over a wide pH range is advantageous to obtain a high chelation yield of the desired metal ion. When it is necessary to displace divalent cations such as calcium prior to chelation of the desired metal ion, yields are variable due to the pH sensitive nature of the displacement reaction. Displacement of the calcium ion requires rurning the reaction at a pH sufficiently low to displace the calcium, yet the pH cannot be too low as to adversely affect the chelation of the desired metal ion. Utilizing the procedure of the s-snaa~ ~aas~s Iii~~e WO 93/20852 PCT/US93/03483 present invention allows the chelating reaction to proceed with high yields over a broad pH range.

The ability to activate the chelating agent priorto forming the chelate and the rapid formation of the chelate with a high yield also has the advantage in that it reduces the purification necessary prior to the conjugation step. When the metal ion is chelated prior to activation using thiophosgene, it is necessary to extract the unreacted thiophosgene with an organic solvent and then purify the chelate prior to conjugation. The low concentration of the chelate in the elution volume obtained after purification using an ion exchange column necessitates concentrating the chelate prior to conjugation. During the process of extracting the thiophosgene and concentrating the eluent by reduced pressure and/or heat, some degradation of the product occurs along with radiolysis creating additional impurities.

Following the chelation step of the present invention, the activated chelate can be rapidly purified by column chromatography and the eluent used directly, without concentration, in the conjugation step.

The bifunctional chelate is preferably conjugated to a biological molecule which carries out a specific target function. In a preferred embodiment, the biomolecule is a monoclonal antibody or fragment thereof which is specific against a selected cell-surface target site. Such antibodies may be commercially available or may be made by standard somatic cell hybridization techniques. An example of a suitable monoclonal antibody is CC49, one of a series of monoclonal antibodies specific for TAG-72 (tumor associated glycoprotein) described in published PCT Application No. WO 89/00692, on January 26, 1989, and published PCT application WO 90/04410, on May 3, 1990. Generally, the chelate and protein are mixed in a molar ratio of greater than 1:10 and less than about 100:1 depending on the protein and protein concentration. Ratios of 0.5:1 to 4:1 are preferred.

Methods for conjugating thiocyanate derivatized chelates to antibodies are well known in the art. The procedure generally involves reacting the functionalized chelating agent or chelate with the antibody from 2 to 18 hours in an aqueous buffered solution at pH 6-10 at room temperature.

The increase in purity of the chelate obtained by the process of the present invention allows the chelate to be conjugated to a biological material, preferably a protein, at temperatures from 25C to 40 0 C, preferably 30°Cto 40 0 C, to obtain a protein/chelate containing an activity from 0.5 millicuries to 30 millicuries per mg protein without the expected increase in degradation of the chelate activated group hydrolysis of the isothiocyanate) or protein.

As the temperature of the chelation reaction is increased, an increase in the rate of formation of protein aggregates and hydrolysis of the chelate activated groups would be expected in addition to an increased conjugation rate. Unexpectedly, elevated temperature results in a much faster conjugation rate increase relative to the rate of increase of undesired by-product formation chelate hydrolysis and protein aggregates). Performing the conjugation at I C~mib 0 IP- arr~l WO 93/20852 IPCr/US93/03483 about pH 9.5 and 37*C, the reaction is approximately 92 percent complete in one hour with leis than 4 percent of the radioactivity being associated with protein aggregates, The formation of fewer by-products during the conjugation coupled with a more complete reaction leads to easier product purification, production of a product with higher purity, and gives a greater overall recovery of the initial radioactivity. Utilizing a conjugation temperature of 37"C also allows a radiochemical yield of greater than 90 percent during purification by column chromatography as compared to a radiochemical yield of approximately percent when the conjugation is done at 20 0

C.

The invention will be further clarified by a consideration of the following examples, which are intended to be purely exemplary of the present invention.

In the following examples, the following terms are used.

E bifunctional chelating agent.

PA-DOTA 1-[2-(4-aminophenyl)ethyl]-1,4,7,10- -tetraazacyclododecane-1,4,7,10-tetraacetic acid.

HEPES N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid.

General Experimental Mass spectra (fast atom bombardment with xenon) were obtained on a Vacuum Generators ZAB HS mass spectrometer. Samples for mass spectral analysis were prepared by dissolution in a 3:1 mixture of dithiothreitol:dithioerythritol (magic bullet) unless otherwise stated.

Analytical high performance liquid chromatograph (HPLC) of non-radioactive amples was performed on a Hewlett Packard 1090 liquid chromatograph with a 4.6 x 100 mm Alltech Econosphere C18 3 p column with a flow rate of 1 mL per minute and detection at 254 nm. Samples were eluted with a gradient of 0.05 M, pH 6.0, sodium acetate/ acetonitrile as given for the samples.

HPLC of radioactive samples those in which '"Lu was incorporated) was performed using a DuPont Zorbax GF-250 (9,4 x 250 mm) column with a flow rate of 1.5 mL per minute and radioactivity detection. The mobile phase was 0.25 M, pH 6.0 sodium percent acetonitrile.

Example 1 Purification of PA-DOTA by Flash Chromatography A 1.5 x 14 inch flash chromatography column was packed with 17.0 g of Merck 60 A silica gel, 230-400 mesh (Aldrich Chemical Co.) which had been acid-washed by Alltech Inc. Crude PA-HaDOTA.3HCI (3.2 g, 75 percent pure by HPLC analysis) was applied to the column and eluted with a mobile phase of 2:2:1 chloroform:metharlol:concentrated ammonium hydroxide. Fractions containing only PA-DOTA(NH 4 2 as assayed by thin layer chromatography were collected and pooled to give PA-DOTA(NH 4 2 which was greater than 98 percent area purity as assayed by HPLC analysis. The absence of calcium irn the recovered -O n I l WO 93/20852 PCI/US93/03483

PA-DOTA(NH

4 2 was confirmed by mass spectrometry and HPLC using an elution buffer gradient of 95/5 sodium acetate (0.05 M, pH 6)/acetonitrile to 30/70 in 15 minutes.

Chelation of yttrium to the PA-DOTA obtained above was performed by dissolving 3 mg of PA-DOTA in 3 mL of 0.5 of sodium acetate buffer (pH 6) and 0.2 mL of 40 millimolar Y(OAC) 3 .4H 2 0, this represents approximately a 5 fold excess of yttrium. The extent of chelation was monitored by HPLC as described above.

Chelation with PA-DOTA which was purified using acid washed silica gel was complete in less than 5 minutes at room temperature (18-25C).

Comparative Example A Chelation with PA-DOTA purified using silica gel which had not been acid washed was 4 percent complete in 15 minutes at room temperature and was 86 percent complete in minutes at These results show acid washing the silica gel to remove extraneous metal ions prior to use for purifying the chelating agent greatly enhances the rate at which the desired metal is sequestered at room temperature.

Example 2 Preparation of SCN-PA-H 4 DOTA.2HCI.2NH 4

CI

To 100 mL of 0.01 M hydrochloric acid was added 250 mg of calcium free

PA-H

2

(NH

4 2 DOTA (0.448 millimoles, 2 of the carboxylated groups are protonated and 2 of the carboxylated groups are ammonium salts) as prepared in Example 1, and the solution placed in a 40-oz. Waring blender. Thiophosgene (170 microliters, 2.23 millimoles) was added and the blender quickly started. After 2 minutes of mixing, the mixture was added to a separatory funnel and excess thiophosgene was extracted with three 50 mL portions of chloroform. The aqueous layer was added to 100 mL of aceton, trile and the solution reduced to dryness on a rotary evaporator (fitted with a vacuum pump) at room temperature. The solid obtained was further dried for 2 hours at room temperature on a vacuum line. The yield was 321 mg percent yield) of an off-white solid.

Characterization of the product by mass spectrometry and by HPLC showed the product was 96 percent by weight an isothiocyanate derivatized PA-DOTA

(SCN-PA-H

4 DOTA.2HCI*2NH 4 CI). The HPLC elution gradient being sodium acetate/acetcnitrile 95/5 to 70/30 in 15 minutes and then to 40/60 in 20 minutes.

Comparative Example B Samples prepared in an identical manner except that the reac'on was carried out by the addition of 20 to 50 percent by volume chloroform prior to mixing were 84 to 89 percent pure as measured by HPLC.

These results show that a high purity isothiocyanato activated chelating agent can be obtained by mixing the thiophosgene and chelating agent in an aqueous system in the absence of an organic solvent.

i I II Is~ UI~- O- QI~XM WO 93/20852 PICT/US93/03483 Example 3 Preparation of [177Lu(SCN-A-DOTA)]' To 20 pL of SCN-PA-DOTA (4.88 x 10-3 moles) prepared as described in Example 2 was added 100 pL of Lu-177 (50 microcurries) to give a molar ratio of chelating agent to Lu-177 of about 1:1. The solution was mixed on a vortex mixer for about 5 seconds, 100 pL of HEPES buffer (0.5 M, pH 7) were added and the solution mixed for 5 minutes. The pH was measured and adjusted to between pH 6-7 with HEPES buffer if necessary. The reaction was allowed to proceed for another 5 minutes at room temperature and the yield of 7 Lu(SCN-A-DOTA)] was 91.4 percent as determined by HPLC.

The [1 7 7Lu(SCN-PA-DOTA)] complex was purified by placing the sample on a PRP- 1 mini-clean" column (80 pL) which had been pretreated with 800 pL acetonitrile, 400 pL of water and 800 pL 10 percent by volume acetonitrile in 20 mM carbonate buffer, pH 9.5. After loading the complex onto the column, the reaction vial was rinsed with a 200 pL and then a 600 pL volume of 10 percent acetonitrile in carbonate buffer, the washes also being placed on the column.

The complex was then eluted from the column using a 1:2 ratio of carbonate buffer (20 mM, pH 9.5):acetonitrile. Approximately 80 percent of the radioactivity was recovered in the second 50 IL elution volume.

Example 4 Conjugation of [1 77 Lu(SCN-PA-DOTA)]-With IgG CC49 at Room Temperature (21-22 0

C)

To 257 pL of an antibody solution (IgG CC49, 14.5 mg/mL in 50 mM carbonate buffer, pH 9.5) was added 25 pL of [1 7 7 Lu(SCN-PA-DOTA)]' prepared as described above, giving a BFC:antibody molar ratio of about 1.36. The activated chelate and antibody solution was mixed on a vortex mixer for about 10 seconds and allowed to stand at room temperature for approximately 2 hours with mixing about every 10 to 15 minutes. The disappearance of the [177Lu(SCN-PA-DOTA)]- was measured by HPLC. After 120 minutes, about 62 percent of the 177 Lu activity was associated with the antibody, about 7 percent was non-antibody associated impurities, and unreacted [1 7 7 Lu(SCN-PA-DOTA)]'was about 30 percent.

Example 5 Conjugation of [1 77 Lu(SCN-PA-DOTA)]- With IGg CC49 at 37°C To an antibody solution (15.7 mg CC49 IgG (30 nanomoles) in 286 pL of carbonate buffer, 20 mM, pH 9.5) was added 21 nanomoles of [177Lu(SCN-PA-DOTA)] complex (9.7 millicuries of activity) in 11 IL of the eluent from Example 3. The mixture was mixed on a vortex mixer and allowed to stand in a 37"C oven for one hour with mixing about every 10 to minutes. The progress of the reaction was monitored by HPLC analysis for the disappearance of the complex as described in Example 4. After 1 hour the 17 7 Lu activity associated with the antibody as measured by HPLC was 91 percent, less than 4 percent of the 177Lu activity was associated with impurities, and 5 percent was unreacted [177Lu(SCN-PA-DOTA)]-.

p~slr~u-o~ ll--l I WO 93/20852 PCT/US93/03483 The conjugation reaction was terminated by isolation and purification of the conjugate on a PD-10column (Sephadex G-25, medium, 9 mL) which had been equilibrated with phosphate buffered saline.

Homogeneity of the labeled antibody was examined by HPLC and by SDS PAGE electrophoresis (sodium dodecyl sulfate-polyacrylamide electrophoresis) coupled with autoradiography. Immunoreactivity was determined by IRMA and affinity column binding and was found to be comparable to that conjugated at 25 0 C. In vivo biodistribution (in rats) of the conjugate prepared at37 0 C was not significantly different from that prepared at The results show that the chelate can be rapidly conjugated to an antibody at 37°C without affecting the immunoreactivity or biodistribution of the conjugate.

Other embodiments of the invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, win the true scope and spirit of the invention being indicated by the following claims.

II--

WO 93/20852 PIr/US93/03483 1. A process for preparing a chelate-antibody conjugate which comprises reacting an isothiocyante activated chelate with an antibody at between 30°C and wherein the chelate is a chelating agent complexed with a metal ion; the chelating agent is of S the formula

Q

R3 X Q 1

R

2 /C

(CH

2 C- (CH 2 )r N-Q N/ m N

Q

(I)

wherein: each Q is independently hydrogen, (CHR5)pCO 2 R or (CHR5)pPO 3

H

2 Q1 is hydrogen or (CHR 5

),CO

2

R;

each R independently is hydrogen, benzyl or C-C 4 alkyi; with the proviso that at least two of the sum of Q and Q1 must be other than hydrogen; each R5 independently is hydrogen, Ci-C 4 alkyl or -(C 1

-C

2 alkyl)phenyl; X and Y are each independently hydrogen or may be taken with an adjacent X and Y to form an additional carbon-carbon bond; n is 0 or 1; m is an integer from 0 to 10 inclusive; p 1 or 2; r 0or 1; w Oor 1; wi Lh the proviso that n is only 1 when X and/or Y form an additional carbon-carbon bond and the sum of r and w is 0 or 1;

R

2 and R 4 are independently hydrogen, or amino;

R

3 C1-C4 alkoxy, -OCH 2

CO

2 H, hydroxy and hydrogen; with the proviso that R 2 and R 4 cannot both be hydrogen but one of R 2 and R4 must be hydrogen; and the metal ion is selected from the group consisting of 1 53 5m, 166 Ho, 1 7 7 7 Lu, 159Gd, 14 0La, 14 2 Pr, 1 49 Pm, 90Y and 111In.

2. The process of Claim 1 wherein the metal ion is 177 Lu.

3. The process of Claim 1 or 2 wherein the chelating agent is 1-[2-(4-aminophenyl)ethyl]-1,4,7,10--tetraazacyclododecane- 1,4,7,10-tetraacetic acid.

4. In a process for preparing polyaza conjugates having at least the steps of -19- 9 r

Claims (13)

1. A process for preparing a chelate-antibody conjugate which comprises reacting an isothiocyanate activated chelate with an antibody at a temperature between 3ooC and 0 C wherein the chelate is a chelating agent complexed with a metal ion; the chelating agent is of the formula R Q X Q 1 R O (CH 2 )n (CH 2 )r-N N-Q Y H N R4 Q (I) wherein: each Q is independently hydrogen, (CHR 5 )pCO 2 R or (CHR 5 )pPO 3 H 2 Q 1 is hydrogen or (CHR 5 )wCO 2 R; each R independently is hydrogen, benzyl or C 1 -C 4 alkyl; with the proviso that at least two of the sum of Q and Q1 must be other than hydrogen; each R 5 independently is hydrogen, C 1 -C 4 alkyl or -(C 1 -C 2 alkyl)phenyl; X and Y are each independently hydrogen or may be taken with an adjacent X and Y to form an additional carbon-carbon bond; n is 0 or 1; m is an integer from 0 to 10 inclusive; p =1 or 2; S* r =0 or 1; 20 w or 1; with the proviso that n is only 1 when X and/or Y form an additional carbon-carbon bond and the sum of r and w is 0 or 1; R 2 and R 4 are independently hydrogen, or amino; R 3 is C 1 -C 4 alkoxy, -OCH 2 CO 2 H, hydroxy or hydrogen; 25 with the proviso that R 2 and R 4 cannot both be hydrogen but one of R 2 and R 4 must be hydrogen; and the metal ion is selected from 153 Sm, 1 66 Ho, 175 Yb, 177 Lu, 159 Gd, 140 La, 142 Pr, 149 m, 90 Y and 11 ln. V 2. A process of claim 1, wherein said temperature is between 30 0 C and 40 0 C.

3. A process of claim 1 or 2 wherein the metal ion is 177 Lu.

4. A process of any one of the preceding claims, wherein the chelating agent has been activated by contact with thiophosgene is an aqueous environment in the absence of an organic solvent at a pH from 1 to 7. A process of any one of the preceding claims, wherein the chelating agent has been purified by chromatography on a silica gel column wherein the silica gel has been R4As, acid washed prior to loading with the chelating agent. [N:\libZ]00593:SAK -~I BB~gr o o o r o D o D 21

6. A process of any one of the preceding claims, wherein the chelating agent is 1-[2-(4-aminophenyl)ethyl]-1,4,7,10 -tetraazacyclododecane-,4,7,10-tetraacetic acid.

7. A process for preparing polyaza conjugates having at least the steps of purifying a polyaza chelating agent as defined in Formula I of claim 1, by chromatography on a silica gel column, wherein the silica gel has been acid washed prior to loading the polyaza chelating agent onto the column; contacting the purified polyaza chelating agent with thiophosgene in an aqueous environment in the absence of an organic solvent at a pH from 1 to 5 to form an isothiocyanato activated polyaza chelating agent, and recovery of the isothiocyanato 1o activated polyaza chelating agent; adding a metal ion to the isothiocyanato activated polyaza chelating agent before or after step to form an isothiocyanato activated polyaza chelate; and contacting the isothiocyanato activated polyaza chelate with a biological molecule at between3doC to 40 0 C to form a conjugate.

8. A process of claim 7, wherein step is carried out after step

9. A process of claim 7 or 8 wherein the biological molecule is a protein and the metal ion is 153 Sm, 166 Ho, 1 75 Yb, 177 Lu, 1 59 Gd, 140 La, 1 42 pr, 149 pm, 90 y or 111 1n. A process of claim 9 wherein the protein is an antibody.

11. A process of any one of claims 7 to 10 wherein the chelating agent is 20 aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid and the metal ion is 17 7 Lu.

12. A process of any one of claims 1 to 5 or 7 to 10 wherein r of formula I is 0 and each Q is (CHR 5 )pCO 2 R.

13. A process for preparing a chelate-antibody conjugate, substantially as 25 hereinbefore described with reference to Example

14. A chelate-antibody conjugate prepared by the process of any one of claims 1 to 13. Dated 30 July, 1996 The Dow Chemical Company e cc Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [N:\libZ]00593:SAK n ~R INTERNATIONAL SEARCH REPORT International Application No PCT/US 93/03483 I. CLASSIFICATION OF SUBJECT MATTER (If several classification symbols apply, Indicate all)' According to International Patent Classification (IPC) or to both National Classification and IPC Int.Cl. 5 A61K49/02 I. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols Int.Cl. 5 A61K Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched 1 I1. DOCUMENTS CONSIDERED TO BE RELEVANT' Category 0 Citation of Document, I with Indication, where appropriate, of the relevant passages 12 Relevant to Claim No.t3 Y BIOCONJUGATE CHEMISTRY 1-10 vol. 2, MAY-JUNE 1991 pages 180 186 C. H. CUMMINS ET AL 'A convenient synthesis of bifunctional chelating agents based on DTPA and their coordination chemistry with Yttrium(iii)' See p. 180, general methods lines 1-2 X,Y EP,A,0 353 450 (THE DOW CHEMICAL COMPANY) 1-10 7 February 1990 cited in the application see page 3, line 49 line 58 see page 5, line 1 line 32 see page 5, line 52 line 58 see page 6 page 7; claims; figures II,IV see page 19, line 38 line 54 see scheme I-V o Special categories of cited documents :10o "T later document published after the international filing date or priority date and not in conflict with the application but document defining the general state of the art which is not cited to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but published on or after the international Ix document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the International filing date but in tin art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 14 OCTOBER 1993 22 10. 93 International Searching Authority EUROPEAN PATENT OFFICE Signature of Authorized Officer BERTE M.J. i LjL-ziG_ fibet) (inmiry I re_ I- PCT/US 93/03483 l nternational Apipllca:Ion No [in. DOCUMENTS CONSIDERED TO BE RELEVANT (CON~TINUED IFROM THE SECOND SHIEET) Ctegory Citation of Document, with Indication, where appropriate, of the relemat passages Relevant to Claim No. X EP,A,0 374 947 (THE DOW CHEMICAL COMPANY) 1-10 27 June 1990 see page 2, line 1-8 see page 2, line 22 line 24; claims; tables X EP,A,O 296 522 (THE DOW CHEMICAL COMPANY) 1-10 28 December 1988 see claims Y CHEMICAL ABSTRACTS, vol. 113, no. 25 1-10 Columbus, Ohio, US; abstract no. 231345j, see abstract PURE APPL. CHEM. vol. 62, no. 6, 1990, pages 1115 1118 R. M. IZATT ET AL. 'Macrocycle-metal cation interactions involving polyazamacrocycles bonded to silica gel via a nitrogen donor atom.' see the whole document X EP,A,O 292 689 R. SQUIB SONS) 1-10 November 1988 see page 7, line 29 page 8, line 4; claims Form FcI10 (e~dra LI~dI (.lmt=Y M93) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. 9303483 73577 This annex ists the patent Family members relating to the patent documents cited in the abovv~nentioned international search report. The mlembhers are as contained in the European Patent Office EDP file oa The Europeani Patent Office is in Do way liable for thiem particulars which ame merely given for the purpose of information. 14/10/93 Patent document Publication Patent family PublicatioD cited in search report I date I member(s) Iat EP-A-0353450 07-02-90 AU-A- AU-B- AU-A- EP-A- JP-T- WO-A- US-A- 3705889 634167 3979489 0420942 4504247 8912631 5064956 04-01-90

18-02-93 12-01-90 10-04-91

30-07-92 28-12-89 12-11-9 1 EP-A-0374947 27-06-90 US-A- 5006643 09-04-9 1 JP-T- 3502937 04-07-91 WO0-A- 9007342 12-07-90 EP-A-0296522 28-12-88 US-A- AU-B- AU-A- JP-A- US-A- US-A- 4994560 614973 1833288 1026586 5006643 5064956 19-02-91 19-09-91 05-01-89 27-01-89 09-04-91 12-11-9 1 EP-A-0292689 30-11-88 US-A- 4885363 05-12-89 CA-A- 1296715 03-03-92 JP-A- 1052764 28-02-89 SFor more details about this annex :see Official Journal of the European Patent Office, No. 1Z/82