United States Patent (19) 11 Patent Number: 4,880,935 Thorpe 45 Date of Patent: Nov. 14, 1989 (54. HETEROBIFUNCTIONAL LINKING Wang et al., Israel Journal of Chem., vol. 12, 1974, pp. AGENTS DERVED FROM 375-389. N-SUCCNMDO-DTHO-ALPHIA Vallero et al., Science, 222, 1983, pp. 512-515. METHYL-METHYLENE-BENZOATES Camber et al., Method of Enzymol, 112, 1985, pp. 201-225. 75 Inventor: Philip E. Thorpe, London, England Langone et al., Method of Enzymol, 93, 1983, p. 280. Masuho et al., J. Biochem, 91, 1982, pp. 1583-1591. 73 Assignee: ICRF (Patents) Limited, London, Carlsson et al., Biochem. J., 1978, 173, pp. 723-737. England Calombatti et al., J. Immunol, 131, 1983, pp. 3091-3095. Brown et al., Cancer Res., vol. 45, 1985, pp. 1214-1221. 21 Appl. No.: 90,386 Ramakrishnonet al., Cancer Res., 44, 1984, pp. 201-208. 22 Filed: Aug. 27, 1987 Gras et al., J. of Immunol Methods, 81, 1985, pp. 283-297. Related U.S. Application Data Primary Examiner-Alan L. Rotman Attorney, Agent, or Firm-Nixon & Vanderhye 63 Continuation of Ser. No. 884,641, Jul 11, 1986, aban doned. (57 ABSTRACT The efficacy of immunotoxins having an antibody that 51) Int. Cl.".................. C07D 207/46; CO7D 207/48; recognizes a tumour associated antigen linked to a cyto CO7D 401/12 toxin through a heterobifunctional agent of the disul 52 U.S. Cl. ..................................... 546/281; 548/542 phide type is improved by providing in the heterobi 58 Field of Search ......................... 548/542; 546/281 functional agent a molecular grouping creating steric 56 References Cited hindrance in relation to the disulphide link. This steric hindrance can be provided by a methyl substituted PUBLICATIONS methylene group located in the heterobifunctional link Thorpe et al., Chemical Abstracts, vol. 108, (11), Abst. adjacent to the disulphide bond. No. 87663p, Mar. 14, 1988. Lutter, FEBS Letters, 48(2), 1974, pp. 288-292. 4. Claims, 1 Drawing Sheet U.S. Patent Nov. 14, 1989 4,880,935 REACTION SCHEME Ho-K)-c.2 CH 3 O CH3 HO- KX- CH-Br O CH3 o HO- C CH-S-SO - No 3. O CH3 to-(). SH (IV) 4,880,935 1. 2 directed simply to the provision of bulky groups that HETEROBFUNCTIONAL LINKING AGENTS will not interfere with the biological properties of the DERVED. FROM immunotoxin but which will afford a measure of shield N-SUCCNMDO-DTHO-ALPHA ing or protection to the disulphide link during the pas METHYL-METHYLENE-BENZOATES 5 sage of the immunotoxin from the point of administra tion to the site of action. It will therefore be seen that This is a continuation of application Ser. No. 884,641, the selection of the sterically hindering group is gov filed July 11, 1986, now abandoned. This invention relates to immunotoxins and is particu erned largely by considerations of biological inertness larly concerned with the vision of immunotoxins of 10 and ease of chemical synthesis and such considerations improved efficacy in vivo. point to the use of one or more hydrocarbyl groups, e.g. The use of immunotoxins in drug targeting is now containing from 1-6 carbon atoms, since they have been well recognised. Such immunotoxins essentially com found to provide an adequate degree of protection to prise an antibody that recognises a tumour associated the disulphide link during its transport to the target cell antigen that is linked to a suitable cytotoxin. The exact 15 but still permit cleavage of the disulphide bond in the nature of the link is important in that it must provide a target cell. linkage between the antibody and the toxin that will Our experiments indicate that a useful measure of remain intact during the delivery of the toxin through protection of the disulphide group can be achieved by the patients body to the intended site of action and, at the incorporation of one methyl substituent onto a the same time, it must not interfere with the immunolog 20 methylene group adjacent to the disulphide link. How ical specificity of the antibody or the cytotoxic proper ever, improved protection would be expected by the ties of the toxin. provision of larger substituents, e.g. 2 methyl groups or A group of linking agents known as heterobifunc one or two C2-C4 alkyl groups which may be the same tional agents have been developed in recent years for or different. From the practical point of view, one must this purpose. Two compounds that have achieved ac 25 balance the additional longevity of the immunotoxin in ceptability are N-succinimidyl-3-(2-pyridyldithio)pro vivo against the additional difficulty and cost of synthe pionate which has become known as SPDP and 2 sising the more sterically hindered heterobifunctional iminothiolane hydrochloride which has become known agents. as 2IT. These compounds, when used as heterobifunc The improvement of the present invention is applica tional linking agents, produce immunotoxins of the 30 ble to all heterobifunctional agents of the type which Structure form a chemical bond with the antibody and which link with the texin through the disulphide link. It is particu O () larly suited to the modification of the existing heterobi SPDP: IgG NHC,CH2CH2.SS Toxin functional agents of the SPDP and 2IT type by the 35 introduction of the selected steric hindering groups on -- (II) the methylene adjacent to the disulphide group. For H. example, when SPDP is modified in accordance with 2IT: IgG NH.C.CH2CH2CH2.SS Toxin the present invention, use may be made of linking agents The disulphide bond appears to be necessary for cy having the formula totoxic activity, probably because the toxic component has to be released from the antibody by reduction inside O (III) the cell in order to inactivate the cells machinery for A. protein synthesis. The problem with such linking agents SH: is that the essential disulphide bond is labile in the blood 45 N-O-CO CH-SSO-Na+ stream or extravascular tissues of mammals and experi ments that have been carried out in mice indicate that W there is degradation of the disulphide bond at a rate O such that the immunotoxin has a half life of approxi O (IV) mately 8 hours. This premature degradation of the im 50 A. munotoxin is undesirable from the therapeutic point of SH: view because the released antibody component from NorCarCO CHSS O the immunotoxin is relatively stable in the mammal and can so mask the target antigen sites on the tumour from W N subsequently administered immunotoxin. 55 O It has been found, in accordance with the present invention, that the rate of premature degradation of the In these compounds, it is preferred that X is methyl, Y immunotoxin can be reduced by structural modification is hydrogen and Z is sodium. of the heterobifunctional linking agent to provide a It is also possible to incorporate the concept of steric Substituent masking or protecting the disulphide bond. 60 hindrance of the disulphide link in other heterobifunc The present invention therefore provides an im tional reagents, for example analogues of comounds III proved immunotoxin in which the heterobifunctional and IV above in which the succinimide ring is substi agent includes a molecular grouping that will create tuted by a sulphonic acid group and/or in which the steric hindrance in relation to the disulphide link. Steric pyridyl ring of SPDP is replaced by a 3-carboxy-4- hindrance is most conveniently provided by a substi 65 nitrophenyl ring. tuted methylene group adjacent to the disulphide link. The antibody component of the immunotoxins of the The exact nature of the substituents is not of critical invention may be any of the antibodies that recognise importance, the concept of the present invention being tumour associated antigens. The antibody will normally 4,880,935 3 4 be a monoclonal antibody and will be selected in accor dance with the nature of the tumour to be treated. EXAMPLE 2 The cytotoxin component of the immunotoxins of the invention can again be any one of those cytotoxic mate This example illustrates linking of a monoclonal anti rials customarily used in immunotoxins and in this con body MRC-OX7 via linking agent IV to the A chain of nection, reference is made to Myers et al, Blood, 63, ricin. OX7 is a mouse IgG1 sub-class antibody produced 1178-1185, (1984) and Thorpe et al., JNC 75, 151-159 from a hybridoma cell line MRC-OX7 which is de (1985). These papers describe the incorporation into scribed in Mason et al, Biochem. J. 187, 1-20 (1980) and immunotoxins of various ribosome inactivating proteins is available from the Medical Research Council Cellular which are of particular interest in this invention. Typi 10 Immunology Unit, University of Oxford, Oxford, En cal ribosome inactivating proteins are the A chain of gland. OX7 recognises an antigen Thy 1.1 which is the ricin and abrin. Other A chain like proteins which may antigen expressed by the lymphoma cell lines AKR-A be incorporated in the immunotoxins of this invention and BW5147 which are obtainable respectively from are bryodin, momordin, saporin, gelonin and PAP. Prof. I Maclennan of The Department of Experimental The immunotoxin of the present invention may be 15 used in the same way as prior art immunotoxins to com Pathology, Birmingham University, Birmingham, En bat tumour growth in a host by administering an effec gland and Dr. J. Hewitt of the Wellcome Research tive amount of the immunotoxin to the host. The immu Laboratories, Beckenham, Kent, England. notoxins are also particularly useful for purging bone 1. To 22.5 mg of OX7 antibody at a concentration of marrow in vitro in connection with bone marrow trans 20 7.5 mg/ml in nitrogen-flushed borate-saline buffer (0.05 plantation.