IMMUNOGLOBULIN CONJUGATES

Abstract: There is described an immunoglobulin conjugate com­ prising an immunoglobulin or immunoglobulin fragment modified by one or more groups of the following formula which are covalently linked to it: The conjugates are useful in the treatment of cancers and are made by reaction of the immunoglobulin or immunoglobulin fragment with desacetylvinblastine acid azide.

Description: IMMUNOGLOBUL IN CONJUGATES This invention relates to novel immunoglobulin conjugates which are anti-cancer agents. In the chemotherapeutic treatment of cancer it is sometimes necessary to employ drugs that are limited in their effectiveness by unwanted side effects on the patient and efforts are continually being made to minimise such effects. One such drug is vindesine, a compound of the following formula (I) EMI1.1 Vindesine is disclosed, for example, in British Patent 1,463,575 which describes its preparation and biological activity. This invention provides a novel conjugate comprising an immunoglobulin or immunoglobulin fragment modified by one or more groups of the following formula which are covalently linked to it: EMI2.1 The' immunoglobulin or fragment is an antibody or a fragment of an antibody with antigen recognising properties. The e preferred immunoglobulin material is an antibody or a fragment of an antibody adapted for recognition of antigens on the surface of malignant cells of the type occurring in the human body. However immunoglobulin materials of other kinds are also included within the scope of the invention since they may be of use in treatment of animals and in control and assay experiments. The novel conjugates are useful in the treatment of cancers. They are potentially more effective and have fewer side effects by virtue, inter alia, of their ability to increase the concentration of the cytotoxic drug at the site of action. It is also to be understood that the invention includes conjugates for use in an indirect system in which they are used to recognise an antibody specific to the cell surface antigen. Immunoglobulins specific to antigens on the surface of cells to be killed, and techniques for their production from the serum of immunised animals or by culturing hybridomas secreting monoclonal products, are well known. The preferred type of antibody is an immunoglobulin of the IgG class. Some representative immunoglobulins are as follows (i) Ig from goats or sheep immunised with carcinoembryonic antigen (ii) Ig from rabbit anti-acute lymphoblastic leukemia serum (iii) Ig from various primate antisera raised against acute lymphoblastic leukemia, acute myleoblastic leukemia, chronic lymphoblastic leukemia and chronic granulocytic leukemia (iv) Ig from goats or sheep immunised with lung carcinoma material (v) monoclonal Ig from mouse hybridomas secreting anti-human colorectal carcinoma antibodies (vi) monoclonal Ig from mouse hybridomas secreting anti-human melanoma antibodies (vii) monoclonal Ig from mouse hybridomas secreting antibodies reacting with human leukemia cells (viii) monoclonal Ig from mouse hybridomas secreting antibodies reacting with human neuroblastoma cells (ix) monoclonal Ig from mouse hybridomas secreting antibodies reacting with human breast cancer antigens (x) monoclonal Ig from mouse hybridomas secreting antibodies reacting with human ovarian carcinoma cells (xi) monoelonal Ig from mouse hybridomas secreting antibodies reacting with human osteosarcoma cells. As indicated above, the conjugate can also be made with immunoglobulin fragments, referred to as Fab, Fab' or F(ab')2 or IgM monomer derived from an antibody by, for example, proteolytic enzyme digestion. Such materials and methods of preparation are well known and it may be mentioned that preferred proteolytic enzymes are pepsin and papain. Vindesine of formula (I) can be bound to the immunoglobulin material via the carbon atom of the carboxyl residue at the 3-position by reaction with the appropriate azide derivative and the invention includes, as a further aspect, a process for preparing an anti-cancer agent which comprises reacting an immunoglobulin or immunoglobulin fragment with an azide compound of the formula (III) EMI5.1 This process represents a convenient way of linking the drug vindesine to immunoglobulin under mild conditions whilst maintaining the structural integrity of the immunoglobulin. The process is preferably carried out in an aqueous medium and at a temperature of from 50C to 250C, for example at room temperature, and at a pa of 8.5 to 9.5, preferably 9.0, and results in the attachment by covalent linkage of one or more vinca residues at the free amino groups of the immunoglobulin molecule, for example, amino groups derived from lysine residues. The number of residues attached will depend on the concentration of the reactants and the duration of the reaction but the average number is usually from 1 for example from 2 to 5. For example in carrying out the reaction, a solution of the azide in a suitable solvent such as dioxan is slowly added dropwise to a buffered solution of immunoglobulin in for example 0.34 M borate buffer at pH 9. The conjugate is isolated by gel filtration and stored in saturated ammonium sulphate solution being readily brought back into solution by dialysis with borate buffer, or alternatively it can be stored in a refrigerator at 40C or frozen at for example -2O0C. The azide reactant of formula (III) can be readily prepared from vinblastine, vinblastine sulphate or desacetylvinblastine by preferential hydrazinolysis of the C3- ester followed by diazotisation as for example described in J.Med.Chem., 1978, Vol. 21, No. 1 pages 88 to 96, and in British Patent 1,463,575 referred to above. Hydrazinolysis of vinblastine at moderate temperatures in methanol solution proceeds yielding predominantly the monohydrazide and reaction of this product at temperatures of OoC to 100C with hydrochloric acid and sodium nitrite in a suitable aqueous solvent, yields the desired azide of formula (III) (desacetylvinblastine acid azide). Alternatively the azide of formula (III) can be prepared at a temperature of below SOC as described above, and, without isolating the azide, the pH is adjusted to 9 and a water miscible solvent, such as for example dioxan, added. The immunoglobulin in borate buffer pH9 is then added and the reaction mixture allowed to rise to room temperature. Excess azide is destroyed with dilute ammonia and the conjugate purified by gel filtration. EXAMPLE 1 PreParation of vindesine-sheep-anti-carcinoembryonic antigen conjugate Desacetyl vinblastine acid hydrazide (6 mg) was dissolved in IN HC1 (340 p1) and the solution cooled to 40C. Sixty microlitres of a 10 mg/ml aqueous solution of sodium nitrite were added and the reaction stirred at 40C. After 5 minutes, IN NaOH (300 pl) and dioxan (120 > 1) were added with vigourous stirring, following by 280 pl of a 21.3 mg/ml solution of sheep-anti-CEA in 0.34 M borate buffer pH 8.6. The mixture was stirred at room temperature for 2 hours maintaining pH 9 using N/10 NaOH. Excess azide was destroyed by adding IN NH4 OH(60 pl) and stirring for a further hour. The product was isolated by gel filtration on a 1 x 25.5 cm(20 ml) column of Bio-GelP-6 equilibrated with phosphate buffered saline. The excluded peak was collected (2.66 ml), and assayed for protein content by the Lowry method and for vindesine by difference spectroscopy. The conjugate so prepared contained 4.6 moles vindesine per mole of Ig. EXAMPLE 2 PreParation of vindesine-mouse-monoclonal anti-carcinoembryonic antigen conjugate. Desacetylvinblastine acid hydrazide (10 mg) was dissolved in 1N HC1 (0.56 ml) and the solution cooled to 40C. One hundred microlitres of a 10 mg/ml solution of sodium nitrite were added and the reaction stirred at 40C. After 5 minutes, 1N NaOH (0.5 ml) Evaluation of the conjugate can be carried out using well known techniques such as affinity chromotography. The efficacy of the conjugate can be estimated by counting the number of viable cells after treatment of a suspension of tumour cells with the conjugate, or from measurements of the uptake of tritiated uridine. Protein and drug concentrations are determined by measuring optical densities of conjugate solutions at two wavelengths, for example 270 and 280 nm, and relating the values obtained to those for the free drug and unconjugated immunoglobulin at the same two wavelengths. The novel conjugates of the invention are useful in the treatment of cancers and as such are preferably prepared for use in formulations suitable for injection. Thus the invention includes a pharmaceutical formulation, for example an injectable preparation, comprising a conjugate of the invention together with a pharmaceutically-acceptable carrier or diluent such as are well known in the art. It is preferably in unit dosage form each dosage containing for example from 0.01 to 2 mg of the active ingredient (in terms of the vindesine moiety). The novel conjugates are effective over a wide dosage range and for example for the treatment of adult humans dosages per week will normally fall within the range of 1 to 10 mg/kg (vindesine moiety) more usually in the range of from 3 to 9 mg/kg. However it will be understood that the amount of compound actually administered will be determined by a physician in the light of the relevant circumstances, including the condition to be treated and the chosen route of administration. The invention is illustrated by the following Examples and dioxan (0.2 ml) were added with vigorous stirring, followed by 0.5 ml of a 15.6 mg/ml solution of mouse monoclonal anti-CEA in 0.34 N borate buffer pE 8.6. The mixture was stirred at room temperature for 2 hours maintaining pH 9 using N/10 NaOH. Excess azide was destroyed by adding 1N NH4OH (0.1 ml) and stirring for a further hour. The product was isolated by gel filtration on a 1 x 27 cm (21 ml) column of Bio-Gel R P-6 equilibrated with phosphate buffered saline. The excluded peak was collected (4.07 ml) and assayed for protein and vindesine by spectroscopy at 270 nm and 280 nm. The conjugate so prepared contained 2.1 moles vindesine per mole of Ig. EXAMPLE 3 Preparation of vindesine-rabbit anti-mouse Ig conjugate. Desacetylvinblastine acid hydrazide (12 mg) was dissolved in IN HC1 (0.67 ml) and the solution cooled to 40C. One hundred and twenty microlitres of a 10 mg/ml solution of sodium nitrite were added and the reaction sirred at 40C. After 5 minutes, 1N NaOH (0.6 ml) and dioxan (0.24 ml) were added with vigorous stirring, followed by 1.0 ml of an 11.7 mg/ml solution of rabbit anti-mouse Ig in 0.34 M borate buffer pH 8.6. The mixture was stirred at room temperature for 2 hours maintaining pH 9 using N/10 NaOH. Excess azide was destroyed by adding 1N NH4OH (0.12 ml) and stirring for a further hour. The product was isolated by gel filtration on a 1.5 x 26 cm (46 ml) column of Bio-GelP-6 R P-6 equilibrated with phosphate buffered saline. The excluded peak was collected (6.22 ml) and assayed for protein and vindesine by spectroscopy at 270 nm and 280 nm. The conjugate so prepared contained 3.2 moles vindesine per mole of Ig. EXAMPLE 4 A preparation of cells growing in culture was dispensed into microtitre culture trays at a level of 104 cells per well. After allowing the cells 24 hours for establishment, replicate wells were treated with a range of concentrations of a conjugate or a control preparation. After six days in culture, the number of cells present in the wells was assessed by direct counting, using an image analyser linked to an inverted microscope. In one example, a human colon adenocarcinoma cell line was treated with vindesine linked to a monoclonal anti-carcinoembryonic antigen antibody in a molar ratio of 2.1 moles vindesine per mole of Ig (this was the conjugate of Example 2) and the cytostatic effect measured by cell counts, as follows: Conjugated drug concentration Number of cells/well % of (vindesine, pg/ml) after six days control (mean + standard deviation) 4 replicates 0 16.3 + 4.8 x 104 100 0.08 13.8 + 2.5 x 104 84.6 0.40 12.6 + 1.9 x 104 77.4 2.00 10.8 + 3.9 x 104 66.7 10.00 1.9 + 0.6 x 104 11.9

Claims: CLAIMS 1. A conjugate comprising an immunoglobulin or immunoglobulin fragment modified by one or more groups of the following formula which are covalently linked to it EMI11.1 2. A conjugate according to claim 1 utilising an IgG immunoglobulin. 3. A conjugate according to either of claims 1 and 2 in which the immunoglobulin or immunoglobulin fragment is modified by an average of from 2 to 5 groups of formula (II). 4. A process for preparing a conjugate according to claim 1 which comprises reacting an immunoglobulin or immunoglobulin fragment with an azide compound of the formula EMI12.1 5. A pharmaceutical formulation comprising a conjugate according to any of claims 1 to 3 together with a pharmaceutically-acceptable carrier or diluent therefor. CLAIMS 1. A process for producing a conjugate comprising an immunoglobulin or immunoglobulin fragment modified by one or more groups of the following formula which are covalently linked to it EMI13.1 which comprises reacting an immunoglobulin or immunoglobulin fragment with desacetylvinblastine acid azide. 2. A process according to claim 1 for the preparation of a conjugate comprising an IgG immunoglobulin. 3. A EMI13.2 according to either of claims 1 and 2 in which the immunoglobulin or immunoglobulin fragment is modified by an average of from 2 to 5 groups of formula (II).

Inventors: ROWLAND, GEORGE FERDINAND, SIMMONDS, ROBIN GEORGE
Applicants: LILLY INDUSTRIES LIMITED
Application year: 1982
Main classification: C07K15
IPC codes: C07D519, A61K31, C07B43, A61K39, A61P35, A61K47