Skip to main content
SpringerLink
Log in

Synthesis of site-specific methotrexate-IgG conjugates

Comparison of stability and antitumor activity with active-ester-based conjugates

  • Original Articles
  • Published:
Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Summary

With a view to increasing drug incorporation without loss of antibody activity, tritium-labeled methotrexate (MTX) was covalently linked to a polyclonal rabbit IgG antibody against bovine serum albumin and a monoclonal mouse IgG antibody against human renal cancer (Dal K20) by a site-specific method based on hydrazone bond formation between MTX hydrazide and the aldehyde groups generated by periodate oxidation of carbohydrate moieties in IgG (which are uncommon in the antigen-binding region). These conjugates were compared with the corresponding non-site-specific MTX-IgG conjugates produced by the N-hydroxysuccinimide active-ester method with regard to synthesis, stability, retention of antibody activity, inhibition of the target enzyme dihydrofolate reductase and antitumor effect. Incorporation levels achieved with the hydrazide method were no greater than with the active-ester method, typically 6–7 mol MTX/mol IgG. Approximately the same dihydrofolate-reductase-inhibitory capacity was observed for MTX bound by either method. Hydrazide conjugates lost bound drug more rapidly than active-ester conjugates on freezing and thawing, on incubation at 37° C and 51° C, and in the presence of serum or rat liver homogenates. Exposure to rat liver homogenates at 37° C, pH 4.6, for 24 h led to the loss of 50%–60% of the bound drug from hydrazide conjugates compared to 20%–30% from the active ester conjugates. Bio-Gel P-2 chromatography of low-molecular-mass fractions, obtained after exposure of each of the conjugates to liver homogenates, revealed the presence of a compound that had the same elution volume and R F on thin-layer chromatography as free MTX. Enzyme-linked immunosorbent assay showed loss of antibody activity of both types of conjugates at 51° C and on freezing and thawing. In a clonogenic assay, the active-ester conjugate of Dal K20 appeared to be equally effective or slightly better as a tumor inhibitor than the corresponding hydrazide conjugate. The hydrazide method may be useful in linking MTX to those monoclonal antibodies that tend to denature when subjected to the active-ester method of linkage.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Baldwin RW, Embleton MJ, Garnett MO, Pimm MV (1987) Conjugates of monoclonal antibody 791T/38 with methotrexate in cancer therapy. NCI Monogr 3:95

    Google Scholar 

  2. Bayer EA, Wilchek M (1980) The use of avidin-biotin complex as a tool in molecular biology. Methods Biochem Anal 26:1

    Google Scholar 

  3. Belinska M, Nimec Z, Galivan J (1982) Characteristics of methotrexate polyglutamate formation in cultured hepatic cells. Arch Biochem Biophys 216:466

    Google Scholar 

  4. Blair AH, Ghose T (1983) Linkage of cytotoxic agents to immunoglobulins. J Immunol Methods 59:28

    Google Scholar 

  5. Bough CM, Krumdieck CL, Nair MG (1973) Polygammaglutamyl metabolites of methotrexate. Biochem Biophys Res Commun 52:27

    Google Scholar 

  6. Chua MM, Fan ST, Karush F (1984) Attachment of immunoglobulin to liposomal membrane via protein carbohydrate. Biochim Biophys Acta 800:291

    Google Scholar 

  7. Clemo GR, Smith JM (1928) The reduction of p-dimethylaminobenzaldehyde and the preparation of p-dimethylaminobenzyl alcohol. J Chem Soc 2423

  8. Deguchi T, Chu TM, Leong SS, Horoszewicz JS, Lee CL (1986) Effect of methotrexate-monoclonal anti-prostatic acid phosphatase antibody conjugate on human prostate tumor. Cancer Res 46:3751

    Google Scholar 

  9. Dopp D, Dopp H (1985) Carbonsaure-hydrazide. In: Falbe J (ed) Methoden der Organischen Chemie. Houben-Weyl, Georg Thieme, Stuttgart, Band E5, p 1154

    Google Scholar 

  10. Embleton MJ, Garnett MC, Baldwin RW (1984) Use of an antitumor monoclonal antibody for targeting MTX. Protides Biol Fluids 32:429

    Google Scholar 

  11. Embleton MJ, Nabib NA, Garnett MC, Wood C (1986) Unsuitability of monoclonal antibodies to oncogene proteins for antitumor drug targeting. Int J Cancer 38:821

    Google Scholar 

  12. Endo N, Kato Y, Takeda Y, Saito M, Umemoto N, Kishida K, Hara T (1987) In vitro cytotoxicity of a human serum albumin-mediated conjugate of methotrexate with anti-MM46 monoclonal antibody. Cancer Res 47:1076

    Google Scholar 

  13. Endo N, Takeda Y, Kishida K, Kato Y, Saito M, Umemoto N, Hara T (1987) Target selective cytotoxicity of methotrexate conjugated with monoclonal anti-MM46 antibody. Cancer Immunol Immunother 25:1

    Google Scholar 

  14. Fields R, Dixon HBF (1968) A spectrophotometric method for the microdetermination of periodate. Biochem J 108:883

    Google Scholar 

  15. Fogh J, Trempe G (1975) New human tumor cell lines. In: Fogh J (ed) Human tumor cells in vitro. Plenum Press, New York, p 115

    Google Scholar 

  16. Fry DW, Yolowich JC, Goldman ID (1982) Rapid formation of poly-γ-glutamyl derivatives of methotrexate and their association with dihydrofolate reductase as assessed by high pressure liquid chromatography in the Ehrlich ascites tumor cell in vitro. J Biol Chem 257:1890

    Google Scholar 

  17. Galivan J (1979) Transport and metabolism of methotrexate in normal and resistant cultured rat hepatoma cells. Cancer Res 39:735

    Google Scholar 

  18. Garnett MC, Baldwin RW (1986) An improved synthesis of a methotrexate-albumin 791T/36 monoclonal antibody conjugate cytotoxic to human osteogenic sarcoma cell lines. Cancer Res 46:2407

    Google Scholar 

  19. Garnett MC, Embleton MJ, Jacobs E, Baldwin RW (1983) Preparation and properties of a drug-carrier-antibody conjugate showing selective antibody-directed cytotoxicity in vitro. Int J Cancer 31:661

    Google Scholar 

  20. Gewitz DA, White JC, Randolph JK, Goldman ID (1979) Formation of methotrexate polyglutamates in rat hepatocytes. Cancer Res 39:2914

    Google Scholar 

  21. Ghose T, Blair AH, Kralovec J, Mammen M, Uadia P (1988) Synthesis and testing of antibody-antifolate conjugates for drug targeting. In: Rodwell JD (ed) Antibody-mediated delivery systems. Marcel Dekker, New York, p 81

    Google Scholar 

  22. Ghose T, Blair AH, Kulkarni PN (1983) Preparation of antibody-linked cytotoxic agents. Methods Enzymol 93:280

    Google Scholar 

  23. Hurwitz E, Levy R, Maron R, Wilcheck M, Arnon R, Sela M (1975) The covalent binding of daunomycin and adriamycin to antibodies with retention of both drug and antibody activities. Cancer Res 35:1175

    Google Scholar 

  24. Hurwitz E, Wilchek M, Pitha J (1980) Soluble macromolecules as carriers for daunorubicin. J Appl Biochem 2:25

    Google Scholar 

  25. Ito Y, Yamasaki Y, Seno N, Matsumoto I (1986) Preparation of high capacity adsorbents using new hydrazino-carriers and their use for low and high performance affinity chromatography of lectins. J Biochem (Tokyo) 99:1267

    Google Scholar 

  26. Jallal B, Salesse K, Garnier J (1986) A high-capacity affinity gel for the purification of the testicular lutropin receptor. C R Acad Sci 303:73

    Google Scholar 

  27. Johnson DA, Laguzza BC (1987) Antitumor xenograft activity with a conjugate of a Vinca derivative and the squamous carcinoma-reactive monoclonal antibody PF1/D. Cancer Res 47:3118

    Google Scholar 

  28. Kanellos J, Pietersz GA, McKenzie JFC (1985) Studies of methotrexate-monoclonal-antibody conjugates for immunotherapy. J Natl Cancer Inst 75:319

    Google Scholar 

  29. Karpinski KF, Haywards S, Tryphonas K (1987) Statistical considerations in the quantitation of serum immunoglobulin levels using the enzyme-linked immunosorbant assay. J Immunol Methods 103:189

    Google Scholar 

  30. Kornguth ML, Neidle A, Wealsch H (1963) The stability and rearrangement of ε-N-glutamyl-lysines. Biochemistry 2:740

    Google Scholar 

  31. Kulkarni PN, Blair AH, Ghose T (1981) Covalent binding of methotrexate to immunoglobulins and the effect of antibody-linked drug on tumor growth in vivo. Cancer Res 41:2700

    Google Scholar 

  32. Kulkarni PN, Blair AH, Ghose T, Mammen M (1985) Conjugation of methotrexate to IgG antibodies and their F(ab)2 fragments and the effect of conjugated methotrexate on tumor growth in vivo. Cancer Immunol Immunother 16:127

    Google Scholar 

  33. Lathan B, von Hoff DD, Elslager E (1984) Use of a human tumor cloning system to evaluate analogs of methotrexate and mitoxantrone. Cancer Treat Rep 68:733

    Google Scholar 

  34. Lopes AD, Radcliffe RD, Coughlin DJ, Lee LS, McKearn TJ and Rodwell JD (1987) Site-selective methotrexate-antibody conjugates yield a superior therapeutic effect in tumor xenograft-bearing nude mice. In: Prozanski W, Seligmann M (eds) Clinical immunology. Elsevier, New York, p 303

    Google Scholar 

  35. Lowry OH, Rosebrough NJ, Farr AL, Randall R (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:165

    Google Scholar 

  36. Luner SJ, Ghose T, Chatterjee S, Cruz HN, Belitsky P (1986) Monoclonal antibodies to kidney and tumor-associated surface antigens of human renal cell carcinoma. Cancer Res 46:5816

    Google Scholar 

  37. Manabe Y, Tsubota T, Haruta Y, Kataoka K, Okazaki M, Haisa S, Nakamura K and Kimura I (1984) Production of a monoclonal antibody-methotrexate conjugate utilizing dextran-T40 and its biological activity. J Lab Clin Med 104:445

    Google Scholar 

  38. Mancini G, Carbonara AO, Heremans JF (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235

    Google Scholar 

  39. McKearn TJ, Lopes AD, Radcliffe RD, Coughlin DJ, Hrubiec RT, Rodwell JD (1989) In vivo efficacy of site-specific antifolate monoclonal antibody conjugates. Antibody Immunoconjugates and Radiopharmaceuticals (in press)

  40. McKinney M, Parkinson A (1987) A simple non-chromagraphic procedure to purify immunoglobulins from serum and ascites fluid. J Immunol Methods 96:271

    Article  CAS  PubMed  Google Scholar 

  41. Murayama A, Shimada K, Yamamoto T (1978) Modification of immunoglobulin G using specific reactivity of sugar moiety. Immunochemistry 15:523

    Google Scholar 

  42. O'Shannessy DJ, Quarles RH (1985) Specific conjugation of the oligosaccharide moieties of immunoglobulins. J Appl Biochem 7:347

    Google Scholar 

  43. O'Shannessy DJ, Hoffman WL (1987) Site-directed immobilization of glycoproteins on hydrazide-containing solid supports. Biotech Appl Biochem 9:488

    Google Scholar 

  44. O'Shannessy DJ, Dobersen MJ, Quarles RH (1984) A novel procedure for labelling immunoglobulins by conjugation to oligosaccharide moieties. Immunol Lett 8:273

    Google Scholar 

  45. Peterson DL, Gleisner JM, Blakley RL (1975) Bovine liver dihydrofolate reductase: Purification and properties of the enzyme. Biochemistry 14:5261

    Google Scholar 

  46. Pimm MV, Clegg JA, Garnett MC, Baldwin RW (1988) Biodistribution and tumor localization of a methotrexate monoclonal-antibody 791T/36 conjugate in nude mice with human tumor xenografts. Int J Cancer 41:886

    Google Scholar 

  47. Piper JR, Montgomery JA (1982) Synthesis of α- and γ-substituted amides, peptides and esters of methotrexate and their evaluation as inhibitors of folate metabolism. J Med Chem 25:182

    Google Scholar 

  48. Poser RG, Sirotnak FM, Chello PL (1980) Extracellular recovery of methotrexate-polyglutamates following efflux from L1210 leukemia cells. Biochem Pharmacol 29:2701

    Google Scholar 

  49. Rodwell JD, Alvarez VL, Lee C, Lopes AD, Goers JWF, King HD, Powsner MJ, McKearn TJ (1986) Site specific covalent modification of monoclonal antibodies. In vitro and in vivo evaluations. Proc Natl Acad Sci USA 83:2632

    Google Scholar 

  50. Rosowsky A, Yu C-S, Uren J, Lazarus H, Wick M (1981) Methotrexate analogues 13. Chemical and pharmacological studies on amide, hydrazide and hydroxamic acid derivatives. J Med Chem 24:559

    Google Scholar 

  51. Scheidegger JJ (1955) Une micro-methode de l'immunoelectrophorese. Int Arch Allergy Appl Immunol 7:103

    Google Scholar 

  52. Shen W-C, Ryser HJ-P (1984) Selective killing of Fc receptor-bearing tumor cells through endocytosis of a drug-carrying immune complex. Proc Natl Acad Sci USA 81:1445

    Google Scholar 

  53. Shih LB, Sharkey RM, Primus FJ, Goldenberg DM (1988) Site specific linkage of methotrexate to monoclonal antibodies using an intermediate carrier. Int J Cancer 41:832

    Google Scholar 

  54. Singh M, Kralovec J, Mezei M, Ghose T (1989) Inhibition of human renal cancer by methotrexate linked to a monoclonal antibody. J Urol (in press)

  55. Sirotnak FM, Chello PL, DeGraw JI, Piper JR, Montgomery JR (1981) In: Sartorelli AC (ed) Molecular action and targets for chemotherapeutic agents. Academic Press, New York, p 349

    Google Scholar 

  56. Uadia PO, Blair AH, Ghose T (1988) Hydrolysis of methotrexate-immunoglublin conjugates by liver homogenates and characterization of catabolites. Cancer Chemother Pharmacol 22:175

    Google Scholar 

  57. Weber K, Osborn M (1969) The reliability of molecular weight determination by dodecyl sulphate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406

    Google Scholar 

  58. Wilchek M, Bayer EA (1987) Labeling glycoconjugates with hydrazide reagents. Methods Enzymol 138:429

    Google Scholar 

  59. Wilchek M, Bayer EA (1987) Labeling glycoconjugates with hydrazide reagents. Methods Enzymol 138:437

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departments of Biochemistry and Pathology, Dalhousie University, B3H 4H7, Halifax, Nova Scotia, Canada

    J. Kralovec, M. Singh, M. Mammen, A. H. Blair & T. Ghose

Authors
  1. J. Kralovec
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Mammen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. H. Blair
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T. Ghose
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Abbreviations used: aBSA, rabbit anti-(bovine serum albumin) IgG; EDCI, 3-ethyl-1-(3-dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbant assay; IC50, concentration giving 50% inhibition; MTX, methotrexate; MTXAE, N-hydroxy-succinimide-based active ester of MTX; MTXAE-IgG, MTX-IgG conjugate prepared by the active-ester method; MTXH, methotrexate hydrazide; MTXH-IgG, MTX-IgG conjugate prepared by the hydrazide method; PBS, 0.01 M sodium phosphate, pH 7.1, containing 0.145 M sodium chloride; TLC, thin-layer chromatography

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kralovec, J., Singh, M., Mammen, M. et al. Synthesis of site-specific methotrexate-IgG conjugates. Cancer Immunol Immunother 29, 293–302 (1989). https://doi.org/10.1007/BF00199218

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00199218

Keywords

Search

Navigation