Monoclonal Antibodies to Human Fibrin: Interaction with other Animal Fibrins

Summary

Four monoclonal antibodies have previously been raised in our laboratory for possible use in thrombus imaging and the targeting of thrombolytic agents. These antibodies were raised to various human fibrin-related immunogens and each antibody had been selected for its specificity towards fibrin and not fibrinogen. To study further these antibodies in animal circulation models both in vivo and in vitro, their selectivity towards human fibrin as opposed to other animal fibrins was examined. In this study dissociation constants for each antibody with each of six species fibrins (human, baboon, pig, dog, sheep, and rabbit) were estimated using both fibrin clots and monolayers. Some limited data were also obtained with Sepharose-fibrin. Of the antibodies two (denoted 12B3B10 and 12B3A11) are seen to bind almost exclusively to human fibrin with dissociation constants of about 8 × 10⁻¹⁰ M using fibrin clots and monolayers. These same two mabs bound to baboon fibrin with a dissociation constant of 2 × 10⁻⁹ M, while neither displayed significant levels of binding to the fibrins from dog, pig, sheep and rabbit. The other two antibodies investigated (1H10 and 5F3) were found to bind well to fibrins of human, baboon, pig and dog, with dissociation constants in the range of 1.4-4.2 × 10⁻⁹ M. However neither 1H10 nor 5F3 displayed significant recognition of sheep and rabbit fibrins. Both 1H10 and 5F3 were also found by means of competitive ELISA’s to retain their selectivity to baboon, dog and pig fibrins in the presence of their respective fibrinogens.

• References

• 1 Kudryk BJ, Bini A, Rosebrough SF, Schaible TF. Fibrinogen-fibrin: preparation and use of monoclonal antibodies as diagnostics. In: Biotechnology of blood Goldstein J. (ed). Butterworth-Heinmann, Stoneham, MA; 281-313
• 2 Tymkewycz PM, Creighton LJ, Gascoine PS, Zanelli GD, Webbon PM, Gaffney PJ. Imaging of human thrombi in the rabbit jugular vein: comparison of two fibrin specific monoclonal antibodies. In: Radiolabelled antifibrin antibody in the detection of venous thrombosis: preliminary results. Erger HJ Radiology; 1990. 175 79-85
• 3 Knight LC, Maurer AH, Ammar IA, Epps LA, Dean RT, Pah KY, Berger HJ. Tc-99m Antifibrin Fab1 fragments for imaging venous thrombi. Evaluation in a canine model. Radiology 1989; 173: 163-169
• 4 Hui KY. An in vivo chicken model for peripheral intravascular human fibrin clot detection. J Pharm Methods 1988; 20: 79-84
• 5 Wasser MNJM, Koppert PW, Arndt JW, Emeis JJ, Feitsma RIJ, Pauwels EKJ, Nieuwenhuizen W. An antifibrin monoclonal antibody useful in the immunoscintigraphic detection of thrombi. Blood 1989; 74 (02) 708-714
• 6 Wasser MNJM, Cleyndert P, Camps JAJ, Feitsma RIJ, Nieuwenhuizen W, Pauwels EK. J. An in vitro model for the immunoscintigraphic detection of thrombi using a ^99mTc-labelled antifibrin monoclonal antibody. Nucl Med Comm 1989; 10: 653-659
• 7 Runge MS, Bode C, Matsueda GR, Haber E. Conjugation to an antifibrin monoclonal antibody enhances the fibrinolytic potency of tissue plasminogen activator in vitro. Biochemistry 1988; 27: 1153-1157
• 8 Runge MS, Bode C, Matsueda GR, Haber E. Antibody-enhanced thrombolysis: capture of tissue plasminogen activator by a bispecific antibody and direct targeting by an antifibrin-tissue plasminogen activator conjugate in vivo. Proc Natl Acad Sci USA 1987; 84: 7659-7662
• 9 Dewerchin M, Vandamme AM, Holvoet P, De Cock F, Lemmens G, Lijnen HR, Stassen J-M, Collen D. Thrombolytic and pharmacokinetic properties of a recombinant chimeric plasminogen activator consisting of a fibrin fragment D-dimer specific humanized monoclonal antibody and a truncated single-chain urokinase. Thromb Haemost 1992; 69 (02) 170-179
• 10 Holvoet P, Dewerchin M, Stassen J-M, Lijnen HR, Tollenaere T, Gaffney PJ, Collen D. Thrombolytic profiles of clot targeted plasminogen activators: parameters determining potency and initial and maximal rates. Circulation 1993; 87: 1007-1016
• 11 Tymkewycz PM, Creighton-Kempsford LJ, Hockley D, Gaffney PJ. Screening for fibrin specific monoclonal antibodies: the development of a new procedure. Thromb Haemost 1992; 68 (01) 48-53
• 12 Tymkewycz PM, Creighton-Kempsford LJ, Gaffney PJ. Generation and partial characterisation of five monoclonal antibodies with high affinities for fibrin. Blood Coag Fibrinolysis 1992; 4: 211-222
• 13 Edgell TA, Gaffney PJ, Hockley D, Webbon P, Walker J. Fibrin aggregation. The effect of a fibrin specific monoclonal antibody on the fibrin aggregation process. Abstracts of Xllth International Congress on Fibrinolysis. Fibrinolysis 1994 1. 37 (abstract).
• 14 Kudryk B, Rohoza A, Ahadi M, Chin J, Wiebe ME. Specificity of a monoclonal antibody for the NH2-terminal region of fibrin. Mol Immunol 1984; 21: 89-94
• 15 Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods 1985; 77: 305-319
• 16 Procyk R, Kudryk B, Callender S, Blomback B. Accessibility of epitopes on fibrin clots and fibrinogen gels. Blood 1991; 77: 1469-1475
• 17 Ashcom JD, Tiller SE, Dicherson K, Cravens JL, Argraves WS, Strickland DK. The human Β₂-macroglobulin receptor: identification of a 420-KD cell surface glycoprotein specific for the activated conformation of Β₂-macro-globulin. J Cell Biol 1990; 10: 1041-1048
• 18 Hunter WM, Greenwood FC. Preparation of Iodine-131 labelled human growth hormone of high specific activity. Nature 1962; 194: 495-496
• 19 Lindmo T, Boven E, Cuttitta F, Fedorho J, Bunn Jr. PA. Determination of the immunoreactive fraction of radiolabelled monoclonal antibodies by linear extrapolation to binding to infinite antigen excess. J Immunol Meth 1984; 72: 77-89
• 20 Rosebrough SF, McAfee JG, Grossman ZD, Schemancik LA. Immunoreac-tivity of ¹¹¹In and ¹³¹I fibrin specific monoclonal antibody used for thrombus imaging. J Immunol Meth 1989; 116: 123-129
• 21 Holm B, Brosstad F, Kierulf P, Godal HC. Polymerization properties of two normally circulating fibrinogens, HMW and LMW. Evidence that the COOH-terminal end of the a-chain is of importance for fibrin polymerization. Thromb Res 1985; 39: 595-606
• 22 Vehlich YI, Gorhun OV, Medved LV, Nieuwenhuizen W, Weisel JW. Carboxyterminal portions of the a chains of fibrinogen and fibrin. J Biol Chem 1993; 268 (18) 13577-13585
• 23 Koopman J, Haverkate F, Grimbergen J, Lord ST, Mosesson MW, Diorio JP, Siebenlist KS, Legrand C, Soria J, Soria C, Caen JP. Molecular basis for fibrinogen Dusart (AΑ554 Arg → Cys) and its association with abnormal fibrin polymerization and thrombophilia. J Clin Invest 1993; 91: 1637-43
• 24 Cierniewski CS, Budzynski AZ. Involvement of the a chain in fibrin clot formation. Effect of monoclonal antibodies. Biochemistry 1992; 31: 4248-4253
• 25 Gaffney PJ, Mahmoud M. The influence of function on the recent evolution of fibrinogen. J Mol Med 1977; 2: 351-369
• 26 Lanza GM, Wallace KD, Scott MJ, Sheehan CK, Cacheriss WP, Christy DG, Starkey AM, Miller JG, Gaffney PJ, Wickline SA. Development and validation of a novel ligand targeted acoustic contrast agent for imaging vascular thrombi. (Submitted to Circulation).