Synthesis and biological evaluation of immunoconjugates of adriamycin and a human IgM linked by poly[N5-2-hydroxyethyl)-l-glutamine]

Abstract

The synthesis and purification of radiolabelled immunoconjugates, composed of a human IgM monoclonal antibody (IgM 16.88) directed against an intracellular tumour-associated antigen, the drug carrier poly [NS-(2-hydroxyethyl)-L-glutamine] (PHEG) and the cytostatic drug adriamycin (ADR) are described. The immunoconjugates were constructed to allow selective release of ADR in the putatively acidic environment of the tumour through a novel acid-labile maleamic acid linker. The conjugate of PHEG and the acid-labile ADR derivative effectively released ADR in cytotoxic amounts at a pH of 6.0 as judged from incubation in buffer and from inhibition of the growth of HT-29 colon tumour cells in vitro. Immunoconjugates were prepared by coupling of PHEG-ADR having a hydrolytically stable amide bond with J3q-labelled antibody through tbioether bond formation involving a single thiol group at the C-terminus of the polymer chain and maleimido groups introduced onto the * Corresponding author. Abbreviations: AcOSu,N-succinimidyl acetate; ADR, adriamycin; AEC, anion-exchange chromatography; ASI, active specific immunotherapy; BOC, tert-butox ycarbonyl ; (BOC) 20, di-tert-butyldicarbonate; BOC-AB, N-tert-butox ycarbonyl-1,4-diaminobutane; BOC-OEA, N-tert-buto xycarbonyl-2,2'-oxybis(ethylamine); CDI, N,N'-carbonyldiimidazole; CHC13, chloroform; CH2CIz, dichloromethane; CTA-I, colon tumourassociated antigen; Cys, cysteine; d. doublet; dd, double doublet; dt, double triplet; DAB, 3,3'-aminobenzidine; DMF. N,N'-dimethylformamide; DMSO, dimethylsulfoxide; DPn, number-average degree of polymerization; DPw, weight-average degree of polymerization; DSS, 4,4-dimethyl-4-silapentane sodium sulphonate; DTNB, 5,5'-dithiobis ( 2-nitrobenzoic acid) ; DTP, 4,4'-dithiodipyridine; DTT, dithiothreitol; e, molar absorbance coefficient; EEDQ, N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline; EIA, enzyme-linked immunoassay; Et20, diethyl ether; FABMS, fast atom bombardment mass spectrometry; FCS, fetal calf serum; GABA, y-aminobutyric acid; GMB, y-maleimidobutyryl; GMBS, N-(ymaleimidobutyryloxy)succinimide; HOAc, acetic acid; HONSu, N-hydroxysuccinimide; HPMA, N-(2-hydroxypropyl)methacrylamide; HP-SEC, high-performance size exclusion chromatography; HRP, horseradish peroxidase; LALLS, low-angle laser light scattering; m, multiplet; M,. number-average molecular weight; Mw, weight-average molecular weight; MRW, mean residue weight; MoAb, monoclonal antibody: MSA, methanesulphonic acid; MTT, 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NAN,, sodium azide; NCA, N-carboxyanhydride; PBLG, poly( T-benzyl-L-glutamate); PBS, phosphate-buffered saline; PDT, 2-pyridyldithio; PGA, poly Hoes et al. / Journal of Controlled Release 38 (1996) antibody. The immunoreactivity of IgM-PHEG-ADR conjugate was almost fully preserved. Tumour uptake and biodistribution of ~251-1abelled PHEG-ADR and of ~3q-labelled IgM-PHEG-ADR, which was co-administered with ~H-labelled IgM 16.88, in nude mice carrying MRI-W207 human ovarian tumour xenografts were s...

Synthesis of soluble polymers for medicine that degrade by intramolecular acid catalysis

“…These studies defined a structural motif which we have incorporated into a polymer mainchain with the expectation that degradation will occur at acidic pH values (Scheme 2). Cis-aconityl acid [14][15][16][31][32][33][34][35][36][37][38] derived linkers have been used to conjugate bioactive molecules (e. g. a low molecular weight drug or a protein) to macromolecules and are thought to degrade by this mechanism in the acidic environment of the lysosome. The "enforced propinquity" 30) of the pendent carboxylic acid at C-4 is necessary to ensure intramolecular interactions facilitate hydrolysis at the C-4 amide.…”

“…Up to a molecular weight known as the renal threshold, plasma clearance is primarily governed by the rate of kidney glomerular filtration and liver uptake. The drug conjugating pendent chains degrade by either proteolytic [7][8][9][10][11][12][13] or hydrolytic [14][15][16] mechanisms in the lysosome resulting in the intracellular release of the drug [17][18][19][20] . As the solution size of a molecule increases, extended blood clearance times result.…”

Professor Jan Feijen: A pioneer in biomedical polymers and controlled drug release

“…He has also investigated the release of proteins and macromolecules from albumin-heparin microspheres [53,54], synthesis and biodistribution of immuno-conjugates of a human IgM [51], heparin release from thermosensitive hydrogels [55,56], and delivery of antibacterial proteins from gelatin-chondroitin sulfate hydrogels [57]. He has prepared and explored porous membranes for drug delivery [58], studied the biological properties of adriamycin bound to biodegradable polymeric carriers [49,51], and developed albumin-heparin conjugate microspheres for loading and release of adriamycin [31,59]. These adriamycin-loaded albumin-heparin conjugate microspheres were applied for intraperitoneal chemotherapy [60].…”

“…These adriamycin-loaded albumin-heparin conjugate microspheres were applied for intraperitoneal chemotherapy [60]. The immuno-conjugates of adriamycin and a human IgM were obtained by using poly[N5-(2-hydroxyethyl)-L-glutamine] as a linker [51]. The local release of growth factor from heparinized-collagen matrices was shown to improve endothelialization of vascular grafts [61,62].…”

“…As from late 1970s, Prof. Feijen reported macromolecular prodrugs of adriamycin based on poly(L-glutamic acid) for cancer therapy [49][50][51], biodegradable hollow fibers for the controlled release of anti-conceptive hormones [52], and biodegradable polymeric prodrugs of naltrexone [4,5]. He has also investigated the release of proteins and macromolecules from albumin-heparin microspheres [53,54], synthesis and biodistribution of immuno-conjugates of a human IgM [51], heparin release from thermosensitive hydrogels [55,56], and delivery of antibacterial proteins from gelatin-chondroitin sulfate hydrogels [57]. He has prepared and explored porous membranes for drug delivery [58], studied the biological properties of adriamycin bound to biodegradable polymeric carriers [49,51], and developed albumin-heparin conjugate microspheres for loading and release of adriamycin [31,59].…”