Adriamycin(hydrazone)-antibody conjugates require internalization and intracellular acid hydrolysis for antitumor activity

Abstract

Adriamycin hydrazone (ADM-Hzn) immunoconjugates have previously been shown to exhibit antibody-directed antitumor activity in vitro and in vivo. In this report, the biological and biochemical properties of the mAb and linker were investigated. Conjugates prepared with two antibodies 5E9 [anti-(transferrin receptor)] and G28.1 (anti-CD37), (which internalize from the surface of target cells following binding) were more cytotoxic in vitro and had greater antitumor activity against Daudi B lymphoma tumor xenografts than a non-internalizing immunoconjugate prepared with mAb 2H7 (anti-CD20). In addition, the 13-acylhydrazone bond linking the drug to the mAb was labile at pH 5 and released unmodified ADM at a rapid rate (t1/2 = 2.5 h). Immunoconjugates prepared with an oxime linkage at the C-13 position were stable to acid and were not cytotoxic. These findings suggest that internalization of ADM-Hzn immunoconjugates and release of free ADM from the mAb in acidic intracellular compartments were important steps in the mechanism of action of ADM-Hzn immunoconjugates.

Efficient Elimination of B-Lineage Lymphomas by Anti-CD20–Auristatin Conjugates

“…However, these improvements are not curative, and resistance or relapse after treatment remains significant in CD20-positive disease. Anti-CD20 mAbs linked to cytotoxics (21), plant toxins (23), or to immunoliposomes containing Dox (22) were ineffectual avenues for drug delivery to the cell interior. Surprisingly, anti-CD20 conjugated to the antimitotic MMAE has showed significant antitumor activity.…”

“…Antibody-drug conjugates of anti-CD20 have been evaluated and generally found to be ineffective. Anti-CD20 Dox conjugates (21), and more recently a rituximab-liposomal Dox (22) and anti-CD20-ricin A-chain conjugate, were not effective against CD20 positive cells (23). These results were partially attributed to differences in CD20 trafficking because other conjugates could induce modulation and internalization of mAb/antigen com-plexes, whereas a comparable effect was not observed with anti-CD20 (24).…”

Enhanced cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin–GnRH-III bioconjugates

“…Interestingly, a fragment was detected by mass spectrometry at m/z 581.2 (1+), which might be assigned to <EHWK-OH peptide, indicating the cleavage of the amide bond between the side chain of 4 Lys and the fatty acids [10,27]. However, the unambiguous identification of this fragment and of the free SCFA release requires further investigations.…”

“…In the case of the reported bioconjugates, an oxime bond was formed between the C13 keto group of daunorubicin and the aminooxyacetyl group of the GnRH-III derivatives. The oxime bond is chemically stable between pH 3 and 8 [25], as well as under in vitro and in vivo biological experimental conditions [26]. Moreover, the daunorubicin-GnRH-III bioconjugates must possess increased stability in the presence of digestive enzymes present in stomach and intestine in order to be suitable for oral administration.…”

Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis

“…In the same year Pastan and colleagues described the delivery of Pseudomonas exotoxin-antiTfR antibody conjugates into cellular endosomes [49]. Subsequent stu-dies have confirmed that the Tf delivery pathway is very flexible in accommodating transport of both small drugs [16][17][18][19][20][21][22][23][24][25][26], such as adriamycin, and structures [72][73][74][75][76] such as Tf-coated liposomes.…”