Differential Cellular Internalization of Anti-CD19 and -CD22 Immunotoxins Results in Different Cytotoxic Activity

Du, Xiaohong; Beers, Richard; FitzGerald, David J.; Pastan, Ira

doi:10.1158/0008-5472.can-08-0461

Cited by 111 publications

(118 citation statements)

References 41 publications

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“…This concept, however, has recently been challenged, as potent and selective anticancer activity has been observed with ADCs and SMDCs specific to antigens, which do not internalize [19,20,[28][29][30][31].…”

Section: Introductionmentioning

confidence: 99%

Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma

Cazzamalli

Corso

Neri

2016

Molecular Cancer Therapeutics

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In most cases, cytotoxic drugs do not preferentially accumulate at the tumor site, causing unwanted toxicities and preventing dose escalation to therapeutically active regimens. Here, we show that acetazolamide derivatives, which bind to carbonic anhydrase IX (CAIX) on the surface of kidney cancer cells, selectively deliver payloads at the site of disease, sparing normal organs. Biodistribution studies, performed in tumor-bearing mice with acetazolamide derivatives bearing a technetium-99m chelator complex or a red fluorophore as payload, revealed a preferential tumor accumulation of the compound at doses up to 560 nmol/Kg. The percentage of injected dose per gram in the tumor was dose-dependent and revealed optimal tumor:organ ratios at 140 nmol/Kg, with a tumor:blood ratio of 80:1 at 6 h. Acetazolamide, coupled to potent cytotoxic drugs via a dipeptide linker, exhibited a potent antitumor activity in nude mice bearing SKRC-52 renal cell carcinomas, while drug derivatives devoid of the acetazolamide moiety did not exhibit any detectable anticancer activity at the same doses. The observation of tumor regression with a noninternalizing ligand and with different cytotoxic moieties (MMAE and PNU-159682) indicates a general mechanism of action, based on the selective accumulation of the product on tumor cells, followed by the extracellular proteolytic release of the cytotoxic payload at the neoplastic site and the subsequent drug internalization into tumor cells. Acetazolamide-based drug conjugates may represent a promising class of targeted agents for the treatment of metastatic kidney cancer, as the majority of human clear cell renal cell carcinomas are strongly positive for CAIX.

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Section: Introductionmentioning

confidence: 99%

Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma

Cazzamalli

Corso

Neri

2016

Molecular Cancer Therapeutics

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“…For some native toxins such as ricin, intracellular delivery is achieved through lectin binding, followed by internalization and toxin release with membrane fusion or retrograde trafficking (5). Immunotoxins attempt to recreate this scenario by replacing the indiscriminate lectin binding with cancer-specific antigen binding as a means of targeting and internalization (6). Subsequent intracellular trafficking, release, and endosomal escape are often achieved using existing toxin characteristics, translocation domains, protease cleavage sites, disulfide bonds, and/or signaling peptides (7)(8)(9)(10).…”

mentioning

confidence: 99%

Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties

Pirie¹,

Hackel²,

Rosenblum

et al. 2011

Journal of Biological Chemistry

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Gelonin-based immunotoxins vary widely in their cytotoxic. Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ϳ5 ؋ 10 6 toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.Immunotoxins are a promising approach to the targeted delivery of highly potent, cancer-specific, cytotoxic agents. Immunotoxins are frequently composed of a targeting moiety (derived from antibodies or other cell-binding proteins) either chemically conjugated or genetically fused to highly cytotoxic plant or bacterial protein toxins. Clinical success for immunotoxins has been mostly limited to hematological malignancies due to transport limitations in solid tumors (1). Such limitations have been extensively studied experimentally (2) and with several computational models (3, 4).The potency of a particular immunotoxin is dependent on the ability to deliver the toxin to the cytoplasm, which is commonly considered to be the rate-limiting step. For some native toxins such as ricin, intracellular delivery is achieved through lectin binding, followed by internalization and toxin release with membrane fusion or retrograde trafficking (5). Immunotoxins attempt to recreate this scenario by replacing the indiscriminate lectin binding with cancer-specific antigen binding as a means of targeting and internalization (6). Subsequent intracellular trafficking, release, and endosomal escape are often achieved using existing toxin characteristics, translocation domains, protease cleavage sites, disulfide bonds, and/or signaling peptides (7-10). However, the inclusion of toxins with domains facilitating cytoplasmic access can also lead to increased nonspecific toxicity in vivo (11,12).Gelonin is a plant toxin and classified as a type I ribosomeinactivating protein because it lacks any cell-binding or cytoplasmic delivery domains. Recombinant gelonin (rGel) 2 is an ϳ30-kDa N-glycosidase with activity similar to the ricin A chain but exhibiting better stability and lower immunogenicity (13,14). The use of rGel in tumor-targeted cytotoxic agents has been well studied (15, 16). Furthermore, rGel has been shown to be active without cleavage from the binding domain and without negative impact on the targeting agent's pharmacokinetics (...

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“…There is no competing ligand for CD19 as there is for CD21. Importantly, it is known that the majority of CD19-antibody complexes remain on or recycle to the surface of B cells even after extended periods (30,31). Furthermore, coligation of CD19 to the BCR lowers the activation threshold of B cells (15).…”

Section: Discussionmentioning

confidence: 99%

A vaccine directed to B cells and produced by cell-free protein synthesis generates potent antilymphoma immunity

Jia

Patel³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Clinical studies of idiotype (Id) vaccination in patients with lymphoma have established a correlation between the induced anti-Id antibody responses and favorable clinical outcomes. To streamline the production of an Id vaccine, we engineered a small diabody (Db) molecule containing both a B-cell-targeting moiety (anti-CD19) and a lymphoma Id. This molecule (αCD19-Id) was designed to penetrate lymph nodes and bind to noncognate B cells to form an antigen presentation array. Indeed, the αCD19-Id molecule accumulated on B cells in vivo after s.c. administration. These noncognate B cells, decorated with the diabody, could then stimulate the more rare Id-specific B cells. Peptide epitopes present in the diabody linker augmented the response by activating CD4 + helper T cells. Consequently, the αCD19-Id molecule induced a robust Id-specific antibody response and protected animals from tumor challenge. Such diabodies are produced in a cell-free protein expression system within hours of amplification of the specific Ig genes from the B-cell tumor. This customized product can now be available to vaccinate patients before they receive other, potentially immunosuppressive, therapies.immunotherapy | tumor-specific antigen | bispecific antibody fragments I diotype (Id), the unique Ig molecule of each lymphoma tumor, is a good target for the immune system. Passively administered monoclonal antibodies (mAbs) against this target are effective in therapy (1). Furthermore, studies of Id vaccination had suggested a correlation between induced anti-Id antibody responses and progression-free survival and overall survival of patients (2-4). Despite these encouraging results, phase III trials have not established a clinical benefit from Id vaccination, except for a possible subset of patients who have prolonged remissions after initial chemotherapy (5-7). One possible problem may have been the chemical conjugation of Id to the carrier protein, keyhole limpet hemocyanin (KLH). Antigenic determinants on the Id could have been damaged in this process (8). Recombinant vaccines that do not require chemical conjugation may lead to improved immunogenicity and clinical outcomes.Recent studies on antigen (Ag) acquisition by B cells have provided new insights for vaccine design. The majority of B cells reside in follicles within secondary lymphoid organs. Foreign Ags in the form of immune complexes are transported into lymph node follicles by subcapsular sinus macrophages (9-11), and into spleen follicles by marginal zone B cells (12). In the follicles, nonspecific B cells retain immune complexes on their cell surfaces. Some complexes are transferred to follicular dendritic cells (9-11), whereas others may directly cross-link the Ag-specific receptors (BCRs) on cognate B cells (10, 11). These roles played by noncognate B cells in the generation of specific antibody responses were previously not appreciated. In addition to forming immune complexes that facilitate entering the follicles and presenting on the cell surface, foreign Ags may also b...

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Differential Cellular Internalization of Anti-CD19 and -CD22 Immunotoxins Results in Different Cytotoxic Activity

Cited by 111 publications

References 41 publications

Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma

Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma

Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties

A vaccine directed to B cells and produced by cell-free protein synthesis generates potent antilymphoma immunity

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