Antibody-drug conjugates: Current status and future perspectives.

Conventional cytotoxic agents used for the pharmacotherapy of cancer do not selectively localize at the tumor site, which may prevent dose escalation to therapeutically active regimens and may lead to undesired side effects and toxicity to normal organs. There has been a growing interest in the use of monoclonal antibodies as vehicles for the pharmacodelivery of potent cytotoxic drugs to neoplastic lesions. This novel class of targeted biopharmaceutical agents has the potential of improving activity and selectivity of cytotoxic agents.

Antibody Format and Drug Release Rate Determine the Therapeutic Activity of Noninternalizing Antibody-Drug Conjugates.

The development of antibody-drug conjugates (ADC), a promising class of anticancer agents, has traditionally relied on the use of antibodies capable of selective internalization in tumor cells. We have recently shown that also noninternalizing antibodies, coupled to cytotoxic drugs by means of disulfide linkers that can be cleaved in the tumor extracellular environment, can display a potent therapeutic activity. Here, we have compared the tumor-targeting properties, drug release rates, and therapeutic performance of two ADCs, based on the maytansinoid DM1 thiol drug and on the F8 antibody, directed against the alternatively spliced Extra Domain A (EDA) domain of fibronectin.

Antibody-Drug Conjugates and Small Molecule-Drug Conjugates: Opportunities and Challenges for the Development of Selective Anticancer Cytotoxic Agents.

Conventional cancer chemotherapy heavily relies on the use of cytotoxic agents, which typically do not preferentially localize at the tumor site and cause toxicity to normal organs, preventing dose escalation to therapeutically active regimens. In principle, antibodies and other ligands could be used for the selective pharmacodelivery of cytotoxic agents to the neoplastic mass. For many years, the availability of ligands, capable of selective internalization into tumor cells, has been considered to be an essential requirement for the development of targeted cytotoxics.

Noninternalizing targeted cytotoxics for cancer therapy.

Conventional cancer chemotherapy is limited by the fact that small organic cytotoxic agents typically do not preferentially localize at the tumor site, causing unwanted toxicities to normal organs and limiting dose escalation to therapeutically active regimens. In principle, antibodies and other ligands could be used for the selective pharmacodelivery of cytotoxic agents to the tumor environment. While traditionally internalizing ligands have been used for such targeting applications, increasing experimental evidence suggests that the ligand-based delivery of anticancer drugs to the extracellular space in the tumor, followed by suitable release strategies, may mediate a potent anticancer activity.

Small targeted cytotoxics from DNA-encoded chemical libraries.

Conventional chemotherapeutic drugs do not selectively localize to tumors, causing undesired toxicities to healthy organs, and precluding the escalation to therapeutically active regimens. The selective delivery at sites of disease of potent effector molecules represents a promising strategy for the treatment of cancer and other diseases. High affinity antibodies towards disease-associated antigens are currently the vehicles of choice for the targeted delivery of payloads.

A chemically defined trifunctional antibody-cytokine-drug conjugate with potent antitumor activity.

The combination of immunostimulatory agents with cytotoxic drugs is emerging as a promising approach for potentially curative tumor therapy, but advances in this field are hindered by the requirement of testing individual combination partners as single agents in dedicated clinical studies, often with suboptimal efficacy. Here, we describe for the first time a novel multipayload class of targeted drugs, the immunocytokine-drug conjugates (IDC), which combine a tumor-homing antibody, a cytotoxic drug, and a proinflammatory cytokine in the same molecular entity. In particular, the IL2 cytokine and the disulfide-linked maytansinoid DM1 microtubular inhibitor could be coupled to the F8 antibody, directed against the alternatively spliced EDA domain of fibronectin, in a site-specific manner, yielding a chemically defined product with selective tumor-homing performance and potent anticancer activity in vivo, as tested in two different immunocompetent mouse models.

Antibody-based delivery of IL2 and cytotoxics eradicates tumors in immunocompetent mice.

Antibody-drug conjugates are increasingly being used for cancer therapy, but little is known about their ability to promote anticancer immunity, which may lead to long-lasting remissions. We investigated the therapeutic effect of antibody-based pharmacodelivery of cemadotin, a cytotoxic drug, and IL2, a strong proinflammatory cytokine. Using the F8 antibody, which selectively localizes to the tumor neovasculature, combination treatment led to tumor eradication, in a process dependent on CD8(+) T cells and natural killer cells in the C1498 syngeneic mouse model of acute myelogenous leukemia.

Curative properties of noninternalizing antibody-drug conjugates based on maytansinoids.

It is generally thought that the anticancer efficacy of antibody-drug conjugates (ADC) relies on their internalization by cancer cells. However, recent work on an ADC that targets fibronectin in the tumor microenvironment suggests this may not be necessary. The alternatively spliced extra domains A and B (EDA and EDB) of fibronectin offer appealing targets for ADC development, because the antigen is strongly expressed in many solid human tumors and nearly undetectable in normal tissues except for the female reproductive system.

Site-specific chemical modification of antibody fragments using traceless cleavable linkers.

Antibody-drug conjugates (ADCs) are promising agents for the selective delivery of cytotoxic drugs to specific cells (for example, tumors). In this protocol, we describe two strategies for the precise modification at engineered C- or N-terminal cysteines of antibodies in IgG, diabody and small immunoprotein (SIP) formats that yield homogenous ADCs. In this protocol, cemadotin derivatives are used as model drugs, as these agents have a potent cytotoxic activity and are easy to synthesize.

Fucose-specific conjugation of hydrazide derivatives to a vascular-targeting monoclonal antibody in IgG format.

We describe a method that enables specific and efficient conjugation of hydrazide-moieties to an IgG targeting the tumor neovasculature. The resulting chemically defined, homogeneous hydrazone-linked IgG conjugates remain immunoreactive and have a half-life of approximately 18 hours at physiological pH and temperature suitable for localized delivery of toxic drugs.

Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery.

Aldehyde drugs are gaining increasing research interest, considering that aldehyde dehydrogenases overexpression is characteristic of cancer stem cells. Here, we describe the traceless site-specific coupling of a novel potent drug, containing an aldehyde moiety, to recombinant antibodies, which were engineered to display a cysteine residue at their N-terminus, or a 1,2-aminothiol at their C-terminus. The resulting chemically defined antibody-drug conjugates represent the first example in which a thiazolidine linkage is used for the targeted delivery and release of cytotoxic agents.

Antibody-drug conjugates: basic concepts, examples and future perspectives.

Conventional anticancer therapeutics often suffer from lack of specificity, resulting in poor therapeutic indexes and substantial toxicities to normal healthy tissues. Monoclonal antibodies have demonstrated considerable utility in cancer medicine, but their curative potential is often limited. Antibody-drug conjugates represent an innovative therapeutic approach that combines the desirable properties of monoclonal antibodies, with the cell killing activity of cytotoxic drugs, reducing systemic toxicity and increasing the therapeutic benefit for patients.

Reversible intramolecular hydrogen transfer between cysteine thiyl radicals and glycine and alanine in model peptides: absolute rate constants derived from pulse radiolysis and laser flash photolysis.

The intramolecular reaction of cysteine thiyl radicals with peptide and protein alphaC-H bonds represents a potential mechanism for irreversible protein oxidation. Here, we have measured absolute rate constants for these reversible hydrogen transfer reactions by means of pulse radiolysis and laser flash photolysis of model peptides. For N-Ac-CysGly6 and N-Ac-CysGly2AspGly3, Cys thiyl radicals abstract hydrogen atoms from Gly with k(f) = (1.

Selenoglutaredoxin as a glutathione peroxidase mimic.

Glutaredoxin (Grx1) from Escherichia coli is a monomeric, 85-amino-acid-long, disulfide-containing redox protein. A Grx1 variant in which the redox-active disulfide was replaced with a selenocysteine (C11U/C14S) was prepared by native chemical ligation from three fragments as a potential mimic of the natural selenoenzyme glutathione peroxidase (Gpx). Selenoglutaredoxin, like the analogous C14S Grx1 variant, shows weak peroxidase activity.

Reinvestigation of a selenopeptide with purportedly high glutathione peroxidase activity.

A 15-amino acid long selenopeptide (15SeP) was recently reported to possess nearly the same catalytic activity as glutathione peroxidase (Gpx) for the reduction of hydrogen peroxide by glutathione (Sun, Y., Li, T. Y.

Intramolecular addition of cysteine thiyl radicals to phenylalanine in peptides: formation of cyclohexadienyl type radicals.

The intra-molecular addition of peptide cysteine thiyl radicals to phenylalanine yields alkylthio-substituted cyclohexadienyl radicals for the peptides Phe-Cys and Phe-Gly-Cys-Gly, i.e. even when Phe and Cys are separated by a Gly residue, and presents a possible free radical pathway to thioether-containing peptide and protein cross-links.

Convergent protein synthesis.

Methods for the chemical synthesis of proteins have advanced considerably over the past decade. In many instances, laboratory synthesis can now be considered a viable alternative to ribosomal biosynthesis, especially when custom modifications of a protein are desired; chemical approaches guarantee virtually unlimited and tunable variation of the covalent structure of a polypeptide.

3,6-dibromocarbazole piperazine derivatives of 2-propanol as first inhibitors of cytochrome c release via Bax channel modulation.

There is compelling evidence that Bax channel activity stimulates cytochrome c release leading ultimately to cell death, which is a key event in ischemic injuries and neurodegenerative diseases. Here 3,6-dibromocarbazole piperazine derivatives of 2-propanol are described as the first small and potent modulators of the cytochrome c release triggered by Bid-induced Bax activation in a mitochondrial assay. Furthermore, a mechanism of action is proposed, and fluorescent derivatives allowing the localization of such inhibitors are reported.