Miniaturised 'antibody'-drug conjugates for solid tumours?

With Antibody-Drug Conjugate strategies firmly focussed on the precise conjugation to the large protein Immunoglobulin-G format, it is easy to miss the more recent technological innovations in small-format drug conjugates. Here, the targeting ligand can be at 50-95% reduced in size, or even smaller if peptidic in nature. Antibody domains or alternative binding scaffolds, chemically-modified with ultra-potent cytotoxic payloads offer an alternative approach for oncology therapeutics, promising a wider therapeutic window by virtue of superior solid tumour penetration properties and more rapid system clearance. Many of the traditional ADC concepts still apply, but as these miniaturised ADCs enter the clinic over the next 2-3 years, we will learn whether these new features translate to patient benefits.

Production and binding analyses of a humanised scFv against a cryptic epitope on tumour-associated fibronectin.

Tumour-associated splice variants of fibronectin are a major source of tumour-matrix associated targets and are proving very successful in the development of clinical agents to treat cancer. One of the first monoclonal antibodies to be produced to this target, murine BC-1, recognises a cryptic epitope in domain 7 of the B-form splice variant (EDB-FN). Antibody fragments based on this immunoglobulin (IgG) were unstable, but BC-1 humanisation provided an opportunity to produce a more stable single-chain Fv (scFv). The variable domains of the humanized BC-1 IgG were sub-cloned and constructed into a scFv (HuBC-1 scFv) which was successfully expressed in Escherichia coli. The scFv retained its conformationally-sensitive epitope recognition and demonstrated a good affinity to the target of around 50 nM as measured by ELISA, Surface Plasmon Resonance and Flow Cytometry. Furthermore, the scFv was thermostable and stable in serum allowing substantial localisation to human tumours grown in mouse xenograft models. This scFv could form the basis of future tumour-specific biopharmaceuticals.

Glycoengineering approach to half-life extension of recombinant biotherapeutics.

The potential for protein-engineered biotherapeutics is enormous, but pharmacokinetic modulation is a major challenge. Manipulating pharmacokinetics, biodistribution, and bioavailability of small peptide/protein units such as antibody fragments is a major pharmaceutical ambition, illustrated by the many chemical conjugation and recombinant fusion approaches being developed. We describe a recombinant approach that leads to successful incorporation of polysialic acid, PSA for the first time, onto a therapeutically valuable protein. This was achieved by protein engineering of the PSA carrier domain of NCAM onto single-chain Fv antibody fragments (one directed against noninternalizing carcinoembryonic antigen-CEA and one against internalizing human epidermal growth factor receptor-2-HER2). This created novel polysialylated antibody fragments with desired pharmacokinetics. Production was achieved in human embryonic kidney cells engineered to express human polysialyltransferase, and the recombinant, glycosylated product was successfully fractionated by ion-exchange chromatography. Polysialylation was verified by glycosidase digestion and mass spectrometry, which showed the correct glycan structures and PSA chain length similar to that of native NCAM. Binding was demonstrated by ELISA and surface plasmon resonance and on live cells by flow cytometry and confocal immunofluorescence. Unexpectedly, polysialylation inhibited receptor-mediated endocytosis of the anti-HER2 scFv. Recombinant polysialylation led to an estimated 3-fold increase in hydrodynamic radius, comparable to PEGylation, leading to an almost 30-fold increase in blood half-life and a similar increase in blood exposure. This increase in bioavailability led to a 12-fold increase in tumor uptake by 24 h. In summary, recombinant polysialylation of antibody fragments in our system is a novel and feasible approach applicable for pharmacokinetic modulation, and may have wider applications.

Generation of a selectively cytotoxic fusion protein against p53 mutated cancers.

BACKGROUND: A significant number of cancers are caused by defects in p21 causing functional defects in p21 or p53 tumour-suppressor proteins. This has led to many therapeutic approaches including restoration by gene therapy with wild-type p53 or p21 using viral or liposomal vectors, which have toxicity or side-effect limitations. We set out to develop a safer, novel fusion protein which has the ability to reconstitute cancer cell lines with active p21 by protein transduction. METHODS: The fusion protein was produced from the cell-translocating peptide Antennapedia (Antp) and wild-type, full-length p21 (Antp-p21). This was expressed and refolded from E. coli and tested on a variety of cell lines and tumours (in a BALB/c nude xenograft model) with differing p21 or p53 status. RESULTS: Antp-p21 penetrated and killed cancer cells that do not express wild type p53 or p21. This included cells that were matched to cogenic parental cell lines. Antp-p21 killed cancer cells selectively that were malignant as a result of mutations or nuclear exclusion of the p53 and p21 genes and over-expression of MDM2. Non-specific toxicity was excluded by showing that Antp-p21 penetrated but did not kill p53- or p21- wild-type cells. Antp-p21 was not immunogenic in normal New Zealand White rabbits. Recombinant Antp peptide alone was not cytotoxic, showing that killing was due to the transduction of the p21 component of Antp-p21. Antp-p21 was shown to penetrate cancer cells engrafted in vivo and resulted in tumour eradication when administered with conventionally-used chemotherapeutic agents, which alone were unable to produce such an effect. CONCLUSIONS: Antp-p21 may represent a new and promising targeted therapy for patients with p53-associated cancers supporting the concept that rational design of therapies directed against specific cancer mutations will play a part in the future of medical oncology.

Natural killer cell signal integration balances synapse symmetry and migration.

Natural killer (NK) cells discern the health of other cells by recognising the balance of activating and inhibitory ligands expressed by each target cell. However, how the integration of activating and inhibitory signals relates to formation of the NK cell immune synapse remains a central question in our understanding of NK cell recognition. Here we report that ligation of LFA-1 on NK cells induced asymmetrical cell spreading and migration. In contrast, ligation of the activating receptor NKG2D induced symmetrical spreading of ruffled lamellipodia encompassing a dynamic ring of f-actin, concurrent with polarization towards a target cell and a "stop" signal. Ligation of both LFA-1 and NKG2D together resulted in symmetrical spreading but co-ligation of inhibitory receptors reverted NK cells to an asymmetrical migratory configuration leading to inhibitory synapses being smaller and more rapidly disassembled. Using micropatterned activating and inhibitory ligands, signals were found to be continuously and locally integrated during spreading. Together, these data demonstrate that NK cells spread to form large, stable, symmetrical synapses if activating signals dominate, whereas asymmetrical migratory "kinapses" are favoured if inhibitory signals dominate. This clarifies how the integration of activating and inhibitory receptor signals is translated to an appropriate NK cell response.

Current strategies for the discovery and bioconjugation of smaller, targetable drug conjugates tailored for solid tumor therapy.

Introduction: Antibody-Drug Conjugates (ADCs) have undergone a recent resurgence with 5 product approvals over the last 2 years but for those close to the field, it's been repeated cycles of setbacks and new innovations. A new wave of innovation is in the type of format used to deliver the cytotoxic payloads, with smaller bio-molecules being designed to have more optimal penetration and elimination properties tailored for solid tumors.Areas covered: In this review, the authors cover many of the recently described smaller-format drug conjugates (including formats such as diabodies, Fabs, scFvs, domain antibodies) with an emphasis on the types of conjugation technologies used to attach the chemical linker-payload.Expert opinion: Smaller formats are highly influenced by the structure of the linker-payload, arguably more-so than larger ADCs, so careful consideration is needed where solublising and pharmacokinetic modulation is required. High-quality conjugates are being developed with in vivo tumor efficacy and tolerability properties competitive with ADCs and with a few formats already in clinical development, we expect the pipeline to expand and to reach the market.

Novel photosensitisers derived from pyropheophorbide-a: uptake by cells and photodynamic efficiency in vitro.

Photodynamic Therapy (PDT) is a minimally invasive procedure used for treating a range of neoplastic diseases, which utilises combined action of light and a PDT drug called a photosensitiser. The efficiency of this treatment depends crucially on the properties of the photosensitiser used, namely on its efficient uptake by cells or by the surrounding vasculature, intracellular localisation, minimal dark toxicity and substantial phototoxicity. In this report we compare the spectroscopic properties, cell uptake and in vitro phototoxicity of two novel hydrophilic photosensitisers derived from pyropheophorbide-a (PPa). Both new photosensitisers have the potential to form bioconjugates with antibody fragments for targeted PDT. We find that the photophysical properties of both new photosensitisers are favourable compared to the parent PPa, including enhanced absorption in the red spectral region and substantial singlet oxygen quantum yields. Both molecules show efficient cellular uptake, but display a different intracellular localisation. Both new photosensitisers exhibit no significant dark-toxicity at concentrations of up to 100 microM. The phototoxicity of the two photosensitisers is strikingly different, with one derivative being 13 times more efficient than the parent PPa and another derivative being 18 times less efficient in SKOV3 ovarian cancer cells. We investigate the reasons behind such drastic differences in phototoxicity using confocal fluorescence microscopy and conclude that intracellular localisation is a crucial factor in the photodynamic efficiency of pheophorbide derivatives. These studies highlight the underlying factors behind creating more potent photosensitisers through synthetic manipulation.

Tackling solid tumour therapy with small-format drug conjugates.

The pharmacokinetic-pharmacodynamic relationship is extremely complex and tumour drug penetration is one key parameter influencing therapeutic efficacy. In the context of antibody-drug conjugates (ADCs), which has undergone many innovation cycles and witnessed many failures, this feature is being addressed by a number of alternative technologies. Immunoglobulin-based ADCs continue to dominate the industrial landscape, but smaller formats offer the promise of more-effective cytotoxic payload delivery to solid tumours, with a higher therapeutic window afforded by the more rapid clearance. To make these smaller formats viable as delivery vehicles, a number of strategies are being employed, which will be reviewed here. These include identifying the most-appropriate size to generate the larger therapeutic window, increasing the amount of functional, cytotoxic payload delivered through conjugation or half-life extending technologies or other ways of extending the dosing without inducing toxicity.

Targeted photodynamic therapy with multiply-loaded recombinant antibody fragments.

Current photodynamic therapy (PDT) of cancer is limited by inefficiencies involved in specifically targeting photosensitizers to tumors. Although antibodies are being explored as targeting vehicles, they present significant challenges, particularly in terms of pharmacokinetics and drug-coupling. We describe here a novel and effective system to covalently attach multiple photosensitizer molecules (both preclinical, pyropheophorbide-a and clinically approved, verteporfin photosensitizers) to single-chain Fvs. Further, we demonstrate that not only do the resulting photoimmunoconjugates retain photophysical functionality, they are more potent than either free photosensitizer, effectively killing tumor cells in vitro and in vivo. For example, treatment of human breast cancer xenografts with a photoimmunoconjugate comprising an anti-HER-2 scFv linked to 8-10 molecules of pyropheophorbide-a leads to significant tumor regression. These results give an insight into the important features that make scFvs good carriers for PDT drugs and provide proof of concept of our unique approach to targeted photodynamic therapy (tPDT). This promises to significantly improve on current photodynamic therapies for the treatment of cancer.

Single-chain Fv phage display propensity exhibits strong positive correlation with overall expression levels.

BACKGROUND: Single chain Fvs (scFvs) are widely applied in research, diagnostics and therapeutic settings. Display and selection from combinatorial libraries is the main route to their discovery and many factors influence the success of this process. They exhibit low thermodynamic stability, resulting in low levels of premature cytosolic folding or aggregation which facilitates sec YEG-mediated translocation and phage in E. coli. However, there is little data analysing how this is related to and influenced by scFv protein expression. RESULTS: We characterised the relationship between overall scFv expression and display propensity for a panel of 15 anti-tetanus toxin scFvs and found a strong positive correlation (Rho = 0.88, p < 0.005) between the two parameters. Display propensity, overall expression and soluble localisation to the periplasm and extracellular fractions were clone specific characteristics which varied despite high levels of sequence homology. There was no correlation between display of scFv or its expression in non-fused (free) form with soluble scFv localisation to the periplasm or culture supernatant. This suggests that divergence in the fate of scFv-pIII and non-fused scFv after translocation to the periplasm accounts for the observed disparity. Differential degrees of periplasmic aggregation of non-fused scFv between clones may affect the partitioning of scFv in the periplasm and culture supernatant abrogating any correlation. We suggest that these factors do not apply to the scFv-pIII fusion since it remains anchored to the bacterial inner membrane as part of the innate phage packaging and budding process. CONCLUSION: We conclude that in the absence of premature cytosolic aggregation or folding, the propensity of a scFv to be displayed on phage is directly related to its overall expression level and is thus indirectly influenced by factors such as codon bias, mRNA abundance or putative DNA motifs affecting expression. This suggests that scFvs capable of high overall expression and display levels may not produce high yields of non phage-fused soluble protein in either the periplasmic or extracellular fractions of E. coli. This should be considered when screening clones selected from combinatorial libraries for further study.

Phage display of chelating recombinant antibody libraries.

High affinity and discriminating specificity are important parameters for any successful antibody based targeting strategy. We herein describe a system for the construction and subsequent selection of affinity-optimised chelating recombinant antibodies (CRAbs) from a randomised filamentous phage-display inter-scFv linker library. Using a simple, robust and highly degenerate tandem scFv cloning strategy a phage-display library of CRAbs with varied inter-scFv linkers was constructed and characterised. The library consisted of two single-chain Fvs (scFvs) of well characterised anti-lysozyme antibodies D1.3 and HyHEL-10(TF), specific for distinct non-overlapping epitopes, separated by flexible polypeptide linkers of varying lengths and sequences. The use of a stringent affinity-based selection strategy quickly led to the enrichment of CRAbs with a restricted set of linker lengths (16-21 amino acids) which agrees very closely with previously described crystal structure data, affinity measurements and mathematical modelling. This CRAb linker phage-display selection strategy is a widely applicable approach for the selection of very high affinity CRAbs for pairs of scFvs against potentially any target antigen, complementing the more arbitrary affinity maturation approaches based on random mutagenesis.

Small-Format Drug Conjugates: A Viable Alternative to ADCs for Solid Tumours?

Antibody-Drug Conjugates (ADCs) have been through multiple cycles of technological innovation since the concept was first practically demonstrated ~40 years ago. Current technology is focusing on large, whole immunoglobulin formats (of which there are approaching 100 in clinical development), many with site-specifically conjugated payloads numbering 2 or 4. Despite the success of trastuzumab-emtansine in breast cancer, ADCs have generally failed to have an impact in solid tumours, leading many to explore alternative, smaller formats which have better penetrating properties as well as more rapid pharmacokinetics (PK). This review describes research and development progress over the last ~10 years obtained from the primary literature or conferences covering over a dozen different smaller format-drug conjugates from 80 kDa to around 1 kDa in total size. In general, these agents are potent in vitro, particularly more recent ones incorporating ultra-potent payloads such as auristatins or maytansinoids, but this potency profile changes when testing in vivo due to the more rapid clearance. Strategies to manipulate the PK properties, whilst retaining the more effective tumour penetrating properties could at last make small-format drug conjugates viable alternative therapeutics to the more established ADCs.

Using antibody directed phototherapy to target oesophageal adenocarcinoma with heterogeneous HER2 expression.

Early oesophageal adenocarcinoma (OA) and pre-neoplastic dysplasia may be treated with endoscopic resection and ablative techniques such as photodynamic therapy (PDT). Though effective, discrete areas of disease may be missed leading to recurrence. PDT further suffers from the side effects of off-target photosensitivity. A tumour specific and light targeted therapeutic agent with optimised pharmacokinetics could be used to destroy residual cancerous cells left behind after resection. A small molecule antibody-photosensitizer conjugate was developed targeting human epidermal growth factor receptor 2 (HER2). This was tested in an in vivo mouse model of human OA using a xenograft flank model with clinically relevant low level HER2 expression and heterogeneity. In vitro we demonstrate selective binding of the conjugate to tumour versus normal tissue. Light dependent cytotoxicity of the phototherapy agent in vitro was observed. In an in vivo OA mouse xenograft model the phototherapy agent had desirable pharmacokinetic properties for tumour uptake and blood clearance time. PDT treatment caused tumour growth arrest in all the tumours despite the tumours having a clinically defined low/negative HER2 expression level. This new phototherapy agent shows therapeutic potential for treatment of both HER2 positive and borderline/negative OA.

Upregulation of mucin glycoprotein MUC1 in the progression to esophageal adenocarcinoma and therapeutic potential with a targeted photoactive antibody-drug conjugate.

BACKGROUND: Mucin glycoprotein 1 (MUC1) is a glycosylated transmembrane protein on epithelial cells. We investigate MUC1 as a therapeutic target in Barrett's epithelium (BE) and esophageal adenocarcinoma (EA) and provide proof of concept for a light based therapy targeting MUC1. RESULTS: MUC1 was present in 21% and 30% of significantly enriched pathways comparing BE and EA to squamous epithelium respectively. MUC1 gene expression was x2.3 and x2.2 higher in BE (p=<0.001) and EA (p=0.03). MUC1 immunohistochemical expression increased during progression to EA and followed tumor invasion. HuHMFG1 based photosensitive antibody drug conjugates (ADC) showed cell internalization, MUC1 selective and light-dependent cytotoxicity (p=0.0006) and superior toxicity over photosensitizer alone (p=0.0022). METHODS: Gene set enrichment analysis (GSEA) evaluated pathways during BE and EA development and quantified MUC1 gene expression. Immunohistochemistry and flow cytometry evaluated the anti-MUC1 antibody HuHMFG1 in esophageal cells of varying pathological grade. Confocal microscopy examined HuHMFG1 internalization and HuHMFG1 ADCs were created to deliver a MUC1 targeted phototoxic payload. CONCLUSIONS: MUC1 is a promising target in EA. Molecular and light based targeting of MUC1 with a photosensitive ADC is effective in vitro and after development may enable treatment of locoregional tumors endoscopically.

Emerging formats for next-generation antibody drug conjugates.

INTRODUCTION: Antibody drug conjugates now make up a significant fraction of biopharma's oncology pipeline due to great advances in the understanding of the three key components and how they should be optimised together. With this clinical success comes innovation to produce new enabling technologies that can deliver more effective antibody-drug conjugates (ADCs) with a larger therapeutic index. AREAS COVERED: There are many reviews that discuss the various strategies for ADCs design but the last 5 years or so have witnessed the emergence of a number of different antibody formats compete with the standard whole immunoglobulin. Using published research, patent applications and conference disclosures, the authors review the many antibody and antibody-like formats, discussing innovations in protein engineering and how these new formats impact on the conjugation strategy and ultimately the performance. The alternative chemistries that are now available offer new linkages, stability profiles, drug:antibody ratio, pharmacokinetics and efficacy. The different sizes being considered promise to address issues, such as tumour penetration, circulatory half-life and side-effects. EXPERT OPINION: ADCs are at the beginning of the next stage in their evolution and as these newer formats are developed and examined in the clinic, we will discover if the predicted features have a clinical benefit. From the commercial activity, it is envisaged that smaller or fragment-based ADCs will expand oncological applications.

Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding.

An approach for enhancing antibody affinity is to engineer Chelating Recombinant Antibodies (CRAbs) which consist of two tandemly linked single-chain Fvs (scFvs) that bind to distinct non-overlapping epitopes on the antigen molecule leading to a synergistic decrease in K(D). In order to develop this technology, the aim of this present study was to identify scFvs which can simultaneously bind to the tetanus toxin heavy chain C-terminal sub-domain (H(c)), characterise their bio-physical properties and determine their functional efficacy. Over 50 antibodies specific for Hc were isolated from a human scFv phagemid library and found to bind specifically to the C-terminal sub-domain of H(c) (H(c)C clones), the N-terminal sub-domain (HcN clones) or junctional epitopes on the whole Hc fragment only (HcJ clones). Fifteen clones were assayed in a pairwise competition binding study. The revealed, with few exceptions, that H(c)C clones were able to simultaneously bind to the toxin with H(c)N or H(c)J clones. All other combinations competed for binding. Interestingly, we also observed cooperative binding with many non-competing scFv pairings which may impact upon the binding mechanism of CRAbs. We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity. This included clones that did not bind to the H(c)C sub-domain which is responsible for direct interaction with gangliosides on nerve cells. For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency. The diverse array of clones characterised in this paper can be used to construct CRAbs and will prove useful in further characterisation of toxin biology and in measuring the effects of polyclonal antibody therapy.

Antibodies targeting cancer stem cells: a new paradigm in immunotherapy?

Antibody targeting of cancer is showing clinical and commercial success after much intense research and development over the last 30 years. They still have the potential to delivery long-term cures but a shift in thinking towards a cancer stem cell (CSC) model for tumor development is certain to impact on how antibodies are selected and developed, the targets they bind to and the drugs used in combination with them. CSCs have been identified from many human tumors and share many of the characteristics of normal stem cells. The ability to renew, metabolically or physically protect themselves from xenobiotics and DNA damage and the range of locomotory-related receptors expressed could explain the observations of drug resistance and radiation insensitivity leading to metastasis and patient relapse.Targeting CSCs could be a strategy to improve the outcome of cancer therapy but this is not as simple as it seems. Targets such as CD133 and EpCAM/ESA could mark out CSCs from normal cells enabling specific intervention but indirect strategies such as interfering with the establishment of a supportive niche through anti-angiogenic or anti-stroma therapy could be more effective.This review will outline the recent discoveries for CSCs across the major tumor types highlighting the possible molecules for intervention. Examples of antibody-directed CSC therapies and the outlook for the future development of this emerging area will be given.

Recombinant antibodies for cancer therapy.

BACKGROUND: Recombinant antibodies have evolved into successful therapeutics with 10 approved for cancer and more in the pipeline. Four of the top ten cancer therapy drugs are recombinant antibodies. OBJECTIVES: To survey the current state-of-the-art highlighting the reasons for this success and looking ahead to the next generation of antibody therapy. METHODS: An analysis was carried out to identify preclinical and clinical examples and the underlying concepts and mechanisms that have shown how to design better therapies. RESULTS/CONCLUSIONS: Greater understanding of the molecular basis of cancer has led to improved antibodies and a greater selection of targets. Fine tuning of successful antibodies through modification of glycosylation, affinity, size and other parameters are paying dividends. Fc-engineering is likely to be predominant in the near future but conjugates, fragments and fusion proteins will continue to be developed and find their place in the arsenal of antibody therapeutics.

Modulation of antibody pharmacokinetics by chemical polysialylation.

Chemical coupling of a variety of polymers to therapeutic proteins has been studied as a way of improving their pharmacokinetics and pharmacodynamics in vivo. Conjugates have been shown to possess greater stability, lower immunogenicity, and a longer blood circulation time due to the chemicophysical properties of these hydrophilic long chain molecules. Naturally occurring colominic acid (polysialic acid, PSA) has been investigated as an alternative to synthetic polymers such as poly(ethylene glycol) (PEG) due to its lower toxicity and natural metabolism. Antibodies and their fragments are a good example of the types of proteins which benefit from pharmacokinetic engineering. Here, we chemically attached differing amounts and differing lengths of short (11 kDa) and longer (22 kDa) chain colominic acid molecules to the antitumor monoclonal antibody H17E2 Fab fragment. Different coupling ratios and lengths were seen to alter the electrophoretic mobility of the Fab fragment but have a minor effect on the antibody immunoreactivity toward the placental alkaline phosphatase (PLAP) antigen. Polysialylation generally increased Fab fragment blood half-life resulting in higher tumor uptake in a KB human tumor xenograft mouse model. One H17E2 Fab-PSA conjugate had over a 5-fold increase in blood exposure and over a 3-fold higher tumor uptake with only a marginal decrease in tumor/blood selectivity ratio compared to the unconjugated Fab. This conjugate also had a blood bioavailability approaching that of a whole immunoglobulin.

Antibody-guided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells.

A number of enzyme/prodrug activation approaches for the treatment of cancer have been reported to date with varying success. We describe progress in the development of a system based on a beta-glucosidase enzyme in combination with a naturally occurring "prodrug," the sugar linamarin, which releases the cytotoxin cyanide. A recombinant fusion protein, composed of an scFv (MFE-23) reactive against carcinoembryonic antigen (CEA) and a plant-derived beta-glucosidase (linamarase), was produced and its cytotoxic potential was investigated. The fusion protein was expressed in a supersecretory mutant strain of Saccharomyces cerevisiae and purified by affinity chromatography. Extensive functional in vitro characterisation of the fusion protein showed that it retained antigen binding activity but that its catalytic activity was impaired, a problem not related to its fusion with the scFv. Nevertheless, we demonstrated complete tumour cell killing at doses of prodrug that are completely nontoxic to nontargeted cells. Preliminary in vivo characterisation showed that extensive glycosylation of the fusion protein caused its rapid clearance through the hepatic route. Aggregational properties also led to poor pharmacokinetics. Furthermore, we present some data analysing the mode of cell death resulting from exposure to this system. Enzymic catalysis of the substrate generates cyanide, a metabolic poison that asphyxiates cells and leads them to a necrotic-like cell death. This system has been called antibody-guided enzyme nitrile therapy (AGENT).