Antibody-directed Enzyme Prodrug Therapy Research Articles

Kenneth Dawson Bagshawe. 17 August 1925—27 December 2022

Kenneth Bagshawe was a physician, a scientist and an architect of modern oncology. He was first to show that combination chemotherapy can cure a non-haematological cancer, choriocarcinoma. He demonstrated how a tumour marker in blood and urine can give a quantitative representation of the total amount of tumour, providing a monitor of response to treatment or relapse and an indicator of prognosis. He enhanced understanding and treatment of gestational trophoblastic diseases (GTD), a group of which choriocarcinoma is the most lethal member. The most common form of GTD is hydatidiform mole and he developed a national system for management, resulting in a cure rate of virtually 100%. For choriocarcinoma, the figure is today approaching 98%. His system is used in many countries, and over several decades has contributed to the cure and preservation of fertility in hundreds of thousands of women who previously had little or no hope. He used the same principles to treat testicular and ovarian germ cell tumours, again achieving high levels of cure in patients whose disease had spread and would previously have been fatal. He innovated throughout his career, seeking improved tumour specificity in diagnosis and treatment of GTD and inventing antibody-directed enzyme prodrug therapy for more common cancers. He founded the first department of oncology in the UK and devoted much time to developing the speciality, directing research funding organizations and supporting the development of the speciality worldwide. He was a dedicated, original and hugely productive pioneer of oncology.

Human GST P1-1 Redesigned for Enhanced Catalytic Activity with the Anticancer Prodrug Telcyta and Improved Thermostability.

Protein engineering can be used to tailor enzymes for medical purposes, including antibody-directed enzyme prodrug therapy (ADEPT), which can act as a tumor-targeted alternative to conventional chemotherapy for cancer. In ADEPT, the antibody serves as a vector, delivering a drug-activating enzyme selectively to the tumor site. Glutathione transferases (GSTs) are a family of naturally occurring detoxication enzymes, and the finding that some of them are overexpressed in tumors has been exploited to develop GST-activated prodrugs. The prodrug Telcyta is activated by GST P1-1, which is the GST most commonly elevated in cancer cells, implying that tumors overexpressing GST P1-1 should be particularly vulnerable to Telcyta. Promising antitumor activity has been noted in clinical trials, but the wildtype enzyme has modest activity with Telcyta, and further functional improvement would enhance its usefulness for ADEPT. We utilized protein engineering to construct human GST P1-1 gene variants in the search for enzymes with enhanced activity with Telcyta. The variant Y109H displayed a 2.9-fold higher enzyme activity compared to the wild-type GST P1-1. However, increased catalytic potency was accompanied by decreased thermal stability of the Y109H enzyme, losing 99% of its activity in 8 min at 50 °C. Thermal stability was restored by four additional mutations simultaneously introduced without loss of the enhanced activity with Telcyta. The mutation Q85R was identified as an important contributor to the regained thermostability. These results represent a first step towards a functional ADEPT application for Telcyta.

Tailoring the AIE Chromogen 2-(2-Hydroxyphenyl)benzothiazole for Use in Enzyme-Triggered Molecular Brachytherapy.

A targeted strategy for treating cancer is antibody-directed enzyme prodrug therapy, where the enzyme attached to the antibody causes conversion of an inactive small-molecule prodrug into an active drug. A limitation may be the diffusion of the active drug away from the antibody target site. A related strategy with radiotherapeutics entails enzymatically promoted conversion of a soluble to insoluble radiotherapeutic agent, thereby immobilizing the latter at the target site. Such a molecular brachytherapy has been scarcely investigated. In distinct research, the advent of molecular designs for aggregation-induced emission (AIE) suggests translational use in molecular brachytherapy. Here, several 2-(2-hydroxyphenyl)benzothiazole substrates that readily aggregate in aqueous solution (and afford AIE) were elaborated in this regard. In particular, (1) the 2-(2-hydroxyphenyl) unit was derivatized to bear a pegylated phosphodiester that imparts water solubility yet undergoes enzymatic cleavage, and (2) a p-phenol unit was attached to the benzo moiety to provide a reactive site for final-step iodination (here examined with natural abundance iodide). The pegylated phosphodiester-iodinated benzothiazole undergoes conversion from aqueous-soluble to aqueous-insoluble upon treatment with a phosphatase or phosphodiesterase. The aggregation is essential to molecular brachytherapy, whereas the induced emission of AIE is not essential but provides a convenient basis for research development. Altogether, 21 compounds were synthesized (18 new, 3 known via new routes). Taken together, blending biomedical strategies of enzyme prodrug therapy with materials chemistry concerning substances that undergo AIE may comprise a step forward on the long road toward molecular brachytherapy.

In-vitro and in-vivo efficacy of hollow gold nanoparticles encapsulating horseradish peroxidase: Oxidative stress-mediated tumor cell killing

This article reports the protocol for the synthesis of hollow gold nanoparticles using harmless template-material calcium phosphate nanoparticles for encapsulating enzyme to overcome the problem associated with antibody-directed enzyme pro-drug therapy (ADEPT) or chemotherapy against cancer. Horseradish peroxidase (HRP) could be effectively loaded inside the cavity of hollow gold nanoparticles. The high-resolution transmission electron microscopy (HRTEM) and dynamic light scattering (DLS) analysis confirmed monodispersed formulation demonstrating spherical morphology with an average diameter of 50 nm. Aqueous dispersion of these nanoparticles was highly stable under cold conditions. HRP loaded in the cavity of hollow gold nanoparticles showed more stability in response to change in temperature as compared to unloaded/free enzyme and followed Michaelis-Menten kinetics. From the in-vitro MTT assay, it is concluded that the prodrug - Indole acetic acid (IAA) is non-toxic, however, after oxidative decarboxylation by HRP, it became active/toxic. The cell death mechanism by IAA/entrapped-HRP combination confirmed apoptosis as demonstrated by DNA fragmentation and AO/PI staining. For assessing in-vivo efficacy, the hollow gold nanoparticles with HRP was topically administered into the xenographted tumor in mice. The benign substrate IAA was given either orally or by intra-peritoneal injection. It reached the tumor tissue through systemic circulation to get oxidized by HRP. This created severe and lethal oxidative stress to the cancer cells resulting in regression of tumor. The uniqueness and novelty of our work lies in the fact that we have used benign template to generate/create hollow formulation loaded with HRP, which could enhance the treatment efficacy by multi-fold as compared to the free HRP or solid formulation as more active sites are available due to internal mobility of the enzyme for interaction with the prodrug. From the survival percent graph, we concluded that all mice died after ∼32 days in case of free HRP/IAA combination but in case of HGNP-HRP/IAA combination, 100% mice were alive after ∼30 days and ∼50% of tumor bearing mice remained alive even after 60 days. In conclusion, both in vitro and in vivo experiments revealed that the combination of IAA/HRP entrapped in hollow gold nanoparticles could be efficiently and effectively used for cancer therapy.

Complementation Dependent Enzyme Prodrug Therapy Enables Targeted Activation of Prodrug on HER2-Positive Cancer Cells.

Antibodies have been explored for decades for the delivery of small molecule cytotoxins directly to diseased cells. In antibody-directed enzyme prodrug therapy (ADEPT), antibodies are armed with enzymes that activate nontoxic prodrugs at tumor sites. However, this strategy failed clinically due to off-target toxicity associated with the enzyme prematurely activating prodrug systemically. We describe here the design of an antibody-fragment split enzyme platform that regains activity after binding to HER2, allowing for site-specific activation of a small molecule prodrug. We evaluated a library of fusion constructs for efficient targeting and complementation to identify the most promising split enzyme pair. The optimal pair was screened for substrate specificity among chromogenic, fluorogenic, and prodrug substrates. Evaluation of this system on HER2-positive cells revealed 7-fold higher toxicity of the activated prodrug over prodrug treatment alone. Demonstrating the potential of this strategy against a known clinical target provides the basis for a unique therapeutic platform in oncology.

Glucuronides: From biological waste to bio-nanomedical applications

Long considered as no more than biological waste meant to be eliminated in urine, glucuronides have recently contributed to tremendous developments in the biomedical field, particularly against cancer. While glucuronide prodrugs monotherapy and antibody-directed enzyme prodrug therapy have been around for some time, new facets have emerged that combine the unique properties of glucuronides notably in the fields of antibody-drug conjugates and nanomedicine. In both cases, glucuronides are utilized as a vector to improve pharmacokinetics and confer localized activation of potent drugs at tumor sites while also decreasing systemic toxicity. Here we will discuss some of the most promising strategies using glucuronides to promote successful anti-tumor therapeutic treatments.

Attaching palladium catalysts to antibodies

Antibody-directed enzyme prodrug therapy (ADEPT) is a powerful concept in which antibody targeting is linked to enzymatic prodrug activation. The work herein describes the first steps in the development of a technology analogous to ADEPT but in which a palladium catalyst is attached of an antibody rather than an enzyme. Antibody–metal conjugates have been used in a variety of contexts including for radiotherapy; however, none of the metals attached to the antibodies have been used for catalytic purposes. This work represents the first example a metal being attached to an antibody for the purposes of carrying a functional catalyst.

An antibody-based enzymatic therapy for cancer treatment: The selective localization of D-amino acid oxidase to EDA fibronectin

In order to generate an antibody directed enzyme prodrug therapy, here we designed a chimeric protein by fusing the F8 antibody that recognizes the EDA of fibronectin (expressed on the tumor neovasculature) and an evolved variant of the ROS-generating enzyme D-amino acid oxidase (DAAO). The F8(scFv)-DAAO-Q144R recombinant protein is expressed by both CHO-S and E. coli cells. The F8(scFv)-DAAO-Q144R from E. coli cells is fully soluble, shows a high specific activity, is more thermostable in blood than the native DAAO, possesses a binding affinity for EDA well suited for efficient tumor accumulation, and localizes in tumor tissues. Notably, the F8(scFv)-DAAO-Q144R conjugate generates a stronger cytotoxicity to tumor cells than the native enzyme, especially when an inhibitor of heme oxygenase-1 (HO-1) is used, making it a promising candidate for a selective antitumor oxidative therapy controlled by the substrate addition, in the so called “activity on demand”, thus sparing normal tissue from damage.

Targeting Toxins toward Tumors.

Many cancer diseases, e.g., prostate cancer and lung cancer, develop very slowly. Common chemotherapeutics like vincristine, vinblastine and taxol target cancer cells in their proliferating states. In slowly developing cancer diseases only a minor part of the malignant cells will be in a proliferative state, and consequently these drugs will exert a concomitant damage on rapidly proliferating benign tissue as well. A number of toxins possess an ability to kill cells in all states independently of whether they are benign or malignant. Such toxins can only be used as chemotherapeutics if they can be targeted selectively against the tumors. Examples of such toxins are mertansine, calicheamicins and thapsigargins, which all kill cells at low micromolar or nanomolar concentrations. Advanced prodrug concepts enabling targeting of these toxins to cancer tissue comprise antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT), lectin-directed enzyme-activated prodrug therapy (LEAPT), and antibody-drug conjugated therapy (ADC), which will be discussed in the present review. The review also includes recent examples of protease-targeting chimera (PROTAC) for knockdown of receptors essential for development of tumors. In addition, targeting of toxins relying on tumor-overexpressed enzymes with unique substrate specificity will be mentioned.

Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site.

Broad-spectrum cytotoxic drugs have been used in cancer therapy for decades. However, their lack of specificity to cancer cells often results in serious side-effects, limiting efficacy. For this reason, antibodies have been used to attempt to specifically target cytotoxic drugs to tumours. One such approach is antibody-directed enzyme prodrug therapy (ADEPT) which uses a tumour-directed monoclonal antibody, coupled to an enzyme, to convert a systemically administered non-toxic prodrug into a toxic one only at the tumour site. Among the main drawbacks of ADEPT is the immunogenicity of the antibody-enzyme complex, which is exacerbated by slow clearance due to size, hence limiting repeated administration. Additionally, the mono-specificity of the antibody could potentially result in drug resistance with repeated administration. We have identified a novel short peptide sequence, p700, derived from a human tissue inhibitor of metalloproteinases-3 (TIMP-3), which binds to and inhibits a number of tyrosine kinase growth factor receptors (VEGFRs1-3, FGFRs 1-4 and PDGFRα) which are known to be upregulated in many tumours and tumour vasculature. In this report, we fused p700 to His-tagged, codon-optimised, carboxypeptidase G2 (CPG2). CPG2 is a bacterial enzyme used in ADEPT, which activates potent nitrogen-mustard pro-drugs by removal of an inhibitory glutamic acid residue. Recombinant CPG2-p700 was highly expressed in Escherichia coli and successfully purified by nickel affinity chromatography. Biolayer interferometry showed that CPG2-p700 had a 100-fold increase in binding affinity for VEGFR2 compared with CPG2 alone and retained its catalytic activity, as determined by methotrexate cleavage. In the presence of CPG2-p700, the ZD2676P pro-drug showed significant cytotoxicity for 4T1 cells compared with prodrug alone or CPG2 alone. p700 is, therefore, a potentially useful alternative to monoclonal antibodies for enzyme pro-drug therapy and could equally be used for effective delivery of other cytotoxic drugs to tumour tissue.

Production of Long-Acting CNGRC-CPG2 Fusion Proteins: New Derivatives to Overcome Drug Immunogenicity of Ligand-Directed Enzyme Prodrug Therapy for Targeted Cancer Treatment.

Objectives: Aminopeptidase N (APN) is an enzyme highly expressed in metastatic cancers and could be used in targeted cancer therapy. Our previous work showed the successful construction of CNGRC–carboxypeptidase G2 (CPG2) and CNGRC–CPG2–CNGRC fusion proteins. Our conjugates and prodrugs were effective in targeting high APN-expressing cancer cells. In the present study, we aim to produce long-acting fusion proteins to overcome 2 of the main drawbacks of antibody-directed enzyme prodrug therapy. Methods: N-terminal and N-, C-terminal fusion CPG2, CNGRC–CPG2, and CNGRC–CPG2–CNGRC, respectively, were PEGylated using polyethylene glycol (PEG) maleimide (40K). We examined the effect of PEGylation on the therapeutic efficacy of the new products. The resulting PEGylated fusion proteins were tested for their stability, ex vivo immunotoxicity, binding capacity to their target on high HT1080, and low A549 APN-expressing cells. The catalytic activity of the resulting PEGylated fusion CPG2 proteins was investigated. Pro-drug “ZD2767P” cytotoxic effect in association with PEG CPG2–CNGRC fusion proteins on cancer cells was studied. Results: Our work demonstrated that the properties of the PEGylated single-fused proteins were significantly improved over that of un-PEGylated fused CPG2, and its kinetic activity and APN-binding affinity were not negatively affected by the PEGylation. Significantly, The PEGylated single-fused CPG2 had lower immunogenicity than the un-PEGylated CPG2. Our results, however, were different in the case of the PEGylated double-fused CPG2. Although its stability in human serum under physiological conditions was not significantly affected, the kinetic activity and its binding affinity to their cellular marker (APN) were substantially reduced. When the study was performed with high and low APN-expressing cancer cell lines, using the prodrug ZD2767p, the PEGylated fusion CPG2 demonstrated cancer cell killing effects. Conclusion: We have successfully produced PEGylated-CNGRC–CPG2, which is bioactive and with lower immunogenicity in ligand-directed enzyme prodrug therapy for cancer treatment.

An update on the toxicity of cyanogenic glycosides bioactive compounds: Possible clinical application in targeted cancer therapy

Cyanogenic glycosides are defined as natural herbal toxins which are found in more than 2000 plant species and most of them consumed by humans. These nitrile-containing compounds do not present toxic effects in intact form while they produce toxic cyanide following enzymatic digestion. Therefore, over-consuming of these compounds can lead to cyanide poisoning. On the other hand, some cyanogenic compounds such as amygdalin and linamarin have therapeutic effects and they have been used as a traditional medicine to treat several diseases such as cancer. It seems that the biological properties of these compounds such as bioavailability, solubility, and stability elevate by glycosylation which causes the increased anti-tumor activity of compounds. However, a major challenge in its various applications such as anti-cancer drugs is cyanide poisoning which has been reported in some patients. Interestingly, different approaches of targeted therapy have been applied to elevate these compounds' pharmaceutical properties in targeting cancer cells, like suicide gene therapy, antibody-directed enzyme prodrug therapy (ADEPT), and nanoporous imprinted polymers (nanoMIPs). In this review, we have focused on the challenges of cyanide toxicity raised from amygdalin and linamarin and on various approaches to their targeted therapy in cancer treatment as well.

Production of “biobetter” variants of glucarpidase with enhanced enzyme activity

Glucarpidase, also known as carboxypeptidase G2, is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels “biobetter” variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. Km, Vmax and Kcat measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase.Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase.

Therapeutic Potential of Prodrugs Towards Targeted Drug Delivery.

In designing of Prodrugs, targeting can be achieved in two ways: site-specified drug delivery and site-specific drug bioactivation. Prodrugs can be designed to target specific enzymes or carriers by considering enzyme-substrate specificity or carrier-substrate specificity in order to overcome various undesirable drug properties. There are certain techniques which are used for tumor targeting such as Antibody Directed Enzyme Prodrug Therapy [ADEPT] Gene-Directed Enzyme Prodrug Therapy [GDEPT], Virus Directed Enzyme Prodrug Therapy [VDEPT] and Gene Prodrug Activation Therapy [GPAT]. Our review focuses on the Prodrugs used in site-specific drug delivery system specially on tumor targeting.

Production of “biobetter” glucarpidase variants to improve drug detoxification and antibody directed enzyme prodrug therapy for cancer treatment

Recombinant glucarpidase (formerly: Carboxypeptidase G2, CPG2) is used in Antibody Directed Enzyme Prodrug Therapy (ADEPT) for the treatment of cancer. In common with many protein therapeutics, glucarpidase has a relatively short half-life in serum and, due to the need for the repeated cycles of the ADEPT, its bioavailability may be further diminished by neutralizing antibodies produced by patients. PEGylation and fusion with human serum albumin (HSA) are two approaches that are commonly employed to increase the residency time of protein therapeutics in blood, and also to increase the half-lives of the proteins in vivo. To address this stability and the immunogenicity problems, ‘biobetter’ glucarpidase variants, mono-PEGylated glucarpidase, and HSA fused glucarpidase by genetic fusion with albumin, were produced. Biochemical and bioactivity analyses, including anti-proliferation, bioassays, circular dichroism, and in vitro stability using human blood serum and immunoassays, demonstrated that the functional activities of the designed glucarpidase conjugates were maintained. The immunotoxicity studies indicated that the PEGylated glucarpidase did not significantly induce T-cell proliferation, suggesting that glucarpidase epitopes were masked by the PEG moiety. However, free glucarpidase and HSA-glucarpidase significantly increased T-cell proliferation compared with the negative control. In the latter case, this might be due to the type of expression system used or due to trace impurities associated with the highly purified (99.99%) recombinant HSA-glucarpidase. Both PEGylated glucarpidase and HAS-glucarpidase exhibit more stability in human serum and were more resistant to key human proteases relative to native glucarpidase. To our knowledge, this study is the first to report stable and less immunogenic glucarpidase variants produced by PEGylation and fusion with HSA. The results suggest that they may have better efficacy in drug detoxification and ADEPT, thereby improving this cancer treatment strategy.

Synthesis, anticancer activity and cytotoxicity of galactosylated epothilone B.

Epothilones are the 16-membered macrolide compounds, exhibit microtubule-promoting activity, have the same anti-tumor mechanism as paclitaxel, and are expected to be the ideal substitutes for paclitaxel. However, natural epothilone compounds have been found to have disadvantages such as high toxicity in vivo, poor selectivity to tumor cells, and susceptibility to drug resistance. Herein, epothilone B was synthesized by fermentation, and it was galactosylated by chemical method. The toxicity in vitro of epothilone B and its galactosylated derivative was investigated by the MTT method. The anticancer activity evaluation in vitro was performed using a method similar to the antibody-directed enzyme-prodrug therapy (ADEPT) method. It indicated that the ratio of cytotoxicity between the free epothilone B and the galactosylated epothilone B was about 150. This would lay the foundation for the targeted treatment of cancer with epothilone glycosides.

Engineering of Escherichia coli β-lactamase TEM-1 variants showing higher activity under acidic conditions than at the neutral pH

Escherichia coli β-lactamase TEM-1 is potentially useful in the antibody-directed enzyme/prodrug therapy (ADEPT), converting nontoxic prodrugs to toxic agents. The produced toxin would kill cancer cells, when the enzyme is attached to a tumor-antigen-specific antibody. However, the off-site reaction possibly occurring in the blood or normal tissues raises safety concern. In the present study, we engineered TEM-1 variants preferentially active at pH 5.8–6.2, near the pH of the acidic microenvironment of tumor. A library of randomly mutagenized variants was screened for the ability to confer an antibiotic resistance on E. coli cells in acidic growth media and not in neutral media, to isolate a variant with a Thr-to-Ile substitution at position 160. An extensive mutagenesis study was then conducted in the proximity of this position, to show that a Leu162Glu mutation also causes the acid preference. Kinetic analyses indicated that the overall activity of the wild-type TEM-1 hardly changes over a pH range from 5.8 to 7.0, whereas TEM-1(T160I) is 1.5-times as active at pH 6.2 than pH 7.0, and TEM-1(T160I) is 3.1-fold as active at pH 5.8 than pH 7.0. A further mutagenesis study suggested that a change in the overall structure of the enzyme underlies the pH dependency of the variants.

Characterisation of the Carboxypeptidase G2 Catalytic Site and Design of New Inhibitors for Cancer Therapy.

The enzyme carboxypeptidase G2 (CPG2) is used in antibody-directed enzyme prodrug therapy (ADEPT) to catalyse the formation of an active drug from an inert prodrug. Free CPG2 in the bloodstream must be inhibited before administration of the prodrug in order to avoid a systemic reaction in the patient. Although a few small-molecule CPG2 inhibitors have been reported, none has been taken forward thus far. This lack of progress is due in part to a lack of structural understanding of the CPG2 active site as well as the absence of small molecules that can block the active site whilst targeting the complex for clearance. The work described here aimed to address both areas. We report the structural/functional impact of extensive point mutation across the putative CPG2 catalytic site and adjacent regions for the first time, revealing that residues outside the catalytic region (K208A, S210A and T357A) are crucial to enzyme activity. We also describe novel molecules that inhibit CPG2 whilst maintaining the accessibility of galactosylated moieties aimed at targeting the enzyme for clearance. This work acts as a platform for the future development of high-affinity CPG2 inhibitors that occupy new chemical space and will advance the safe application of ADEPT in cancer treatment.

Isolation and molecular characterization of novel glucarpidases: Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment.

Repeated cycles of antibody-directed enzyme pro-drug therapy (ADEPT) and the use of glucarpidase in the detoxification of cytotoxic methotrexate (MTX) are highly desirable during cancer therapy but are hampered by the induced human antibody response to glucarpidase. Novel variants of glucarpidase (formal name: carboxypeptidase G2, CPG2) with epitopes not recognized by the immune system are likely to allow repeated cycles of ADEPT for effective cancer therapy. Towards this aim, over two thousand soil samples were collected and screened for folate hydrolyzing bacteria using folate as the sole carbon source. The work led to the isolation and the characterization of three new glucarpidase producing strains, which were designated as: Pseudomonas lubricans strain SF168, Stenotrophomonas sp SA and Xenophilus azovorans SN213. The CPG2 genes of Xenophilus azovorans SN213 (named Xen CPG2) and Stenotrophomonas sp SA (named Sten CPG2) were cloned and molecularly characterized. Both Xen CPG2 and Sten CPG2 share very close amino acid sequences (99%); we therefore, focused on the study of Xen CPG2. Finally, we demonstrated that a polyclonal antibody raised against our new CPG2, Xen CPG2, does not react with the CPG2 from Pseudomonas sp. strain RS-16 (Ps CPG2) that are currently in clinical use. The two enzymes, therefore could potentially be used consecutively in the ADEPT protocol to minimize the effect of the human antibody response that hampers current treatment with Ps CPG2. The identified novel CPG2 in this study will, therefore, pave the way for safer antibody directed enzyme pro-drug therapy for cancer treatment.

Status Quo in Antibody-Drug Conjugates - Can Glyco- Enzymes Solve the Current Challenges?

Over the last years, a novel class of anti-cancer drugs named antibody-drug conjugates (ADCs) has been developed. Due to their limited off-target toxicity but highly potent cytotoxicity at tumor sites, ADCs have proven to be a good alternative to ordinary cancer treatment, such as chemotherapy or combination therapy. Numerous enhancements in antibody-drug engineering led to highly potent tumor targeting drugs with a wide therapeutic window. Two ADCs (Brentuximab vedotin and Trastuzumab emtansine) are already on the market and many others are in clinical trials. However, unstable linkers, low drug potency and unwanted bystander-effects are only some of the drawbacks of ADCs. Enzymes used in combination with prodrugs happen to be a promising alternative. The glyco-enzyme horseradish peroxidase (HRP) has proven to activate the hormone indole-3-acetic acid (IAA) to a highly potent cytotoxic drug. This combination of IAA and HRP has been investigated for the use in strategies such as gene-directed enzyme prodrug therapy (GDEPT) and antibody-directed enzyme prodrug therapy (ADEPT). This article reviews the current state of research in ADC engineering and describes the potential major enhancements through use of glycoenzymes in combination with a prodrug.