Abstract
Guided by a specific monoclonal antibody (mAb), antibody drug conjugates or ADC are a new, emerging, class of drugs able to deliver a drug payload directly to an intended target. This approach has recently been boosted by the U.S. Food and Drug Administration approval of brentuximab vedotin (Adcetris®; Seattle Genetics) to treat Hodgkin’s lymphoma and ado-trastuzumab emtansine (Kadcyla®; Genentech) for metastatic breast cancer. These new biotherapeutic drugs will bring many regulatory issues to the forefront regarding the ADME (Absorption, Distribution, Metabolism and Excretion) profile of each ADC. In this article, the authors discuss this and other important aspects of antibody-drug conjugates.
Keywords
Antibody-drug conjugate; ADC; monoclonal antibody; mAb; cancer; carbon-14; linker; immunotherapy; DAR; drug-to-antibody ratio; linker;
1.0. Introduction
The concept of targeted therapy towards the treatment of disease causing agent was first postulated by Paul Ehrlich over 100 years ago [1]. The idea was to create a therapeutic agent, termed the ‘magic bullet,’ to attack specific cellular targets, in the fight against disease states. Ehrlich’s vision is now finally being realized for cancer treatment with the development of targeted therapies using monoclonal antibodies (mAbs), peptides and/or proteins [2].
The emergence of the new class of antibody-drug conjugate (ADC) chemotherapeutics able to deliver a drug payload to its tumour target, guided by a specific MAb is an exciting opportunity for biopharmaceutical companies and contract manufacturing organizations (CMOs). This approach has recently been boosted by the US Food and Drug Administration approval of Adcetris [3] to treat Hodgkin’s lymphoma and Kadcyla [4] for metastatic breast cancer. These new biotherapeutic drugs will bring many regulatory issues to the forefront regarding the ADME (Absorption, Distribution, Metabolism and Excretion) profile of each ADC [5].
2.0 Regulatory
In order to release the ADC certain analytical methods must be developed and implemented. This is to verify and identify the type of MAb and cytotoxic drug used in its manufacture [6]. Typically in the region of 35 different analytical tests will be required for the release of a clinical batch of ADC [7]. The analytical techniques used will include protein mass spectrometry and capillary electrophoresis. A range of analytical tools can be used to determine the character of the ADC including peptide mapping and sequencing. The structure of the linker-drug combination can further be determined using multi-NMR [8] and FTIR spectroscopy techniques [9]. X-Ray crystallography can be used to assess the peptide or antibody structure and the drug to antibody ratio (DAR) can be evaluated using UV methods [10]. Subsequently, the application of size-exclusion chromatography (SEC) techniques can be used to determine fragmentation and aggregate patterns during the synthesis of the ADC [11].
More recently there has been a drive to produce ADCs with a greater degree of homogeneity, particularly in relation to the drug to antibody ratio (DAR) to help reduce regulatory issues [12]. ADCs typically contain a binomial distribution of cytotoxic drugs per antibody typically spanning anywhere from 0-8 drugs moieties per ADC molecule. Emerging technologies are much better able to fix the DAR ratio more tightly [13].
Furthermore, the antigen binding and biological activity of the MAbs must also be assessed against ELISA, in vitro cell-based assays and in vivo studies [14]. A critical factor is to develop robust analytical methods to determine the level of free cytotoxic drug [15]. In addition, chemical impurities obtained during the synthesis which include the impurity profile from host cell proteins must also be identified [16]. The manufactured ADC must be evaluated as a new molecular entity and not as a separate product (antibody-linker-drug) [5].
This is to elucidate a structure/function relationship towards: the pharmacokinetics profile and low immunogenicity; the cytotoxic drug must demonstrate potent anti-tumour activity towards target cancer therapies; linker has to be stable to enable the delivery of the ADC to target antigen; MAb must have high affinity and selectivity towards the cellular target. The tumour-associated antigen expression ratio must be high in tumours compared to normal tissue and allow the ADC-antigen complex to be internalized [17].
3.0. Linker Technology
Linker technology can fall into two areas [18]: cleavable linkers (peptide, hydrazone, or disulfide) or non-cleavable linkers (thioethers). A critical factor in the manufacture of ADCs is choosing the right linker to attach the antibody to the drug. Today, linker technology is a major focus for biopharma in the development of ADC platforms to maximize the killing of cancer cells. Even developed linkers suffer from stability issues and can be cleaved before entering the tumour cell, reducing the potency of the treatment and releasing the cytotoxic drug into systemic circulation [19]. New advances in linker technology include thioether linkages formed via standard conjugate addition chemistry.
These are derived from engineered cysteine SH groups reacting with a maleimide functionality on the linker which then undergoes a subsequent ring opening reaction to prevent the retro conjugate addition from occurring [20]. This technology has been developed by Seattle Genetics and is leading to ADCs with improved linker stability [21].
4.0. Carbon-14 Labeling
Isotopic labelling of modern active pharmaceutical ingredients (APIs), particularly of complex targeted therapies, is becoming more challenging, and material shortages compound the issue for carbon-14. The synthetic design for an isotopic analogue depends on a number of interdependent factors that must be carefully considered, along with any prior knowledge of the molecule, to ensure that a metabolically stable labelling position is achieved. The selection of appropriate labelled starting materials is limited, and the potential savings from beginning a synthesis with simple materials must be carefully weighed against the increased labour cost of a longer synthesis [22].
Figure 1The application of biocatalytic tools, for example, can introduce complex functionality in a single step and significantly help reduce the cost of labelled biomolecules and chiral compounds [23]. Almac’s isotope chemistry group has benefited from amalgamation with the biocatalysis group (Figure 1) it also interacts with the company’s Physical Sciences and Peptide Protein Technologies groups [24].
The major advantage of radiolabeling with carbon-14 is that it can be incorporated into the drugs’ carbon framework without altering its chemical structure, there by producing an identical copy of the unlabelled drug. This approach removes the risk of the radiolabel being scrambled, in comparison to ‘peripheral’ tritium labelling. Consequently, during the design of the synthetic route, it is vital to locate a feasible, biologically stable position for the carbon-14 label and to identify suitable starting materials which can be commercially available or easily made. [22].
Alternately, carbon-14 labeling in conjunction with accelerator mass spectrometry (AMS) can be used for the pharmacokinetic and metabolic profiling of ADCs [25]. The AMS technique uses micro-doses of labelled API to analyse the ratio of carbon-14 to carbon-12 and can detect extremely low quantities of metabolites (10 kBq per study) in biological samples. These two techniques when combined provide a powerful tool for drug metabolism and pharmacokinetics (DMPK) studies which can circumvent any issues with the relatively low specific activity of carbon-14 when compared to tritium [26]
Figure 2Carbon-14 labeling of ADCs can be executed on the linker region or incorporated into the cytotoxic drug or both (Figure 2) [27]. Cytotoxic drug families involving auristatins, maytansinoids and calicheamicins have complicated chemical structures containing several chiral centers [28]. In these cases, carbon-14 labelling is performed on the linker part of the ADC component. This is particularly appropriate for non-cleavable linkers where the linker remains attached to the payload after release from the antibody. Ideally, the label should be placed in the most metabolically stable position on the linker in order to survive in the systemic circulation before internalization of the ADC-antigen complex into the tumor target [29].
Figure 3Almac’s radiochemists have successfully completed several projects involving carbon-14 labeling of ADCs [30]. The strategy for isotopic labeling of the ADC is to prepare a product with the label in either the linker or in the payload or in both units. An example prepared at Almac is shown in Figure 3. The radiolabeling of this ADC was achieved via the carbon-14 labelled linker.
The cytotoxic drug was attached to the linker via the conjugate addition of a thiol-SH on the drug with a maleimide moiety on the linker. This resulted in a [14C]-labelled linker-drug complex bonded together by a non-cleavable thioether linkage. This then underwent purification using UF/DF to remove unbound [14C]-linker from the reaction mixture.
The [14C]-Drug linker conjugate was attached to the mAb via amide bond formation between a random surface epsilon-amino group of lysine and an activated ester moiety on the [14C]-linker-drug complex. The [14C]-labelled ADC was then purified by HIC chromatography, concentrated using UF/DF, filtered through a 0.22micron filter and formulated in pharmaceutical buffer. Stringent biological handling techniques were maintained throughout the manufacture to minimise endotoxin and bioburden levels. The manufacture resulted in a formulated [14C]-labelled ADC with a specific activity of 110 mCi/mmol.
In another example, the customer required the synthesis of a carbon-14 peptide drug (Figure 4), which was then further elaborated to prepare the [14C]-labelled ADC (Figure 5). The carbon-14 peptide was pegylated and this pegylation step also provided the maleimide functionality for attachment to the mAb to allow ADC assembly. The PEGlyation of peptides can be challenging, and these PEG covalent modifications require a reactive or targetable functional group at one end of the carbon-14 peptide [31]. The simplest method to PEGylate carbon-14 peptides – which have a primary amine linker – is to use a PEG compound that contains an activated ester group at one end.
In this target, the unlabelled resin-bound peptide was first synthesised using the SPPS approach. The terminal Fmoc amino acid protecting group was cleaved and the carbon-14 label introducedvia coupling with N-Boc-[1-14C]-glycine. Cleavage from the resin and purification of the crude peptide by preparative HPLC was followed by PEGlyation. Assembly of the ADC involved thioether formation between the cysteine SH group on the antibody with the maleimide functionality on the [14C]-linker-peptide drug. This resulted in a product with two drug linker moieties per antibody (DAR=2).
Conclusion
The future prospects of drug development is at the forefront of science and is captivating new limits of what is possible at the interface of scientific fields. This in turn is leading to more isotopic labelling challenges. To overcome this, isotope chemistry providers must now work in tandem with different technology fields if they are to be successful in aiding drug development.
This is clearly demonstrated with carbon-14 labelling of ADCs and peptides. The more understanding we have about the drug delivery vehicles such as monoclonal antibodies (MAbs) connecting with chemical linkers and drug molecules the more successful the next generation of ADCs will be. Carbon-14 labelling of ADCs has an important role to play in generating this information.
The number of ADCs in clinical development is just over 30 and a significantly larger number are progressing rapidly through the preclinical phase [32]. The potential sensitivity issues due to the low specific activity of carbon-14 labelling of ADCs compared to tritium labelling is outweighed by many advantages, for example the improved stability profile of the ADC towards radiolysis. The sensitivity problems are also easily circumvented by applying AMS technology on micro-doses of carbon-14 labelled ADCs and peptides to analyse extremely low quantities of metabolites (10 kBq per study) in biological samples. Almac is continuing to diversify its offering in the area of complex isotopic labelling services and has completed the preparation of several carbon-14 labelled ADCs to date.
How to Cite
DOI: https://doi.org/10.14229/jadc.2014.03.07.003
Sean L. Kitson, 1 William Watters, 1 David Speed, 1 Thomas S. Moody 1
Making Hot ADCs – ADC Review / Journal of Antibody-drug Conjugates – March 7, 2014. DOI: 10.14229/jadc.2014.03.07.003.
1 Almac Group
