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Antibody Therapeutics, 2020, Vol. 3, No. 4 237–245 doi:10.1093/abt/tbaa024 Advance Access Publication on 25 November 2020 Review Article Tackling solid tumour therapy with small-format drug conjugates 1,2, 3 Mahendra P. Deonarain and Quinn Xue Antikor Biopharma Ltd, Stevenage Bioscience Catalyst, Gunnels Wood Road, Stevenage, Hertfordshire SG12FX, 2 3 UK, Department of Chemistry, Imperial College London, Exhibition Road, London SW72AZ, UK, and Essex Biotechnology Ltd, Shun Tak Centre, Room 2818, China Merchants Tower, Connaught Road Central, Hong Kong 168-200, SAR China Received: August 26, 2020; Revised: November 11, 2020; Accepted: November 13, 2020 ABSTRACT 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. Statement of Signiﬁcance: Antibody–drug conjugates are a clinically and commercially established modality of cancer therapy with ﬁve new agents approved over the last 2 years. Treating solid tumours remains a major challenge with many failures and small-format drug conjugates offer a solution to the tumour penetration issue. KEYWORDS: antibody–drug conjugate; fragments; scaffolds; solid tumours INTRODUCTION overcoming tumour interstitial fluid pressure, diffusing through dense stroma and passing through tight epithelial Drug penetration into solid tumours as a factor influencing barriers (Fig. 1). This typically results in <1% of the efficacy has been discussed at length over the years, but is injected dose/gram of MAb/ADC reacting the target in it only now being actively addressed . For biological solid tumours in humans [4–6]. therapies in particular, the relationship between drug dosing and tumour uptake is highly complex and very These observations increasingly backed up by preclinical often, the micro-distribution across a whole tumour does and clinical data are motivating researchers to look at not correlate with drug dose or plasma concentration smaller formats of targeted therapeutics, which (due to and this underappreciated variability could explain poor more rapid diffusion kinetics) are known to have superior responses due to suboptimal concentrations of therapeutic tissue-penetrating (perfusion) properties compared with agents in the tumour micro-environment (TME) [1,2]. This large proteins such as immunoglobulins . Of course, is especially true with monoclonal antibodies (MAbs), lower molecular weight (MW) therapeutics brings with which have to overcome numerous biological barriers [3,4] it a whole new set of issues on the positive side (e.g. such as poor vascular supply, crossing the endothelium, reduced side effects due to decreased cross-reaction with To whom correspondence should be addressed. Mahendra P. Deonarain. Email: firstname.lastname@example.org. Published by Oxford University Press on behalf of Antibody Therapeutics 2020. This work is written by US Government employees and is in the public domain in the US. 238 Antibody Therapeutics, 2020 fragment derivatives, which will all have very different pharmacokinetic (PK) and pharmacodynamic properties (Fig. 1). For the smaller formats, the chemical linker– payload has a greater influence on these properties as it can make up 10–30% of the overall conjugate mass compared with a typical 2–3% for an IgG, therefore requires special consideration and bespoke design (Fig. 2; Table 1). This review will focus on non-radioactive and non-liposomal pharmaceutical conjugates. RECOMBINANT ANTIBODY FRAGMENTS Recombinant antibody fragments lend themselves to a wide range of engineering approaches [13,14] to facilitate linker–payload bioconjugation such as the introduction of conjugation friendly thiols. They are normally produced in prokaryotic systems removing the glycan-conjugation option utilized by some in the ADC field. Fragments such as single-chain Fvs (scFv) and single-domain antibodies tend to be more robust and stable having been subjected to stringent selection pressures during discovery compared with Fab-fragments . The resulting antibody fragment drug conjugate (FDC) at a drug:antibody ratio (DAR) of ∼ 2 has the feature of carrying more payload compared with a standard ADC of DAR4 on a mass basis but has a shorter half-life and thus lower systemic bioavailability compared with an ADC. Figure 1. Drug conjugate delivery via the tumour vasculature and pene- Fab-fragments have been superseded by formats such tration can be illustrated with broadly three PK profiles. (A) Conventional as scFvs but examples exist of conjugates demonstrating ADCs with MWs of > 150 kDa accumulate and penetrate into tumours over days and eliminate from the body over weeks requiring less frequent proof-of-principle. Early conjugates with moderately dosing, but a higher risk of off-target/cumulative toxicity. (B) A wide range potent, chemotherapy-approved payload such as paclitaxel of smaller (5–100 kDa), protein-based binding scaffolds such as scFv and and doxorubicin have largely been ineffective, but a DARPins, which have uptake and penetration kinetics lasting hours, but trastuzumab–mono-methyl auristatin E (MMAE) FDC are eliminated more rapidly (days), reducing non-specific exposure time, but may require strategies for higher drug delivery (e.g. higher DAR, DAR1 with 200–500 pM potency in vitro required alternate HLE, more frequent or higher dosing). (C) Very small peptidic conjugates day dosing at 20 mg/kg to see any tumour regression (<5 ka) that have very rapid and more complete uptake and penetration . This high dosing requirement was also seen more kinetics, but are eliminated in a matter of hours also requiring strategies recently with an anti-CD-20 Fab appended with a sortase to improve temporal exposure. conjugation tag used for enzymic conjugation of an MMAE payload . The FDC had to be dosed at Fc-receptors, reduced temporal exposure to normal tissues, 20 mg/kg every 3 days for four doses to obtain 4/6 cures, higher tumour: plasma exposure ratio) and negative side compared with complete cures for an equivalent ADC. (smaller window of bioavailability, reduced overall uptake) Notably, the FDC had ∼6× lower plasma exposure as [8,9], so striking the balance to obtain a favourable window measured by the PK area under curve. The FDC, however, is key and probably none more so than in the field of was better tolerated. A similar but dual-linker-payload antibody–drug conjugates (ADCs) [3,10]. (DAR3, cleavable and non-cleavable auristatin) was also With nine approved products and approaching 100 very potent (IC50 0.7–0.9 nM) but not evaluated in vivo ADCs in clinical trials [10,11], this modality is again on . Trastuzumab Fab-based conjugates based on the ultra- an upward trend after numerous setbacks and innovation potent pyrrolobenzodiazepine (PBD) payload class (IC50 cycles. Effective treatment of solid tumours remains a in the low pM range) were recently described where a significant challenge for the reasons outlined above with novel dual maleimide disulphide rebridging technology greater clinical successes seen in haematological cancers previously applied to ADCs was applied to the native [11,12]. The ADC industry is firmly focused on the cysteines in a Fab . The tesirine payload has been Immunoglobulin format with numerous approaches for used in several clinical-stage ADCs but was also the cause refined conjugation and more a homogeneous product of unacceptable toxicity in the discontinued Rova-T and quality, but an evolving area is the use of smaller formats others subsequently . This was modified to be more (i.e. antibody fragments or binding scaffolds smaller hydrophilic with a symmetrical dual maleimide bridge. In than 150 kDa), which promises to widen the therapeutic vitro potencies were 6–7 pM for high human epidermal window by improving tumour kill efficacy whilst reducing growth factor receptor-2 (HER2)-expressing cells and as normal organ toxicity. This review will focus on the potent as the trastuzumab-based ADC despite the reduced emerging small-format ‘biologics’ from ∼ 2 kDa peptide– avidity and possibly reduced internalization kinetics (not drug conjugates to larger ∼ 80 kDa immunoglobulin determined). In vivo efficacy was not explored . Antibody Therapeutics, 2020 239 Figure 2. A size and format comparison of various drug conjugates. The archetypal IgG is shown with the common conjugation strategies (A) surface lysines, (B) hinge thiols, (C) site-specific thiols, (D) Fc-carbohydrate, (E) genetically engineered tag or non-natural amino acid. The same colour coding is used for the conjugation onto the alternative, smaller formats of decreasing size: bivalent antibody fragment (∼75–80 kDa), Fab or diabody (∼50 kDa), high-DAR ScFv (25 kDa), VH-domains (12.5–25 kDa), many types of scaffolds (10–25 kDa) and a variety of peptides. Table 1. A list of drug-conjugate formats in order of increasing size with examples of the targets addressed and payloads used Format size (kDa) Format name Example target Cancer indication Example payload References 1.5–2 Bicycle (bicyclic MMP14 Breast, lung DM1 vcMMAE [67–69, 71] peptides) EphA2 multiple solid Nectin tumours ∼3–5 Pentarin Somatostatin Neuroendocrine DM1 [62–65] receptor Liver ∼3.5–5 Cysteine knots Integrin, Pancreatic Gemcitabine, [60, 61] MMP2 MMAF, Cis-platin 5–6.5 Affibody HER2 Breast/gastric Idarubicin, [43–45, 46] vcMMAE Photosensitizer ∼10–11 Centyrin Adnectin EGFR Multiple solid vcMMAF [48, 50] Glypican tumours Liver Tubulysin ∼15–18 DARPIn EpCAM Multiple solid MMAF  tumours ∼15 Abdurin EphA2 Prostate vcMMAE  ∼12.5–25 VH (like) domains PSMA Prostate DGN549  ∼25–27 ScFv HER2, EGFR Breast/gastric Photosensitizers [21, 22, 24, 25, 28, CD41/61 MMAF, 29] vcMMAE, Auristain F vcMMAE ∼55–60 Diabody CD30 Lymphoma MMAF  ∼50 Fab CD20, HER2 Lymphoma vcMMAE PBD [15, 18] breast/gastric ∼80 SIP ScFv-Fc Fibronectin, Multiple solid Cemadotin, DM1, [35–37, 39] Tenascin-C tumours vcMMAE FGFR-2 ScFvs are artificially tethered, recombinant antibody systemic circulation before laser illumination . We and structures but represent the preferred format for most others have developed this technology and demonstrated antibody discovery programmes that utilize a display tumour eradication in vivo with very few side effects [21,22], technology [13,14]. In specific applications where time- but the complex nature of such a two-step therapy has critical elimination was necessary (e.g. fast clearance hampered commercial development. This has not put ahead of a second step), they have proven useful. There off some companies combining optically active payloads are many reports on targeted photodynamic therapy and conventional ADCs so that therapeutics can be where a conditionally cytotoxic photosensitizer payload simultaneously imaged and used for treatment, in a is delivered to tumours but must be removed from the theranostic approach . 240 Antibody Therapeutics, 2020 We later extended our work on scFv-targeted photo- idea was exploited to build a nanobody–drug conjugate dynamic therapy to conventional payloads with more with a magnetic resonance imaging (MRI) contrast agent commercial success, broadly calling them ‘FDCs’. Using . A biparatopic anti-EGFR nanobody was fused to a particular scFv VH–VL frameworks predisposed to gadolinium-binding domain (imaging) and a C3 tag for chemical conjugation and high payload loading, DARs of payload conjugation. HLE was also incorporated through 5–10 were obtainable via lysine conjugation whilst retaining an anti-albumin nanobody. A maleimide-functionalized the critical biophysical properties [24,25]. Although cis-platin chemotherapy drug was conjugated to the fusion 3+ heterogeneous in nature, stochastic high DAR FDCs have protein’s C-terminus and Gd incorporated non-covalently fewer permutations than lysine-conjugated ADCs. As via dialysis. Uptake and imaging were demonstrated but, expected, the linker–payload structure had a major impact not unexpected for a relatively moderately potent drug on biophysical properties such as aggregation, binding (IC ∼ 1 mM); only moderate potency was seen in vitro. In affinity and thermal stability leading us to tailor payloads vivo, the conjugate was as potent as free cisplatin (in terms specifically to match the scFv format. Superior tumour of platinum content), but much better tolerated. This was penetration compared with ADCs has been observed due to the 4–5× higher accumulation in tumours, which and nM–pM potencies observed in vitro on cell lines was further supported by the T -weighted MRI contrast using auristatin and maytansine payloads [24–27]. A key images . finding when developing high DAR scFv-based FDCs Smaller antibody fragments such as Variable (V)- was that although the MW was theoretically within the domains (Ablynx’s nanobodies: VHH-domain antibodies range for renal excretion, the chemical–physical properties derived from llamas, Crescendo Biologics’ Humabodies: of the linker–payload became a dominating feature that human VH-domains) require some sort of HLE technology altered the PK to a predominantly hepatic clearance to make them viable candidates. Their Humabody-Drug route and a slower-than-expected systemic elimination Conjugates (HDCs) platform is made up of 15kDa approaching albumin-binding half-life extension (HLE) domains conjugated to a low-DAR, additionally half-life methods [26,27]. This, in turn has made FDCs a viable extended using albumin-binding domains. This retains the option with dosing now approaching that of ADCs. benefits of tumour penetration . CB108, a biparatopic We have used lysine residues to achieve the high DAR, HDC against prostate-specific membrane antigen (PSMA) but site-specific conjugation, more aligned to the conven- has been shown to be effective in vivo. A very nice study by tional ADC field can be achieved using C-terminal cysteine Nessler et al  aimed to tease out some of the important thiols or dedicated conjugation tags to obtain lower DARs features and benefits of smaller format drug conjugates. . One example is the SNAP technology that utilizes Low-affinity monovalent (VH1) and high-affinity, rapidly a small, engineered DNA–alkyltransferase enzyme as a internalizing, biparatopic (VH1–VH2) HDCs were created recognition and conjugation domain to link benzylguanine- with and without HLE domains against PSMA. These modified payloads. Low nM potencies against epidermal were conjugated to a DNA-alkylating payload DGN549, growth factor receptor (EGFR)-expressing cells lines were to a DAR1. In the absence of any drug delivery or mass seen in vitro using the scFv derived from the clinically transport limitations, rapid internalization led to the approved panitumumab MAb . highest in vitro potency, but slower internalization aided Specifically focussing on the TME, Yap et al.  tumour penetration and higher efficacy in vivo. HLE was developed a scFv- based FDC targeting an integrin needed for in vivo efficacy as these low-DAR conjugates glycoprotein (GPIIb/IIIa: CD41/CD61), which is found in would otherwise clear via renal filtration. Alexa-Fluor- an active–high-affinity conformation on activated platelets 680 labelling of the various formats (without the payload) that are increasingly thought to be involved in mediating confirmed the superior penetration of the VH1–HLE tumour growth and metastasis in the TME. Using a format, which was additionally backed up with tumour sortase-recognition tag, valine–citruline (vc)-MMAE with spheroid modelling data . Elasmogen have a similar a Gly linker was conjugated to a DAR1. In vivo, four technology based on shark variable domains from new doses of a 6 mg/kg scFv-GGG-vc-MMAE gave a moderate antigen receptors called soloMER™, which coupled with ∼ 8-day tumour growth delay demonstrating proof-of- its HLE technology NDure™  is being utilized to concept for this novel approach. Targeting the TME was discover and develop soloMER™–drug conjugates. further illustrated using a Cy5 dual-labelled conjugate Bivalent antibody-derived fragments have met with . An interesting twist on using scFvs was described greater preclinical success as seen with small immuno- by Wang et al.  aiming to capitalize on the increased proteins (SIP-Philochem) [35–37] and diabodies (Seattle macro-pinocytosis seen in ras-driven cancers such as Genetics) . Neri’s SIP technology uses the CHε4 pancreatic. An-anti-EGFR scFv recombinantly fused to domain to dimerize scFvs yielding a fragment that is domain III of human serum albumin (for HLE) and the ∼ 50% the size of an IgG, with a faster elimination apoprotein/carrier for the cytotoxic antibiotic lidamycin. time due to the absence of neonatal FcR binding. Using The ∼ 60 kDa conjugate effectively internalized and was primarily non-internalizing, tumour neovasculature targets highly potent across four pancreatic cancer cell lines (IC such as fibronectin and tenascin-C, excellent uptake range 15–70 pM), although clear specificity was not shown. and tumour/blood contrast ratios were obtained and The concept of delivering an ADC via non-clatherin the availability of two C-termini presented two cysteine route was demonstrated and a well-tolerated, moderate thiol conjugation positions . The aim is to destroy tumour growth delay was shown at 0.4 mg/kg given twice tumour vasculature to starve the tumour of nutrients . Higher doses were not used. The modular design and this removes the tumour penetration hurdle, but the Antibody Therapeutics, 2020 241 payload is released extracellularly and diffuses into the (binding site barrier). The IgG, as shown by many, gave nearby cells with a resulting bystander killing effect. If higher overall uptake by 24 h. thiol-bearing payloads are used, practically no linker is required (‘traceless’) as long as the disulphide is hindered or buried/protected within the protein architecture to NON-ANTIBODY SCAFFOLDS reduce the risk of inadvertent release . The release The ‘non-antibody’ binding format field continues to thrive mechanism is via extracellular thiols (e.g. glutathione), because they promise to solve the problems presented by which is amplified upon more cells dying. Using a DM1 conventional antibodies such as expensive manufacturing, payload on an anti-fibronectin–EDA SIP, well-tolerated formulation/concentration, glycosylation, thermostability cured were seen in murine F9 teratocarcinoma animal and tissue penetration. These scaffolds tend to range from models dosed at 7 mg/kg three times . An anti-tenascin ∼ 2 to 20 kDa (smaller than most antibody fragments), can C SIP coupled to a more commonly used vc-MMAE be expressed at exceptionally high yield in Escherichia coli, linker–payload (DAR2) also demonstrated tumour growth selected by in vitro display, demonstrate higher stability and inhibition at 7 mg/kg four times but was not as effecting can be multimerized and built up according to the desired as the IgG version that was more stable. The IgG-based properties . A few scaffold companies have published ADC was again more stable in a side-by-side comparison or disclosed intentions to develop SDCs, but other formats of IgG vs. SIP using the F8 antibody and DM1 payload such as Anticalins, Avimers, Fynomers, Kunitz domains conjugated at a DAR2 as a C-terminal disulphide . and Affilins have not gone down this route. As expected, the SIP–drug conjugate accumulated into the tumour and cleared more rapidly and the 24-h uptake levels were more than four times higher for the IgG ADC. Affibody–Drug conjugates Although the payload on the ADC was ∼10× more stable, Affibodies, based on the 6 kDa Staphylococcus protein- the SIP conjugate was more effective on a molar basis with A, Z-domain can be engineered and displayed by phage to authors attributing this to the faster payload release leading generate high-affinity binders. These are being developed to higher tumour payload exposure over a shorter period of as therapeutics by Swedish enterprise Affibody AB and time compared with the slow-release of an ADC. Toxicity, are in Phase 2 clinical trials with a psoriasis therapeutic which may be higher for a less stable drug-conjugate, was and a breast cancer positron emission tomography imag- not evaluated . A similarly configured scFv–Fc format ing agent . No commercial affibody–drug conjugates ADC was made from an anti-fibroblast growth factor have been disclosed, but conjugates have been described receptor-2 (FGFR2) antibody discovered by phage display. targeting HER2 (ZHER2891) with a vcMMAE payload Using the vc-MMAE payload ∼nM potency was seen . (DAR1) with low nM potencies on high HER2-expressing The most comprehensive analysis of a potent antibody cells lines . Higher affinity, longer half-life, Fc-fusions FDC was described by Seattle Genetics  using an anti- had increased potency in vitro (130 pM) on SKBr3 cells CD30 diabody with four cysteine thiols conjugated to . More recently, the same affibody formats were coupled MMAE and mono-methyl auristatin F (MMAF) payloads to the non-releasable DM1 payload (DAR1) resulting in with maleimide linkers. The diabody ADC (MMAF DAR higher in vitro potencies (270–470 pM, comparable to the ∼ 4) was compared with an equivalent IgG ADC. In this trastuzumab ADC) and significant in vivo efficacy. Con- example, the two formats had comparable DARs and jugates radiolabelled with Tc showed marginally higher valency. The diabody–drug conjugate had a faster blood tumour uptake at 4 h at the expense of higher blood and clearance reflected by its smaller size, but the 30× lower normal organ uptake. Doses of 8.5 mg/kg, weekly five exposure level only led to a 3× drop in in vivo efficacy times were needed to see well-tolerated tumour growth (7.2 vs. 2 mg/kg needed for comparable tumour growth delay of ∼ 20 days but no cures were seen in this first in inhibition). Interestingly, the renal clearance expected for vivo proof-of-principle of this scaffold format . The such fragment sizes was not evident, suggesting that the same affibody was conjugated with photosensitizer payload payload had a major influence diverting the conjugate to pyropheophorbide-a (DAR1) with 12–23 nm IC potency the liver for metabolism . on HER2-expressing cells. Well-tolerated cures were seen The above research and development makes observa- with a single injection of 20nMol of conjugate (∼0.2 mg tions based on therapeutic efficacy without direct evidence dose/8 mg/kg) upon laser illumination with the rapid clear- that tumour penetration is having a significantly positive ance being optimal to allow photo-activation without skin impact. This has been difficult to quantify for drug con- toxicity . jugates. Direct correlations have been made between anti- body size and tumour perfusion  but a more recent Fibronectin type III–drug conjugates analysis in a SKOV3–HER2 model examining uptake and tumour penetration homogeneity of monovalent and biva- These popular scaffolds have been reviewed extensively lent nanobodies (MW ∼ 15–30 kDa) size and affinity was  with a number investigated as drug conjugates. carried out using intravital fluorescence microscopic imag- Immunoglobulin-like centyrins are ∼ 100-residue (11 kDa), ing . This nicely showed that the smaller format gave thermal/chemical stable domains being developed by rapid and more homogeneous tumour uptake, during the 1- Janssen/J&J. Extensive surface cysteine scanning mutage- to 3-h time frame, compared with the trastuzumab IgG that nesis identified suitable conjugation positions and an anti- was restricted to around the vasculature, and also show that EGFR DAR1 MMAF conjugate demonstrated ∼0.2 nM a too high affinity for the nanobodies hindered penetration IC in vitro potency . No in vivo data have been 50 242 Antibody Therapeutics, 2020 presented but a bioanalytic workflow was developed for due to the small size taking an impact upon chemical centyrin–drug conjugate analysis in tissues in a collabora- modification . tion between Janssen and Immunogen . Clinical-stage adnectins are also based on the fibronectin domains and are being developed by BMS. Using a tubulysin analogue PEPTIDE–DRUG CONJUGATES payload with a cleavable cathepsin B linker against the Small peptides have even faster penetration and more rapid hepatocellular carcinoma antigen glypican-3, a DAR1 elimination properties compared with the above examples. (via a maleimide moiety to a C-terminal cysteine thiol) Their totally synthetic nature promises many benefits as adnectin–drug conjugate was made and evaluated . One ◦ drug conjugates. This topic is covered extensively by He candidate conjugate had high thermostability (T ∼ 80 C), et al . There are many reports, for example with low- 32 nM K binding affinity and 0.3 nM IC cell-kill potency d 50 potency payloads such as doxorubicin. These conjugates on Hep3B cells in vitro. The payload conjugation had no have micromolar potencies and are not usually more potent deleterious effect on the conformation of the adnectin than the free drug, but generally more specific . More structure as supported by detailed hydrogen–deuterium recent innovations with potent payloads demonstrate more exchange mass spectrometry . No HLE strategy was promising approaches including some at the clinical stage employed as the authors favoured the rapid renal clearance 1 of development. (half-life approx. / h). Quantitative biodistribution showed very specific tumour uptake with renal exposure in the first few hours but low liver and other normal organ exposure. Cystine knot–drug conjugates By 7 days, it was undetectable in all tissues other than the Cystine knots (30–50 amino acids, also known as knottins) tumour. Most impressive was the well-tolerated, complete are at the larger end of the peptide scale, but like tumour cures at 0.12 mmol/kg (∼1.4 mg/kg), despite the peptides, are amenable to scalable solid-phase synthesis moderate affinity and rapid clearance given three times and incorporation of useful stabilizing and functionalizing weekly. The authors acknowledge that this observation non-natural amino acids. They have enhanced chemical, bucks the trend seen with small-format binders and suggest protease and thermal stability properties compared with that the rapid internalization of the glypican-3 target may conventional antibody domains, due to their highly account for these promising results. It remains to be seen if compact structure and stabilizing disulphide bridges . such frequent dosing remains a viable option. Conjugation to cytotoxic payloads was achieved via solid- phase synthetic incorporation of a non-natural amino acid followed by azide–alkyne conjugation of a gemcitabine DARPin–Drug conjugates payload. The Knottin–drug conjugate was able to over- The Designed Ankyrin Repeat (DARPin) class of scaf- come drug resistance in PANC-1 pancreatic cancer cells, fold proteins are well-established with five clinical-stage increasing the potency of gemcitabine 25-fold . A more products, one (abicipar) recently completing a Phase 3 ‘ADC-like ‘molecule was generated using cell-free protein trial in ophthalmology . Drug conjugates are much synthesis with click chemistry (DAR2) and an appended further away. Using bi-orthogonal chemistry, an anti- Fc-domain. The MMAF payload DAR2 was used resulting EpCAM DARPin with a (i) C-terminal cysteine residue in potencies similar to the gemcitabine conjugates but and a (ii) non-natural amino acid azidohomo-alanine tumour growth delay was seen in vivo at 10 mg/kg given was used to attach a half-life extending albumin-binding twice/week for 3 weeks . domain and MMAF payload (DAR1). This generated a DARPin–MMAF conjugate with an IC 400 pM, which Pentarins–drug conjugates had extended plasma half-life (17.4 h in vivo) . No in vivo or commercial developments have been disclosed, but Pentarins and bicyclic peptides represent the shorter end of there may be issues with this format given a recent FDC the peptide scale (2–5 kDa) and are worth mentioning due rejection setback . to the advanced clinical stage of their drug conjugates. The pentarin (penetrate, target) portfolio developed by Tarveda, consists of small peptides that can be made into Abdurin–Drug conjugates pentarin–drug conjugates (PDC). Their lead compound, Abdurins can be diversified to form libraries of binders as PEN-221, is a somatostatin receptor-2 (SSTR2, expressed they are based on engineered IgG CH2 domains (∼15 kDa); on neuroendocrine tumours) targeted DM1 maytansine. similar to the larger Fcabs being developed by F-Star, The payload is conjugated to the disulphide-cyclized Tyr3- these retain the ability to bind to the neonatal Fc recep- octreotate, which had high affinity (∼51 pM) and rapid tor and thus have an inherent extended serum half-life internalization. In vivo, 1–2 mg/kg PEN221 were enough . Recently, Abdurin–drug conjugates were described to cure HCC33 (liver) and H524MD (lung) cancer tumour using Abzena’s CyPEG and HiPEG conjugation technolo- models given four times on a weekly schedule with maximal gies and vcMMAE payload (DAR1). Moderate in vivo payload uptake achieved within 2 h . Results presented efficacy (tumour regression at 5 mg/kg, six doses) was at the American Society for Clinical Oncology in 2018 seen in PC3 xenograft studies. A DAR2 conjugate led to showed that PEN221 was well-tolerated at doses up to some cures but significant loss of target and FcRn-binding 18 mg every 3 weeks with evidence of efficacy in the Phase affinity was observed in various combinations most likely 1 arm . This product is now in Phase 2 clinical trials Antibody Therapeutics, 2020 243 for SSTR2-expressing neuroendocrine and lung tumours. manufacture control processes afforded by bacterial pro- A follow-up compound, PEN866, is a PDC carrying the duction, higher yields, lack of glycosylation and generally SN38 payload targeting the heat-shock protein chaper- simplified analytics due to the smaller size. It remains to one HSP90. This is currently in a Phase 1/2a clinical trial be seen if these features translate into economic or patient for advanced solid cancers sensitive to topoisomerase I benefits. inhibitors and recent updates at the European Society for Precision medicine is often a buzzword used loosely to Medical Oncology (ESMO) and American Association for describe tailoring a drug therapy to a patient’s genetic Cancer Research conferences suggested good tolerability, profile but is increasingly being used in terms of other signs of clinical efficacy  and promising clinical uptake patient characteristics. Personalized dosing schemes to and good PK profile . improve tumour penetration could be one key element  and having available formats to maximize tumour penetration will add to the clinical armoury. The increasing Bicyclic peptide (Bicycle)–drug conjugates preclinical use of payload imaging technologies such as matrix-assisted laser desorption ionization mass spec- The phage-displayable bicyclic peptide (‘bicycles’) tech- trometry imaging  could inform this at the preclinical nology discovered and developed by Heinis et al.  animal model level. Other strategies to aid penetration, is being commercialized by Bicycle Therapeutics Ltd, such as addition of modulators to enhance penetration including a major programme on Bicycle–drug (toxin) (e.g. RGD (Arginine-Glutamate-Aspartate) peptides, conjugates (BTCs). MT1-matrix metalloprotease (MMP) is ligands to endothelial/epithelial cells that increase vascular overexpressed in multiple cancers including triple negative permeability such as NRP-1, Lys/Arg-rich peptides  breast, non-small cell lung and soft tissue sarcoma. An or TEM8 targeting for targeting stroma in solid tumours anti-MT1-MMP BTC (BT1718) carrying a DM1 payload  or LRRC15, cancer-associate fibroblasts marker ) via a hindered disulphide linker has ∼ 2 nM affinity, rodent will require knowledge of an additional receptor making cynomolgus species cross-reactivity and plasma stability tailoring even more complex. of >20 h. It demonstrated efficacy in tumour models at Collateral exposure through non-targeted deposition 3 and 5 mg/kg BDC given twice weekly for 2–4 weeks. within normal tissues is recognized as a key driver to ADC– Complete cures were seen at 10 mg/kg with good tolerability payload toxicity [76,77] with the well-characterized exam- as measured by body weight . This product is currently ple of dose-limiting toxicity of trastuzumab–emtansine in a Phase 1/2 clinical trial. An update from the ESMO caused by Fc-mediated binding to platelets (thrombocy- identified a recommended Phase 2 dosing of 7.2 mg/m , topenia) . Most of these small-format drug conjugates once weekly with demonstratable tumour uptake and signs promise to overcome this due to abolished Fc-receptor of efficacy . A follow-up clinical candidate, BT5528 binding and reduced chronic exposure, but a clear cor- addresses the ephrin A2 receptor (EphA2) receptor (target relation between improved tolerability and conjugate size for MEDI-547, a discontinued ADC that showed severe would be difficult to demonstrate given the wide variation toxicity). Using a different payload, vc-MMAE, rapid in formats. tumour uptake was seen with persistent accumulation Tumour spheroid technology is becoming more acces- and rapid renal clearance in xenograft models. Payload sible and used in the discovery workflow and evaluating conjugation had no adverse effect on the bicyclic peptide penetration can help to prioritize candidates. It is acknowl- affinity (5.7 vs. 1.9 nM) and a rapid renal clearance edged that in vitro cell kill potency (IC ) is a poor indicator was observed (half-life ∼ 0.4–0.6 h in rodents and non- of tumour cure efficacy as we and others find that it’s not human primates). A weekly dose of 0.5 mg/kg (equivalent necessarily that the most potent conjugates make the best in to 10–15 mg/kg of a similar ADC DAR2) gave rise to 3 vivo candidate . Shah et al  modelled the correlation tumour regressions with tumours as large as 1000 mm between in vitro IC and in vivo ID and shown that 27× 50 50 being treatable at doses of 3 mg/kg demonstration the more ADC was needed in the plasma compared with cell penetration advantage over an ADC. As expected, non- culture medium to achieve tumour growth ‘stasis’. This cleavable variants were ineffective. A nice correlation shows that, in these models, there remain transfer barriers was seen between EphA2 receptor level and tumour cure to solid tumour therapy and that smaller formats could efficacy and none of the previously observed toxicities were make the real difference needed to address some of these seen when compared with a MEDI-547 equivalent ADC in difficult-to-treat solid tumours. rat or non-human primate toxicology studies . BT5528 is in a Phase 1/II trial for solid tumours as a monotherapy and combination with checkpoint inhibitor nivolumab . CONFLICT OF INTEREST STATEMENT Other preclinical targets under commercial development MD is an employee and shareholder in Antikor Biopharma include nectin-4 (BT8009) . Ltd and QX is an employee and shareholder in Essex Biotechnology Ltd. DISCUSSION REFERENCES Antibody–drug conjugates are complex therapeutics to 1. Bartelink, IH, Jones, EF, Shahidi-Latham, SK et al. 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Antibody Therapeutics – Oxford University Press
Published: Nov 25, 2020
Keywords: antibody–drug conjugate; fragments; scaffolds; solid tumours
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