Chem Soc Rev
`
`TUTORIAL REVIEW
`
`Cite this: Chem. Soc. Rev., 2021,
`
`50, 1480
`
`Peptides as a platform for targeted therapeutics
`for cancer: peptide–drug conjugates (PDCs)
`
`a Daniel H. O’ Donovan,
`a Jessica Iegre,
`Bethany M. Cooper,
`Maria O¨ lwegård Halvarssonc and David R. Spring
`*a
`
`b
`
`Peptides can offer the versatility needed for a successful oncology drug discovery approach. Peptide–
`drug conjugates (PDCs) are an emerging targeted therapeutic that present increased tumour penetration
`and selectivity. Despite these advantages, there are still limitations for the use of peptides as therapeutics
`exemplified through their slow progression to get into the clinic and limited oral bioavailability. New
`approaches to address these problems have been studied and successfully implemented to enhance the
`stability of peptides and their constructs. There is great promise for the future of PDCs with two
`molecules already on the market and many variations currently undergoing clinical trials, such as
`bicycle-toxin conjugates and peptide–dendrimer conjugates. This review summarises the entire process
`needed for the design and successful development of an oncology PDC including chemical and nano-
`material strategies to enhance peptide stability within circulation, the function of each component of a
`PDC construct, and current examples in clinical trials.
`
`Received 30th September 2020
`
`DOI: 10.1039/d0cs00556h
`
`rsc.li/chem-soc-rev
`
`Key learning points
`1. A variety of methods to address peptide chemical and enzymatic stability can be implemented.
`2. The design of a PDC requires understanding of the mechanism of action intended and hence environmental stimuli (pH, GSH and enzymes).
`3. The function and rationale behind the design of each component of a PDC.
`4. The stability of the PDC construct can be improved through the use of nanomaterials.
`5. Bicycle-toxin conjugates (BTCs) and peptide-dendrimer conjugates are emerging constructs that fall under the umbrella term PDCs.
`
`Introduction
`
`Traditionally, research within drug discovery falls into two
`groups: small molecules (o500 Da) and biologics (45000 Da).1
`Peptides are placed within the molecular weight range that is
`typically under-represented in the pharmaceutical company
`pipelines. A peptide is defined by the FDA as a polymer
`composed of less than 40 amino acids (500–5000 Da).2 Over
`recent years, the research community is acknowledging the
`many advantages that peptides bring over small molecules and
`biologics. These include simpler design, ability to interact with
`underexplored targets, cheaper synthesis, decreased immuno-
`genicity and enhanced tissue penetration.1 To date in the U.S.,
`
`a Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge,
`CB2 1EW, UK. E-mail: spring@ch.cam.ac.uk
`b Oncology R&D, AstraZeneca, Cambridge, CB4 0WG, UK
`c Medicinal Chemistry, Research and Early Development Cardiovascular,
`Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg,
`Sweden
`
`Europe and Japan markets, there are more than 100 peptide
`drugs used to treat a range of diseases.2 Financially, the peptide
`market is lucrative as it is estimated to be worth d11–16 billion
`annually by 2019.2 However, there is still a significant challenge
`for the pharmaceutical industry to get peptides to market with
`many adopting greener peptide synthesis techniques at
`increased costs than traditional approaches.
`Peptides can offer a multifunctional approach – in addition
`to being biologically active, they are excellent at transporting
`cargos to the desired targets. Their use within targeted ther-
`apeutics is an exciting area of research with great promise in
`the future with particular focus in, but not limited to, oncology.
`Witnessing the current success and investment into many
`antibody–drug conjugates (ADCs), the equivalent peptide–drug
`conjugates (PDCs) show promise for the future of the use of
`peptides within this setting. This review will highlight the
`excellence and limitations of peptides, their use in PDCs for
`advancing targeted cancer therapeutics and will consider how
`the specific tumour microenvironment can aid the design of a
`PDC. In addition, the review provides an examination of how
`
`1480 | Chem. Soc. Rev., 2021,50 , 14801494
`
`This journal is
`
`The Royal Society of Chemistry 2021
`
`View Article Online
`
`View Journal
`
` | View Issue
`
`MPI EXHIBIT 1071 PAGE 1
`
`MPI EXHIBIT 1071 PAGE 1
`
`

`

`Tutorial Review
`
`Chem Soc Rev
`
`the stability of PDCs in circulation can be enhanced through
`both chemical modifications and material science – a
`topic rarely discussed but extremely valuable to the successful
`development of new peptide drugs.
`
`ADME & PK considerations for peptides
`
`When considering therapeutic agents, it is crucial to analyse
`pharmacokinetic (PK) properties such as absorption, distri-
`bution, metabolism and excretion (ADME) as well as pharma-
`codynamic (PD) properties. Traditionally, PK properties of
`peptides tend to differ substantially from those of small drug
`molecules.3 A significant limitation of peptides is their limited
`or non-existent oral bioavailability leading to administration
`
`through intravenous (IV) injection. One of the most convenient
`methods of administration is oral as it allows the patient to
`take the medication independently, unlike IV that often
`requires a clinical setting.
`A small molecule is defined as ‘drug-like’ if it satisfies the
`criteria of Lipinski’s Rule of 5 (Ro5). These rules focus on
`several fundamental factors including the molecular weight
`(o500 Da), r5 H-bond donors, r10 H-bond acceptors and
`log P (o5): molecules that satisfy the Ro5 are likely to be orally
`bioavailable.4 Peptides do not fit into the Ro5 criteria due to
`their relatively large size (500–5000 Da) in comparison to small
`molecules (typically o500 Da). Despite peptides not satisfying
`the Ro5 criteria, the rule does not indicate that a peptide
`cannot become a drug as proven through many peptides on
`the market and in clinical trials.4
`
`Bethany M. Cooper received her
`MChem degree from the Univer-
`sity of Leeds in 2018, having
`completed her 3rd year of study
`at Lubrizol Ltd, Hazelwood. On
`return to the University of Leeds
`her final year was spent under the
`supervision of Professor Steve
`Marsden. In 2019, she started
`her PhD studies at the University
`of Cambridge under the super-
`vision of Professor David Spring
`and industrial
`supervisor Dr
`Maria O¨lwegård-Halvarsson, where
`her research has focused on peptide stapling methodologies for use
`within therapeutics.
`
`Bethany M. Cooper
`
`Jessica Iegre was born in Italy
`and
`obtained
`her MSci
`in
`Medicinal Chemistry and Pharma-
`ceutical
`Technology
`at
`the
`University of Pisa, Italy in 2013.
`The same year, she joined the
`AstraZeneca IMED Graduate pro-
`in Go¨teborg, Sweden
`gramme
`where she spent 2 years working
`across three different departments:
`medicinal chemistry, DMPK and
`computational chemistry. In 2015
`she joined the Spring group at the
`University of Cambridge, and she
`obtained her PhD in chemical biology in 2019. Jessica is currently a
`Postdoctoral Research Associate in the group, and she is developing
`novel stapled peptides to inhibit medicinally relevant protein–protein
`interactions.
`
`Jessica Iegre
`
`Daniel Hillebrand O’ Donovan is
`currently
`Associate
`Principal
`Scientist in Early Oncology R&D
`at AstraZeneca, Cambridge, UK.
`Following PhD studies in medi-
`cinal chemistry at Trinity College
`Dublin, he moved to Germany for
`postdoctoral studies at the Max
`Planck
`Institute
`for Kohlen-
`forschung followed by a Marie
`Curie
`fellowship at
`the Uni-
`versity of Oxford, UK. Since
`joining AstraZeneca in 2016, he
`has worked in a variety of
`including antihormonal
`therapies
`for breast
`research areas
`cancer,
`epigenetics,
`immuno-oncology and targeted protein
`degradation.
`
`Daniel H. O’Donovan
`
`Dr Maria O¨lwegård-Halvarsson is
`a Senior Research Scientist in the
`New Modalities group, Depart-
`ment of Medicinal Chemistry,
`AstraZeneca, Sweden.
`In her
`current role she develops linker
`chemistry and synthesizes drug
`conjugates within the targeting
`delivery platform. She obtained
`her PhD in organic chemistry
`from Gothenburg University
`in
`1990 and then spent four years at
`the Department
`of Medicinal
`Biochemistry, Gothenburg Uni-
`versity. She joined AstraZeneca in 1995, working three years in the
`isotope labelling group and has a 25 year commitment to the company,
`synthesizing drug molecules within the cardiovascular and metabolic
`disease areas in lead optimization and lead generation phase.
`
`Maria O¨lwegård Halvarsson
`
`This journal is
`
`The Royal Society of Chemistry 2021
`
`Chem. Soc. Rev., 2021,50 , 14801494 | 1481
`
`View Article Online
`
`MPI EXHIBIT 1071 PAGE 2
`
`MPI EXHIBIT 1071 PAGE 2
`
`

`

`Chem Soc Rev
`
`Tutorial Review
`
`enzymes that are responsible for the chemical instability and
`the breakdown of peptides in the bloodstream.
`Short half-lives are also experienced due to rapid renal
`clearance, resulting in many hampered peptide in vivo studies
`and ultimately the pursuit of the peptide as a drug. Found
`within the kidney are glomeruli pores that have a size of
`B8 nm; circulating peptides that are less than 25 kDa filter
`through the glomeruli, and are not reabsorbed through the
`renal tubule.3 Considering such limitations, it is not surprising
`that many oral peptide drug candidates have entered clinical
`trials, but with limited success and overall are restricted to the
`area of endocrine disorders. Formulations are helpful in this
`setting and have been successfully used in the development of
`Semaglutide, the most recently FDA approved orally available
`peptide drug used to treat Type 2 diabetes (September 2019).
`Sodium N-[8-(2-hydroxybenzoyl)amino caprylate]
`(SNAC)
`is
`used with Semaglutide to form a co-formulation.7 SNAC works
`as a buffering agent within the stomach, which in turn
`diminishes the activity of proteolyzing enzymes including
`pepsin where the maximum activity is experienced at a pH
`within the range of 2–4.7 Despite Semaglutide’s approval, the
`delivery of peptides orally still has a long way to develop before
`we see more in the clinic.
`Several ways can be used to try and improve the ADME
`properties of peptides such as the improvement of the cell
`permeability, enhancement of chemical and proteolysis stability
`and reduction of renal clearance overall resulting in the
`extension of the circulatory half-life. The extended half-life is
`beneficial both economically and for the patients’ compliance.
`The next part of the review will focus on the ways of modifying a
`peptide to achieve such improvements.
`
`Improving enzymatic and chemical
`stability of peptides by chemical
`modifications
`Cyclisation
`
`Cyclisation techniques have been used widely in the peptide
`field and achieved in several ways from cyclising head to tail,
`head/tail to side chain or side chain to side chain.
`A type of side chain to side chain cyclisation is called
`stapling, a technique that enables the peptide to be locked into
`a desired conformation. Peptide stapling is used commonly to
`enhance a peptide’s secondary structure such as a-helices and
`b-turns, which can improve the binding affinity to the target
`and enhance ADME properties.
`There are two subgroups of peptide stapling (PS): one-
`component (1C) and two-component (2C). In one-component
`peptide stapling (1C-PS) there is an intramolecular linkage
`between often unnatural amino acid side chains and can allow
`cyclisation depending on the secondary structure.
`One of the first examples of 1C-PS was by Blackwell and
`Grubbs through the use of ring-closing metathesis (RCM) of O-
`allyl serine residues.8 1C-PS is not without its limitations. For
`example, modifications would result in the entire peptide being
`
`Fig. 1 A schematic to represent the journey of an orally administrated
`peptide through the gastrointestinal tract.
`
`Santos et al. analysed peptides approved by the FDA between
`2012–2016 to allow comparison to the Ro5.4 They determined
`that the most orally available peptides were indeed up to a MW
`of 1200 Da and displayed a log P within the range of 5–8. Close
`interpretation of the results found that orally available peptides
`had 5 times more H-bond donors and acceptors than what was
`considered as acceptable by Lipinski’s Ro5 for small molecules.4
`Oral administration is challenging for both biologics and
`peptides and in many cases, it may not be feasible. The whole
`journey of oral administration through the gastrointestinal
`tract is problematic, starting with the enzymes amylase and
`lipase found within saliva that break down the peptides into
`smaller molecules (Fig. 1).5 On arrival into the stomach, the
`peptide is subjected to harsh acidic conditions and proteolysis
`by cathepsin and pepsin. Even if the peptide successfully
`remains intact to this point, the lumen of the small intestine
`experiences a pH change and has a vast number of proteolyzing
`enzymes including trypsin, chymotrypsin and carboxypeptidase.5
`Compared to biologics, peptides have a much shorter circu-
`latory half-life (days vs. weeks) resulting in the need for sub-
`optimal frequent drug administrations. The impact of this is
`witnessed through the advancement of ADCs and a slower
`progression of PDCs. The lifetime of a hydrophilic peptide in
`circulation is determined by many soluble enzymes in the
`blood and at membranes. Exopeptidases are a class of enzymes
`that can be split into two subgroups: amino- and carboxypepti-
`dases and target the N- and C-terminal, respectively.6 It is these
`
`currently
`is
`Spring
`David
`of Chemistry
`and
`Professor
`Biology
`at
`the
`Chemical
`University of Cambridge within
`the Chemistry Department. He
`received his DPhil
`(1998) at
`Oxford University under Sir Jack
`Baldwin. He then worked as a
`Wellcome
`Trust
`Postdoctoral
`Fellow at Harvard University
`with Stuart Schreiber
`(1999–
`2001), after which he joined the
`faculty at the University of Cam-
`bridge. His research programme
`is focused on the use of chemistry to explore biology.
`
`David R. Spring
`
`1482 | Chem. Soc. Rev., 2021,50 , 14801494
`
`This journal is
`
`The Royal Society of Chemistry 2021
`
`View Article Online
`
`MPI EXHIBIT 1071 PAGE 3
`
`MPI EXHIBIT 1071 PAGE 3
`
`

`

`Tutorial Review
`
`Chem Soc Rev
`
`resynthesized, which could prove costly in both time and
`materials. However, 1C-PS has proved successful in many cases
`exemplified by the first stapled peptide ALRN-5281 to reach
`clinical trials and complete Phase I.9 ALRN-5281 is used to treat
`adult growth hormone deficiency and was cyclised through the
`use of 1C-PS via RCM.9
`Peptide stapling more recently has moved the focus away
`from 1C-PS to two-component peptide stapling (2C-PS) (Fig. 2).
`The use of 2C-PS offers many advantages over 1C-PS and allows
`synthetic versatility through the use of linear peptides and
`separate staples. 2C-PS allows modifications to be made at a
`late stage if needed to the peptide or the staple, which is highly
`beneficial for an optimization campaign. 2C-PS has been widely
`applied to bicycles therapeutics developed by Christian Heinis
`and Sir Greg Winter.10 The overall concept of bicycles is the
`cross-linking of three cysteine residues to a tri-functionalised
`linker to form a bicycle construct. More details on the application
`of bicycles for use within drug conjugates are detailed later in
`the review.
`
`Moving away from proteinogenic amino acids
`
`Side chains of amino acids offer another excellent source for
`modification with many papers being published recently on
`direct amino acid modification. Increasing the steric bulk of
`the side chains results in increased stability, as enzyme recog-
`nition is disrupted.6
`One way to increase the overall stability of the full peptide is
`to swap L-amino acids to D-amino acids. The D-amino acid
`sequence has a decreased substrate recognition and binding
`
`affinity for proteolytic enzymes.3 An example of increasing the
`half-life of a biologically active peptide is modifying Somatos-
`tatin to Octreotide used to treat gastrointestinal tumours.3,6
`Octreotide’s amino acid sequence incorporates two D-amino
`acids, whereas Somatostatin is only composed of L-amino acids
`(Fig. 2). The resulting half-life increases from a few minutes for
`Somatostatin to 1.5 hours for Octreotide hence enhancing
`favourable PK properties.11 Despite this example highlighting
`the benefits of L- to D-amino acid exchange, there are a few
`cases in which D-amino acid-containing peptides show a
`reduced half-life in comparison to the L-analogue.3 It is important
`to consider the effects that such modifications could have on
`the overall secondary structure of the peptide and on any intra-
`or intermolecular interactions.3 An alternative is the use of
`D-peptides. These peptides are mirror images of the L-amino
`acid-containing counterpart and are composed fully of D-amino
`acids. The Kay group have recently developed D-peptides for use
`as HIV entry inhibitors.12
`
`Slowing down renal clearance by chemical modifications
`
`The overall net charge for the peptide sequence is an important
`consideration for renal clearance. Peptides that acquire a net
`negative charge tend to exhibit a longer half-life in comparison
`to those with a net positive charge.13 The presence of anionic
`carbohydrate moieties found within the kidney’s glomerular
`membrane limit the filtration of anionically charged species
`into the urine.13
`Another approach is the conjugation of peptides with larger
`molecules (450 kDa) to increase lipophilicity and their binding
`
`Fig. 2 Different methods used to improve the enzymatic and chemical stability of peptides including: cyclisations and peptide stapling, formulations,
`moving away from proteinogenic amino acids and chemical modifications.
`
`This journal is
`
`The Royal Society of Chemistry 2021
`
`Chem. Soc. Rev., 2021,50 , 14801494 | 1483
`
`View Article Online
`
`MPI EXHIBIT 1071 PAGE 4
`
`MPI EXHIBIT 1071 PAGE 4
`
`

`

`Chem Soc Rev
`
`Tutorial Review
`
`to albumin, thereby improving the PK and PD properties of
`peptides.13 It is the enhanced steric that prevent the conjugate
`from being filtered out through the kidneys and allow a longer
`circulation period. Modifications at the N- and C-terminus help
`slow down renal clearance. Typically the breakdown by exopepti-
`dases of a peptide sequence occurs at the N- or C-terminus.6
`There are ways this can be prevented by modifying the
`terminus to increase proteolysis resistance. An amide bond
`between the C- and N-terminus achieved through a cyclisation
`reaction has been shown to prevent enzyme degradation.6
`If cyclisation is not the preferred structure for binding, then
`N-acetylation and C-amidation can be an alternative to enhance
`resistance to proteolysis. These modifications have proven
`successful in a range of studies on Somatostatin, where the
`half-life of the modified molecule was extended when com-
`pared to the unmodified peptide.3
`N-Methylation of amide bonds is another modification
`that enhances metabolic resistance. N-Methylation increases
`the steric hindrance and allows tuning of
`the peptide
`conformation.14 Cyclosporin is an example of a naturally occur-
`ring peptide used as an immunosuppressive drug (Fig. 2).
`Cyclosporin is a hepta-N-methylated cyclic undecapeptide, with
`an oral bioavailability of 29%.14
`(PEG) has been vastly
`The use of polyethylene glycol
`explored in slowing down renal clearance and by increasing
`the binding to plasma proteins such as albumins. PEG is an
`ideal candidate for modification: it is cheap, biocompatible,
`hydrophilic, non-toxic and non-immunogenic.6 The promise of
`PEGylation for the modification of peptides is highlighted by a
`number of examples, which are discussed herein.
`RGD is a homing tripeptide (see later), whose sequence is
`able to allow the HM-3 peptide to selectively bind to specific
`target sites that display high levels of
`integrins within
`tumours.13,15 The original HM-3 peptide had limited effect as
`a consequence of its short half-life and required twice a day
`administration. There was a need to enhance the peptide’s
`half-life to reduce the number of administrations needed.
`Methoxy-poly(ethylene glycol)-aldehyde (mPEG-ald) was the PEG
`linker of choice for attachment at the N-terminus. Upon this
`modification, there was a 5.86 fold increase in half-life in male rat
`studies when compared to the unmodified HM-3 peptide.15
`Another PEGylated peptide is PEG-adrenomedullin (PEG-ADM)
`by Bayer used in patients suffering from Acute Respiratory
`Distress Syndrome (ARDS) associated with lung failure.16 The
`peptide was enrolled into Phase 2 clinical trials in August 2020
`with the predicted end date of early 2023.16
`PEG is not the only molecule used in conjugation to the
`peptide approaches to slow down renal clearance. Other widely
`used examples include polysialic acids (PSA), a homopolymer,
`and hydroxyethyl starch (HES), a branched amylopectin.13
`The addition of fatty chains has been an effective method as
`an addition to peptides to increase the half-life. Glucagon-like
`peptide (GLP-1) receptor agonists have been used to control
`blood sugar levels in patients with Type 2 diabetes.17 One of the
`earliest example is Exenatide, an analogue of a nonhuman
`peptide that in 2005 had twice-daily administration and an IV
`
`Fig. 3 The structures of Liraglutide and Semaglutide.
`
`half-life of 30 minutes. In 2009 Liraglutide, a near analogue of
`human GLP-1, had a fatty acid chain with a spacer joined to
`the main peptide backbone for binding to albumin (Fig. 3).
`Liraglutide represented a significant improvement of GLP-1
`with an extended IV half-life of 8–10 hours and once-daily
`administration.17 Semaglutide is a GLP-1 agonist, with a
`gGlu-2xOEG linker to a C18 fatty chain (Fig. 3).17 The determined
`IV half-life was 46.1 hours through studies involving mini pigs,
`enabling once-weekly doses – a vast improvement to the early
`GLP-1 agonists.17
`
`Enhancing bioavailability via formulations
`
`Several methods have been used previously in the literature to
`enhance the oral bioavailability of peptide therapeutics via
`formulations.5 These can include permeation enhancers and
`acid-stable coatings (Fig. 2).5 Permeation enhancers are able to
`transport the peptide through epithelial cells – an alternative
`route is available through intercellular junction and adhesion
`protein interference resulting in a paracellular route.5 The use
`of acid-stable coatings to improve the oral availability of a drug
`is a widely used approach. These coatings are pH active, where
`at low pH in the stomach the coating remains intact; as the
`peptide moves to the intestine the pH rises and the coating
`breaks down to release the contents. The introduction of citric
`acid can be used to help neutralise the optimum basic pH
`conditions for a range of gastrointestinal peptidases, hence
`slowing down degradation caused by peptidases.5
`Formulations can be used to enhance the bioavailability of
`IV administrated peptides. The FDA approved Sandostatin LAR
`is an excellent example of this in which Octreotide is encapsulated
`in a glucose-poly(lactide-co-glycolide) (Glu-PLGA) star-shaped poly-
`mer (Fig. 2).18
`Improving the overall chemical and enzymatic stability of a
`peptide using the techniques discussed is beneficial for the
`discovery of new therapeutics including targeted therapies
`exemplified by peptide–drug conjugates. These concepts will
`be discussed in the following section.
`
`Cancer therapy and targeted drug
`delivery
`
`Cancer is among the leading contributors to human mortality
`and disease, with nearly 50% of people being diagnosed with
`
`1484 | Chem. Soc. Rev., 2021,50 , 14801494
`
`This journal is
`
`The Royal Society of Chemistry 2021
`
`View Article Online
`
`MPI EXHIBIT 1071 PAGE 5
`
`MPI EXHIBIT 1071 PAGE 5
`
`

`

`Tutorial Review
`
`Chem Soc Rev
`
`cancer in their lifetime.19 In 2000 and 2010, two landmark
`publications defined the ‘‘Hallmarks of Cancer’’, characterising
`all cancers with eight common traits: cancer cells stimulate
`their own growth, lack sensitivity to anti-growth signals, evade
`programmed cell death (apoptosis), can divide indefinitely, can
`sustain blood vessel formation (angiogenesis), invade other
`tissues and metastasise, deregulate metabolism and evade the
`immune system.20,21 Our growing understanding of cancer cell
`biology has enabled significant advances in treating this disease.
`Subject to the stage and tumour type, patients are treated
`with one or a combination of the following options: surgery,
`radiotherapy or pharmacotherapy.
`is charac-
`Traditional pharmacotherapy (chemotherapy)
`terised by cytotoxic drug regimens which target rapidly dividing
`cells through inhibiting mitosis and is associated with serious
`side-effects such as bone marrow and gastrointestinal toxicity.
`Even if the tumour is successfully eradicated, lasting damage
`may continue to affect healthy tissues and residual cancer cells
`may result in relapse of the disease.
`Fortunately, newer molecular therapies can improve patient
`outcomes and reduce toxicity by targeting the unique charac-
`teristics of tumour cells including the cell morphology (leaky
`cell membranes), lower pH, increased glutathione (GSH) and
`enzyme presence (Fig. 4).22
`Antihormonal therapy is often effective for tumours whose
`growth is driven by endocrine signalling, such as breast and
`prostate cancers. Drugs which selectively inhibit an oncogenic
`mutant protein while sparing the unchanged (wild-type) protein
`are another successful approach, such as Gleevec (imatinib), a
`tyrosine kinase inhibitor which has revolutionised the treatment
`of Chronic Myelogenous Leukemia (CML).23 By targeting cancer’s
`ability to evade the immune response, checkpoint inhibitor
`antibodies such as Keytruda (pembrolizumab) and Imfinzi
`(durvalumab) can rekindle the immune system’s ability to recog-
`nise and eliminate tumours, providing new treatment options for
`recalcitrant tumours such as non-small lung cell cancer (NSCLC)
`and Hodgkin’s lymphoma. Following treatment with all of these
`
`Fig. 4 A schematic representation of how a targeted therapeutic (green
`square) is specific for the tumour cells in comparison to a non-targeted
`therapeutic (blue triangle).
`
`drugs, cancers may further mutate to develop resistance to
`targeted therapies; these resistance mutations provide challenges
`but also new opportunities for drug discovery.24
`Antibody–drug conjugates (ADCs) are another promising
`line of targeted therapy composed of an antibody which binds
`to a protein highly expressed in tumour cells (a tumour antigen),
`connected via a linker to a cytotoxic small molecule payload.
`In principle, this design enables the targeted delivery of a drug
`to the tumour, killing cancer cells while sparing healthy tissue
`and providing a wider therapeutic margin than traditional
`chemotherapy. In 2000, Mylotarg (gemtuzumab ozogamicin)
`became the first ADC approved by the FDA, combining an
`antibody to the CD33 antigen expressed in leukaemia cells with
`a cytotoxic payload derived from a calicheamicin natural product.
`Over the past 20 years, there have been significant advances in
`this field exemplified by 5 ADCs approved between 2008 to 2018
`and 3 ADCs approved by the FDA in 2019 for various cancers,
`including AstraZeneca and Daiichi’s Enhertu (trastuzumab der-
`uxtecan) for the treatment of HER2 metastatic breast cancer,
`Roche’s Polivy (polatuzumab vedotin) for large B-cell lymphoma,
`and Astella and Seattle Genetics’ Padcev (enfortumab vedotin-ejfv)
`for urothelial bladder cancer.25 The most recent ADC being
`approved in August 2020 was GSK’s Blenrep (belantamab
`mafodotin-blmf) for relapsed or refractory multiple myeloma,
`bring the total to 9 FDA approved ADCs as of September 2020.26
`Despite continuing progress for ADCs, these therapies pre-
`sent several drawbacks. As cytotoxic drugs, ADCs can incur
`significant toxicity; in the case of Mylotarg, the FDA issued a
`black box warning for potentially life-threatening side effects in
`patients who did not receive stem cell transplantation.27 ADCs
`can also generate dangerous immune reactions, yielding toxi-
`city and halting further therapy.28 Furthermore, the compli-
`cated structure of ADCs results in high production costs, in
`some cases restricting access to these drugs as their cost-
`effectiveness may be called into question by health insurers.
`Owing to their high molecular weight and protein-like physico-
`chemical properties, ADCs also suffer from restricted distri-
`bution which can prevent these therapies from penetrating some
`types of solid tumours and limiting their efficacy.
`To overcome these issues, many research groups have
`explored alternative approaches such as peptide-based drugs,
`protein–protein interaction inhibitors (PPIs) and drug delivery
`transporters. This review will focus on the latter and describe
`how peptides can be used within peptide–drug conjugates
`(PDCs) for the targeted treatment of cancer (Fig. 5).
`
`Peptide–drug conjugates (PDCs)
`
`Peptide–drug conjugates are a class of targeted therapeutics,
`with a similar construct to that of ADCs and only differing
`through the homing device. A PDC is composed of three vital
`components: a homing peptide, a linker and a cytotoxic pay-
`load (Fig. 5). All three work in synergy to deliver cytotoxins
`through targeting the selected receptor of a tumour cell. As
`discussed earlier, the ADC market has been fast-paced, but
`
`This journal is
`
`The Royal Society of Chemistry 2021
`
`Chem. Soc. Rev., 2021,50 , 14801494 | 1485
`
`View Article Online
`
`MPI EXHIBIT 1071 PAGE 6
`
`MPI EXHIBIT 1071 PAGE 6
`
`

`

`Chem Soc Rev
`
`Tutorial Review
`
`These PDCs are examples of therapeutics, however, PDCs
`can be used as successful diagnostic tools by employing the use
`of radionucleotides.
`On constructing a PDC, it is key to understand the role of
`each component to fully appreciate the design. Considerations
`include the mechanism of action and how alternative
`approaches can improve the current limitations of this emer-
`ging modality.
`
`Homing peptide
`
`A homing peptide is a selected peptide that is chosen for its
`specific targeting capabilities of protein receptors found over-
`expressed at tumour tissues. In the case of PDCs, the homing
`peptide will direct the whole PDC construct to the targeted cell
`and limit off-target delivery. These homing peptides often have
`precedent in the literature for having a strong binding affinity
`to the target site within the nanomole magnitude. Several
`techniques can be used to determine their binding affinity
`including surface plasmon resonance (SPR), biolayer interfero-
`metry (BLI) and isothermal titration calorimetry (ITC).
`The secondary structure of the homing peptide has a pro-
`nounced effect on its binding affinity. Therefore, structural
`information is important when seeking to increase the binding
`affinity of the homing peptide through stabilisation of the
`secondary structure. The most common examples of peptide
`secondary structure include a-helix, b-sheet and random coil.
`On the attachment of the linker to allow conjugation to the
`cytotoxin, the secondary structure of the homing peptide must
`be retained, and the linker should not disrupt binding. There is
`a vast range of homing peptides for many targets detailed in a
`recent review by Vrettos et al. and summarised in Table 1.19
`Most homing peptides reported to date are linear. Even
`though they show good binding, there are several drawbacks
`including degradation by enzymes at the termini, chemical
`instability and fast renal-clearance. A way to overcome these
`limitations is through the use of cyclisation or peptide stapling
`of the linear peptide as described earlier.
`A study by Lu and co-workers demonstrated how a stapled
`RGD peptide was used as a homing peptide that targets avb3
`integrin (Fig. 6).31 The peptide can be used to modify a
`nanoparticle to effectively deliver a drug to target glioblastoma
`multiforme (GBM), an aggressive CNS tumour with poor
`prognosis.31 The RGD tripeptide has precedent for targeting
`avb3 integrins expressed on glioma cells and overcoming the
`blood brain tumour barrier (BBTB). During the early stages of
`glioma, therapeutics can be hindered as the blood brain barrier
`(BBB) still remains intact. Therefore, a suitable vehicle to
`
`Fig. 5 A schematic of a peptide–drug conjugate construct consisting of a
`homing peptide, linker and payload. The structure of 177Lu-dotatate an
`FDA approved peptide–drug conjugate.
`
`unfortunately, this is not the same case for PDCs. A contributing
`factor is that methodologies that overcome the intrinsic poor PK
`properties of the peptides have only recently emerged, and
`pharmaceutical companies have started expanding their pipelines
`to accommodate peptide therapeutics only in the last decade.
`There is currently only one therapeutic PDC on the market,
`177Lu-dotatate, but many more are in various phases of the
`pipeline. 177Lu-dotatate is used to treat gastroenteropancreatic
`neuroendocrine tumours (GEP-NETs) and was the first FDA
`approved PDC (Fig.