PHOTOSENSITIZER ANTIBODY-DRUG- CONJUGATES: PAST

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Review

PHOTOSENSITIZER ANTIBODY-DRUGCONJUGATES: PAST, PRESENT AND FUTURE Ross W. Boyle, and Jordon Sandland Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00055 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019

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Bioconjugate Chemistry

Photosensitizer antibody-drug-conjugates: Past, present and future Jordon Sandlanda,b, Ross W. Boylea*

a Department

bPositron

of Chemistry and Biochemistry, Cottingham Road, University of Hull, Hull, UK, HU6 7RX.

Emission Tomography Centre, Cottingham Road, University of Hull, Hull, UK, HU6 7RX.

*Ross W. Boyle : [email protected]

NOTE Authors declare no competing financial interest.

KEYWORDS Photosensitizer, photodynamic therapy, antibody, bioconjugation, personalized medicine, antibody-drugconjugates.

ABSTRACT This review aims to highlight key aspects of tetrapyrrole-based antibody-drug-conjugates (ADCs) and the significant developments in the field since 2010. Many new conjugation methods have been developed and employed in the past decade and, associated with this, there has been a rising interest in theranostic conjugates. We have investigated the physicochemical properties that tetrapyrroles need to possess in order to be viable photosensitizers for conjugation to antibodies. Differences in conjugation strategies are discussed, and structureactivity relationships of tetrapyrrole-antibody conjugates are reported, where available. As the elegance of bioconjugation techniques has increased it has paved the way for exceptionally phototoxic, yet highly selective, tetrapyrrole-antibody conjugates, with photocytotoxicities in the nanomolar range, to be synthesised and biologically evaluated.

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INTRODUCTION Porphyrins are ubiquitous in the fields of photodynamic therapy and photodiagnosis and are one of the most prominent classes of photosensitizer in these areas of biomedical science.1, 2 Porphyrins are highly synthetically versatile, and are also found widely in nature. Photophysical and physicochemical properties of porphyrins are constantly being modified and optimised through chemical manipulation and design. Tetrapyrrolic macrocycles can be characterised photochemically by UV-Visible spectroscopy, which for porphyrins reveals a strong absorption with high molar absorptivity in the region of 400 nm, commonly called the Soret band.3 Weaker Q bands are observed in the 500-650 nm region, typically four bands for free base porphyrins and two for metalloporphyrins.4,5 Chlorins are optically similar to porphyrins, however their Q bands appear at 600-700 nm and the fourth Q band typically has a far higher extinction coefficient than that of porphyrins.6 Bacteriochlorin has a Q band at 700-800 nm which has a higher extinction coefficient than that of its Soret.7 Lastly, phthalocyanine have exceptionally high extinction coefficient bands in the NIR-region (600-700 nm).8 Depending on the metal centre, axial ligands can be coordinated, further altering the physiochemical properties.9,

10

Characteristically,

porphyrin, chlorin, (iso)bacteriochlorin and benzoporphyrin (Figure 1) consist of a heterocyclic porphine macrocyclic ring, composed of four pyrrolic units bridged by methine units,11 while porphyrazine and phthalocyanine contain nitrogen heteroatoms. The central cavity in tetrapyrrolic macrocycles has been extensively investigated in relation to altering their optoelectronic and physiochemical properties, notably by the insertion of metal ions to form chelates.12, 13 NH N

N

NH

HN

N

Porphyrin

NH

N

N

HN

Chlorin

NH

N HN

N

N HN

Isobacteriochlorin

Bacteriochlorin

N N NH

HN N

Benzoporphyrin

N

N

N NH

N

N N

HN

N

N NH

N

HN

N Porphyrazine

N

Phthalocyanine

Figure 1. Common tetrapyrrolic photosensitizers used for photodynamic therapy. Since the early investigations of photosensitization of tissues by Raab in 1900, and the subsequent understanding of the cytotoxic mechanism that leads to cell death,14 efforts have been made to increase the triplet


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lifetime,15 enhance quantum yields,15 induce NIR absorbance,16 minimise cytotoxicity and, crucially, increase specificity of photosensitizers for target tissues.15 Porphyrins do, however, benefit from a degree of ‘passive’ targeting of cancerous tumours,17 predominantly through the enhanced permeability and retention (EPR) effect.18 In the past few decades significant efforts have been dedicated to creating so-called ‘third generation’ photosensitizers by the conjugation of porphyrins to active targeting moieties for enhanced selectivity.17, 19, 4 In this review we will focus primarily on research conducted from 2010 to the present day, with the reader directed to a previous review of this field by our group covering literature reports prior to that date. 20 The sections covered herein contain background discussions of photodynamic therapy, personalised medicine, antibody targeting of cancers, physicochemical properties of photosensitizers required for antibody conjugation and conjugation techniques. The main body critically discusses the recent advances in bioconjugation chemistries towards antibodies and an evaluation of the biological data of the ADCs. Photodynamic Therapy Photodynamic therapy requires three key ingredients: light, molecular oxygen and a photosensitizer.17 The ideal photodynamic sensitizer will satisfy several key criteria: i.

Strong absorption with a high extinction coefficient in the red/NIR-region.

ii.

High singlet oxygen quantum yield and long triplet state lifetime.

iii.

Solubility in biological media.

iv.

Minimal dark toxicity in the absence of irradiation.

v.

Rapid clearance from the blood.

vi.

Be a single well-characterised compound.

vii.

Have a simple bio-compatible drug formulation.

viii.

Exhibit high tumor specificity and selectivity.

The process of photosensitization, best described by the simplified Jablonski diagram (Figure 2), is the use of light energy, input into the system in the form of quantised energy packets (photons), by a photosensitizer to generate highly reactive oxidising species. Briefly, a photosensitizer absorbs light of an appropriate wavelength and is promoted to an excited singlet state from a singlet ground state. Naturally, internal conversion and vibrational relaxation can occur within the excited states. These excited singlet states can decay through an energy emissive process in the form of fluorescence, but can also undergo inter-system crossing via the Laporte forbidden inversion of the spin of an electron to give an excited triply degenerate state. In this state two processes can occur;



either emission of a photon by phosphorescence, or generation of cytotoxic species, usually by electron or energy transfer to molecular oxygen. The lifetime of the excited singlet state is short, typically 10-12 – 10-9 s,21 however the excited triplet state is much more long-lived, 10-3-101 s. This limits the photochemistry available via the singlet excited state, but photochemical reactions via the excited triplet state are more versatile.

Figure 2. Simplified Jablonski diagram. Two relevant mechanisms are possible via the excited triplet state: type I - this mechanism occurs through electron transfer to species in close proximity to the photosensitizer and can generate, among other species, hydroxyl radicals, superoxide radicals and peroxy radicals;22 type II - through this mechanism, molecular oxygen in its triplet ground state is converted to singlet oxygen by energy transfer, singlet oxygen is a highly reactive species and is widely reported to be the dominant cytotoxic species in PDT.23 Cell death occurring due to PDT can be by apoptosis, necrosis, or in some cases autophagy.24 However, in tumour tissues, the concentration of molecular oxygen is often depleted due to an inadequate network of vasculature supplying the growing tumour. This leads to hypoxia which can result in a treatment such as PDT or radiotherapy being rendered ineffective. The generation of singlet oxygen, via photosensitization, has also been used to destroy gram-negative bacteria.25, 26 As previously mentioned, for PDT activity to occur a spin-forbidden transition from an excited singlet state to the excited triplet state must occur,27 and this process can be enhanced by the introduction of an atom with high atomic weight, known as the heavy atom effect.27 Heavy-metal cations that the porphyrin tetradentate macrocycle can ligate include palladium,28 silver,29 platinum,28 and gold.30 This can result in a bathochromic shift towards the near-infrared (NIR) region, enhancing tissue transmission of light.27 For this to occur, a spin-orbital perturbation


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must occur, which is enhanced by the presence of the heavy-atom,31 and these metalloporphyrins have been conjugated to monoclonal antibodies.32, 33 Personalized Medicine In January 2015, the UK government pledged an investment of approximately £14 million for research into personalised medicine.34 The main principle of personalised medicine is to treat the individual and the individual’s needs, as opposed to the commonly employed concept of the same drug and treatment for all patients.35 Personalised medicine involves an individualised approach to the needs of the patient at all stages from diagnosis, treatment and prognosis.36 This is important clinically as not all patients respond in the same manner, and understanding the disease, and how to combat it, improves the efficacy of the treatment regime.36 In the past few decades PDT has emerged as a viable therapeutic technique with potential in personalised medicine. For an excellent review of organ-specific PDT clinical trials see D. van Straten et al.37 Generally, targeting moieties for tetrapyrroles have included: small molecules such as folic acid,38 sterols,39 and alkylphosphocholine,40,

41

peptides,42, 43 proteins,44 nanoparticles,45, 46,

47

gels,48, 49 antibody fragments and

antibody species.50 Antibodies are Y-shaped macromolecular proteins produced, naturally, by B-cells in blood upon activation of the immune system.50,

51

ADCs represents an opportunity to personalise the treatment of

cancers by targeting of specific antigens which can be overexpressed by certain cancer cells.50 Cancers which do not respond well to classical catch-all treatments such as radiotherapy and platinum-based chemotherapy make ideal targets.52 A mismatch between the wavelengths of light required to efficiently penetrated tissue and those which effectively activate porphyrins has been a drawback of PDT. This can, however, be overcome by two main methods: changing the chromophore to one which absorbs at longer wavelengths, such as chlorins, bacteriochlorins or phthalocyanines; or secondly by changing the delivery method of the light,53 such as transmitting light into the body using optical fibres.54, 55 Fractionation of the incident light has also been employed to increase the effectiveness of PDT in tissues via the use of optical fibres.56 More recently, the activation of photosensitizers has been carried out with LED arrays.57 Furthermore, luminescence properties of PDT sensitisers can be used to image cancers and obtain bio-distribution profiles.58 This allows clinicians to determine the localisation of tumours, as well as treat them.58 So-called ‘theranostic’ medicines are now emerging, which combine therapy with a diagnostic technique.59 For a comprehensive review of PDT theranostic porphyrins see Sandland et al.60 In the fields of porphyrins and related macrocycles lend themselves to theranostic applications



acting as both photosensitizer and optical imaging probe.61, 62, 63 Theranostic antibody conjugates have also been found to reduce recurrence of pancreatic cancers in orthotopic mouse models.64 Antibody Targeting of Cancers The targeting of cancers is required in order to yield a more personalised treatment in oncology. Often, patients do not respond to the treatment regime administered to them by clinicians, which can lead to a poor prognosis. By better understanding the physiology of the cancer, one can correctly administer an effective treatment. The targeting of cancers begins with the selection of suitable biomarkers. Structural biologists over the past few decades have identified many potential biomarkers for cancers. The targeting of cancers is not only an ideal for cancer therapy but is intrinsic in cancer imaging for diagnostic purposes. 65, 44 Recent examples of this latter strategy are the conjugation of multi-coloured BODIPY fluorophores to proteins,66 and site-specific conjugation of NIR-emitting aza-BODIPY to the clinically approved monoclonal antibody trastuzumab for fluorescent guided surgery of HER-2 positive tumors.67 Also notably is the targeting of cancers by the ligation of a drug-conjugate to trans-membrane proteins such as CXCR4, which is present in over 20 different cancer types.68 Other commonly targeted trans-membrane proteins are the αvβ3 and αvβ6 integrins which recognise linear and cyclic RGD containing peptides,69, 70, 71, 72 human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR),73, 74, 75, 76 folate receptors (FR),45, 77 prostate specific membrane antigen (PSMA),78 and CD40.79 For an excellent review of receptor-mediated delivery systems see C. Allen et al.80 Antibody-drug conjugates (ADC) are of particular interest due to their extremely high selectivity and affinity for their corresponding antigens.81 In mammals, antibodies are divided into five isotypes based on the number of Y units and type of heavy chain (IgG, IgD, IgE, IgD, IgM). To date, antibodies can be classified into four major groups, polyclonal, monoclonal, antibody fragments and nanobodies. The most distinct differences can be seen between polyclonal antibodies and monoclonal antibodies. Polyclonal antibodies are relatively inexpensive, easy to produce and store, high stability, bind to multiple epitopes and good sensitivity, unfortunately, they vary between batches due to production in different animals at different times. Monoclonal antibodies differ in that they are more expensive and possess high specificity towards a single epitope and are extremely homogenous, however, small modifications to the epitopes can render them useless. Monoclonal antibodies were first described by Kohler and Milstein, who demonstrated their exceptional ability to bind to antigen epitopes.82 The therapeutic potential of an antibody conjugate is often dependent on


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internalization of the antibody upon binding to the antigen (Table 1).83, 84 Mew et al. first reported the targeting ability of antibody conjugates of hematoporphyrin bound to the monoclonal CAMEL-1 species.85,

86

Typically,

internalising porphyrin-antibody conjugates achieve better control of tumours and are more phototoxic, compared with those which remain bound to the surface of the cell membrane.87,

88

Receptor-mediated endocytosis is the

typical mechanism for the internalisation of antibodies and related conjugates,89 and it is the most fully understood.90 Endosomes formed during the internalisation process can then be trafficked through the cell by a complex array of recycling or degradative pathways. 90 Internalisation Phagocytosis

Description Phagocytosis is the mechanism by which relatively large (>0.5 μm) particles, such as bacteria, dead cells, or (as in the example here) polystyrene beads, are internalized. During phagocytosis in immune cells such as neutrophils and macrophages, receptors in the cell membrane first recognize antibodies on the target, which causes membrane protrusions called pseudopodia to surround the target in a zipper-like mechanism This is followed by fusion with lysosomes, acidification of the phagosome, and degradation of the target.

Reference 91

Micropinocytosis

During micropinocytosis, plasma membrane ruffling occurs that can lead to membrane fusion of the plasma membrane with itself. This membrane fusion envelops extracellular fluid that enters the cell within 0.5–5.0 μm vacuoles formed during the ruffling and membrane fusion process.

92

Caveolindependant endocytosis

Facilitated by disruption of the actin cytoskeleton, inhibited by the kinase inhibitors staurosporine and genistein, and enhanced by the phosphatase inhibitors: okadaic acid and vanadate. Binding to the cell surface activates a tyrosine kinase-based signalling cascade, disrupting the local actin cytoskeleton, and recruiting dynamin II to the site of internalization where it is endocytosed together with caveolin-1-GFP.

93

Receptormediated endocytosis

Vesicles transport cargo molecules from the plasma membrane of eukaryotic cells into the cytoplasm through the recruitment of endocytic proteins from the cytosol in a highly regulated sequence.

94

Table 1. Internalisation modes commonly associated with antibodies. Physiochemical Properties of Photosensitizers Required for Antibody Conjugation The ideal porphyrin for conjugation would contain only a single functional group, which should react in a facile manner with the antibody without the need for excessive heating or prolonged reaction times. Furthermore, the chemical reaction should, ideally, be regiospecific, giving a homogeneous conjugate. Other groups present on the porphyrin, often for purposes of solubilization, should, therefore, be non-reactive towards any of the target amino acid residues on the antibody. A key property of the porphyrin is its solubility, modulation of a porphyrins solubility can be achieved synthetically in its construction and design.95 Lipid-based delivery vehicles such as liposomes and emulsions can



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allow for the administration of hydrophobic photosensitizers. For successful bioconjugation to an antibody, one of the main properties that a lead PS candidate must possess is solubility in polar media; particularly aqueous buffer solutions.85 A lack of hydrophilicity of the porphyrin can lead to aggregation and precipitation of the antibody conjugate. Non-covalent interactions of hydrophilic porphyrins with amino acid residues can lead to coating of the surface of serum proteins such as BSA,96 and HSA.97 Even highly water-soluble cationic porphyrins can exhibit this behaviour.96 These serum proteins can allow the porphyrin to be chaperoned into the cell by endocytosis.98 Incorporating hydrophilicity into porphyrins can be achieved by three main techniques: anionic, cationic and non-ionic functionalisation. Historically, sulfonation of tetraphenylporphyrin with concentrated sulfuric acid or oleum yielded sulfonated water-soluble tetraphenylporphyrins by the ionization of the sulphonate groups.99 Meso- and β-sulfonation of diphenylporphyrins has been investigated, in some cases with the regioisomers formed requiring extensive purification by HPLC.100 Regioselective sulfonation of tetraphenylporphyrins can also occur using more exotic, but less harsh, chemistry.101 More recently, anionic porphyrins have demonstrated applications in materials and antimicrobial PDT, as well as for Mab conjugation.101,

102, 103

Other anionic groups include

carboxylic acids and the elegant polyalkyl phosphonic acid swallow-tail motifs employed by Lindsey et al. to give millimolar concentration aqueous solutions of hydrophilic porphyrins.104, 105 In the fields of cancer treatment and imaging, synthesis of AB3 porphyrins with pyridyl groups remains a convenient way to confer water solubility by the methylation of the tertiary nitrogens with methyl iodide giving the tri-cationic methyl pyridinium analogue in a single step.106, 107, 108 Quaternisation of primary aryl amines has a similar effect.108 The solubility of these cationic groups can be further modulated by exchanging counter ions from iodide to chloride. Lipophilic cationic tetraphenylporphyrins have been synthesized in excellent yields by quaternerising phosphorous and nitrogenous groups.109 These compounds exhibited good phototoxicity (LD90 = 5.1 μM) which was modulated by the appended lipophilic cationic groups.109 Thiols have been employed for regioselective nucleophilic aromatic substitution of the para-fluorines of meso-aryl pentafluorophenyl porphyrins. 110, 111 This reaction has been utilized to incorporate water solubility through cationic and non-ionic PEG chains.112, 113, 114

Polyhydroxyl groups can be used to solubilize photosensitizing porphyrins through hydrophilic interactions and hydrogen bonding,45 polylysine groups have been used to similar effect.115 Simple sugars can directly induce water solubility on porphyrins,116 while providing the added benefit of increasing specificity


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towards tumours through the Warburg effect.117, 118 Alkyl ether chains and poly amine chains have been used to decorate the periphery of porphyrins and related macrocycles.119,

120, 121

Amino acids and peptides can confer

both targeting abilities as well as modulating the hydrophilicity of macrocycles.99 Bioconjugation Methods Ideal conjugation Bioconjugation is the covalent linkage of two molecules, at least one of which has some biological activity; in the context of this review, a porphyrin derivative and an antibody or antibody fragment.122 Several strategies have been developed over the decades to synthetically produce antibody conjugates.122, 123, 124 There are two main methods to conjugate a compound to an antibody: the first being direct attachment, and the second relying on an intermediate.125 The conjugation method ideally should require as little modification of the native antibody as possible, be high yielding, facile and lead to a conjugate which can be readily purified with limited aggregation.126 Importantly, the conjugation should take place in biologically compatible media such as buffered solutions. Ideally, indiscriminate loading should be avoided, the conjugation of the photosensitisers should be specific and give a homogenous conjugate.127, 128 Lysine conjugation routes Lysine residues are heterogeneously distributed throughout antibodies, which can make them acceptable targets.129 Activated esters can bind to amines on amino acid residues.129 Porphyrin activated NHS esters can be isolated as pure compounds that are relatively stable to hydrolysis. They can be created in situ and used as coupling reagents directly with antibodies with the assistance of carbodiimides as a facilitator to expedite the reaction.130, 131

Isocyanates have been used for lysine conjugation, however, they are rapidly hydrolysed and require the handling of highly toxic chemicals such as phosgene and triphosgene.132 Their sulfurous cousins, isothiocyanates, are now more commonly used in bioconjugation due to their increased stability to hydrolysis in aqueous media.132, 133 Isothiocyanates can be readily generated from the corresponding amines using a variety of thiocarbonyl transfer

reagents, often yielding the desired NCS group in near quantitative yield.132 Cysteine conjugation routes Conjugation to cysteine residues is markedly selective and allows for multiple functional groups to be utilized. In their native form, the thiols present in cysteine residues are often found as disulphide bridges, however,



these are easily reduced and the resulting free thiols can then be utilised,129 allowing for site-specific conjugation.134 Maleimides have been extensively used to conjugate a range of species to free thiols, formed after the reduction of the disulphide bridges linking antibody fragments (Fab) together.129, 135 Sulfhydryl (thiol) groups have been investigated and utilised as excellent nucleophiles for site-specific conjugation.136 Recent improvements in conjugation techniques involving cysteine residues include the hydrolysis of succinimide linkers, giving the succinic acid conjugates, and providing a species which is resistant to the retro-Michael reaction, which can be problematic with this type of conjugation.137 The kinetics of these ring opening reactions have been studied and demonstrate their benefits for the formation of antibody-conjugates.138 ‘Click’ conjugation routes A mild route was developed to reduce disulphide-bridges with tris(2-carboxyethyl)phosphine (TECP) and then cleave the para-azidobenzyl group for irreversible cysteine labelling, ideal for introducing reactive handles.139 These developments over the last decade have allowed for a new bioconjugation technique to be employed with the advent of the copper catalysed alkyne-azide cycloaddition reaction (‘click’ or CuAAC).140 The ‘click’ reaction requires two components: an alkyne and an azide. Azides can be introduced into porphyrins by diazotisation reactions using a mixture of sodium nitrite, TFA and sodium azide resulting in high yields. Similarly, they can be appended on side chains such as PEG linkers. A secondary method involves the use of azide transfer agents, such as imidazole-1-sulfonyl azide (ISA). Recently, a one-pot method of yielding a zinc-protected azide bearing porphyrin from a primary amino substituent was reported.67 Alkyne functionalities can be readily introduced to tetrapyrroles by the utilization of the Williamson ether synthesis reaction of a nucleophile and a propargyl chain with a leaving group in near quantitative yields. The strain-promoted alkyne-azide cycloaddition (SPAAC) is a copper-free variant which has emerged where the use of copper (II) (and reducing agents) or copper (I) is not ideal.141 This reaction has become a reaction of choice for hassle-free bioconjugation due to its broad solvent computability and 100% atom economy.141 However, these conjugation strategies typically require premodification of the antibody species before conjugation takes place to introduce either the alkyne or azide moieties.

ANTIBODY-DRUG-CONJUGATES FOR PDT Amide Conjugation One of the most prevalent conjugation methods for linking two molecules together is through the formation of amide bonds, often by pairing amines with activated esters. NHS ester substituted porphyrins (Figure


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3: 1) (Table 2) can be isolated after purification by column chromatography to give readily conjugatable species. These porphyrins can be synthesized in good yields (ca. 10%) for a [3+1] mixed aldehyde condensation, typically by the Alder-Longo method for the methyl ester porphyrin which can be readily saponified giving the corresponding carboxylic acid. The NHS ester porphyrin can be isolated, not without some difficulty, due to the nature of the activated group reacting with the dichloromethane/methanol eluent required for column chromatography reducing the obtained yield. Conjugation to an anti-CD104 Mab has been achieved through this method in sodium bicarbonate buffered solution followed by purification using gel-permeation chromatography, however low loading ratios were achieved and harsh conditions were required;142 additionally, large molar excesses of porphyrin to antibody were used in this study (40:1 – 80:1).142 In another study comparing the conjugation of a single porphyrin to this antibody, as well as HSA and BSA, it was found that the LD50 of the antibody-conjugate was an order of magnitude lower than that of the protein (HSA, BSA) conjugates.142 Both the protein (HSA, BSA) and antibody conjugates, however, were more photocytotoxic than the free porphyrin alone and showed no deleterious effects on the ROS production of the PS.142

O O

OH

N

HN N

N N

NH

O

N O

N

HN

O

N

N N

NH

1 N

N

Figure 3. Lipophilic carboxylic acid and NHS-activated ester functionalized porphyrins. Similarly, Korsak et al. reported utilizing NHS ester conjugation of photosensitisers to trastuzumab to destroy gastric cancers, 20% of which overexpress the HER2 antigen.143 Good phototoxicity was observed in vitro, albeit with an abnormally high total light dose of 40 J/cm2 (Table 2).143 The cell death mechanism was explored via dual PI/Annexin-V FITC staining and flow cytometry, which was found to be predominantly necrosis with late-stage apoptosis.143 In vitro immunoreactivity analysed by flow cytometry confirmed the selectivity of the conjugate towards HER2. However, it was recognised that overloading of the antibody leads to self-quenching of the excited triplet state due to the close proximity of the porphyrins to one another.143 Immunoreactivity was also be reduced by indiscriminate loading of the porphyrin onto the antibody epitope recognising regions.143 These



limitations have led to several new conjugation methods being developed, specifically to reduce these unwanted side-effects.143 Pyropheophorbide-α (Figure 4: 2) is commercially available and has already demonstrated its efficacy as a novel photosensitizer in its own right against SKOV-3 cells.144 Pyropheophorbide-α is a more ideal PS than a regular porphyrin due to its slightly red-shifted Soret and a higher extinction coefficient final Q band allowing for deeper tissue penetration. In terms of suitability for conjugation, it contains a single carboxylic acid which allows for activated ester conjugation to antibodies which can reduce non-specificity such with asymmetrical dicarboxylic acid porphyrins. This allowed for the creation of a novel photosensitizing antibody-drug-conjugate against mucin glycoprotein 1 (MUC1), which is a glycosylated transmembrane protein expressed on epithelial cells.145 This study demonstrated a proof-of-concept for targeting Barrett’s epithelium and oesophageal adenocarcinoma with ADCs.145 This rare cancer with an incidence rate of

PHOTOSENSITIZER ANTIBODY-DRUG- CONJUGATES: PAST

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