REVIEW REVIEW mAbs 5:1, 13–21; January/February 2013; © 2013 Landes Bioscience Implications of receptor-mediated endocytosis and intracellular trafficking dynamics in the development of antibody drug conjugates Michael Ritchie, Lioudmila Tchistiakova and Nathan Scott* Global Biotherapeutic Technologies; Pfizer Global Research and Development; Cambridge, MA USA Keywords: endocytosis, endosomal sorting, antibody engineering, ADC, lysosome, HER2, affinity, linker payload . e Abbreviations: ADC, antibody drug conjugate; EE, early endosome; RE, recycling endosome; LE, late endosome; MVB, t u multivesicular bodies; ILV, intraluminal vesicle; FcRn, neonatal Fc Receptor; IgG, Immunoglobulin G; TCZ, tocilizumab; b IL6-R, interleukin 6 receptor; PCSK9, proprotein convertase subtilisin kexin type 9; LDL, low density lipoprotein; CDR, i complementarity-determining region; PRL, human prolactin protein; PRLR, human prolactin protein receptor; HER2, human r t epidermal growth factor receptor 2; EGFR, epidermal growth factor receptor; ScFv, single chain variable fragment; CEA, s i carcinoembryonic antigen; Dyn, dynmin d t o n The use of antibody-drug conjugates (ADCs) as a therapeutic engineer antibodies and select appropriate targets has increased. platform to treat cancer has recently gained substantial o Monoclonal antibodies (mAbs) can achieve selective cyto- momentum. This therapeutic modality has the potential D toxic effects against tumors that overexpress a particular tar- to increase the efficacy and reduce the systemic toxicity associated with current therapeutic regimens. The efficacy get. This result can be achieved through multiple mechanisms e. depending on the therapeutic platform used. The mainstay of ADCs, however, relies on the proper exploitation of c of cancer biologic therapies has concerned naked antibodies, intracellular sorting dynamics of the antigen as well as the n specificity, selectivity and pharmacokinetic properties of but with advances in antibody engineering, antibodies con- e the antibody itself. Our understanding of endocytosis and jugated to toxic payloads have become increasingly prevalent. i c endosomal trafficking of receptors has appreciably increased Unconjugated mAbs (also referred to as ‘naked’) do not have s in recent years, as improvements in the assays used to study toxic payloads attached to them. Typically, they can act through o these events have resolved many of the molecular mechanisms a number of different mechanisms including receptor downreg- i regulating these processes. As a result, we now have the ulation, induction of apoptosis through inhibition of receptor- B knowledge necessary to exploit these pathways efficiently to linked signaling pathways, antibody-dependent cell-mediated s improve the efficacy of antibody-based therapy. This review cytotoxicity or complement-dependent immunocytotoxicity.1 e discusses some recent studies that have explored how endo/ Alternatively, conjugated mAbs utilize receptor internalization d lysosomal dynamics can affect the efficacy of engineered and act as a carrier to deliver the toxic payload to the cancer cell. n therapeutic antibodies, including ADCs. The more recently developed ADCs require the successful deliv- a ery of the ADC to the lysosomal compartment for proper release L of the toxic payload to the cell.2 Accordingly, a more compre- 2 Introduction hensive understanding of the molecular mechanisms govern- 1 ing intracellular trafficking, the nuances involved in designing 0 Cells continuously internalize their surface receptors through effective elements of the ADC and the biological interactions 2 receptor-mediated endocytosis. When these internalized recep- that occur between an ADC and a tumor mass is needed for the © tors incorporate into endosomes, they are trafficked throughout successful development of efficacious ADCs. Here, we review a complex array of recycling or degradative pathways. Since the recent studies which have explored the ways an antibody can be discovery of this process, there has been a great deal of emphasis designed to exploit certain aspects of the endolysosomal system, put on identifying ways to efficiently harness receptor-mediated how engineered antibodies interact with a tumor mass and the endocytosis as part of a therapeutic strategy through the use biological implications of the chemistry involved in the design of engineered antibody conjugates and other biologic modali- of an ADC. ties. This idea has gained substantial momentum recently, as our knowledge of the endo/lysosomal system and our ability to Receptor-Mediated Endocytosis and Intracellular Trafficking Dynamics *Correspondence to: Nathan Scott; Email: [email protected] Submitted: 09/19/12; Revised: 11/09/12; Accepted: 11/11/12 Molecules can be internalized from the surface of eukary- http://dx.doi.org/10.4161/mabs.22854 otic cells through a wide array of mechanisms. These include www.landesbioscience.com mAbs 13 regulate receptor internalization is adaptor complex 2 (AP2), which binds to short linear tyrosine- and dileucine-based sequences on the cytoplasmic tails of receptors.6 Once receptors are selected by adaptor proteins for internalization, clath- rin moves from the cytoplasm to adaptor protein-enriched regions of the membrane; the subsequent polymerization of clathrin causes membrane displacement and the . e formation of the budding vesicle.7 t Liberation of the budding vesicle u from the plasma membrane is medi- b ated, in part, by the large GTPase, ri dynamin (Dyn). Dyn is recruited st by Bin–Amphiphysin–Rvs domain- i d containing proteins, such as amphi- physin, endophilin and sorting t o nexin 9, which interact with Dyn’s n proline-rich regions through SRC o homology 3 domains.8-10 The precise D mechanism of vesicle release is pres- ently unclear, but Dyn undergoes a . e GTP hydrolysis-dependent confor- c mational change that likely helps n to mediate scission.11-13 Once indi- e vidual vesicles are liberated from the i c plasma membrane, they fuse with s each other in the cytoplasm and o form the early endosome. i B The endosome is an extraordi- Figure 1. Intracellular trafficking through the endo/lysosomal system. The internalized cargo is initially narily complex and compartmental- s contained within endocytotic vesicles which fuse together to become the early endosome. Cargo can ized system of proteins and lipids e be recycled directly back to the plasma membrane from the early endosome through the “short recy- functioning in concert to regulate d cling loop” or retained within the maturing endosome. As the early endosome matures, cargo can be the intracellular distribution of inter- n trafficked through the trans-golgi network for repackaging or trafficked back to the plasma membrane nalized proteins (Fig. 1). Endosomes a through the “long recycling loop.” As the early endosome matures to multivesicular bodies (MVB), the retained cargo is internalized into intraluminal vesicle (ILVs) formed within the fluid phase of the MVB send cargo through two distinct L and subsequently delivered to the lysosome. A biochemical event that occurs during the maturation pathways. The first is cargo recycling 2 of an early endosome to an MVB is the RAB5 to Rab7 switch. Although the precise role that the Rab5 to that can result in the trafficking 1 Rab7 switch is unknown, it serves as a reliable marker for the transition from an early endosome to a late of the receptor back to the plasma 0 endosome. membrane.14 The second pathway 2 that internalized cargo can traverse © clathrin-independent mechanisms such as phagocytosis, is endolysosomal degradation. This route is entered when inter- macropinocytosis and caveolin-dependent endocytosis or nalized cargo are retained in a maturing endosome (rather than clathrin-dependent mechanisms such as receptor-mediated being recycled back to the plasma membrane) until finally being endocytosis.3,4 Clathrin-dependent endocytosis is the best char- delivered to the lysosome. acterized and predominant mechanism for the internalization The early endosome (EE) is the organelle within a cell that of cell surface receptors and thus provides an ADC with a cell receives the incoming endocytosed cargo and fluid from the specific entry mechanism.3,4 Clathrin-mediated endocytosis plasma membrane.15 EEs are mildly acidic (pH 5.9–6.8) and commences with the recruitment of adaptor proteins, accessory serve as the initial sorting stations for internalized cargo.16- proteins and a clathrin polymeric lattice to phosphatidylinosi- 18 While cargo can be recycled back to the plasma membrane tol-4,5-bisphosphate-enriched plasma membrane regions.5 The directly from the EE through the “short recycling loop,” a dis- clathrin adaptors function to select the cargo proteins that will crete endosomal structure called the recycling endosome has be internalized; the adaptor protein most commonly found to been identified which recycles cargo through the “long recycling 14 mAbs Volume 5 Issue 1 loop.”19-22 Additionally, cargo can be sent to the Golgi complex internalization.35-38 Under these conditions, antibody dissociation for re-packaging through the EE-associated retromer complex.22 from its receptor within the endosome would allow the antibody Before delivering its cargo to the lysosome, maturing EEs to escape lysosomal degradation by binding to FcRn and return- transition into late endosomes (LE), also known as multivesicular ing to the cell surface, effectively increasing the serum half-life bodies (MVBs).23 Endosomal maturation to LEs is characterized of the particular mAb. Accordingly, these IgGs are rescued from by an increase in luminal acidification, movement to the peri- degradation via FcRn and potentially re-bind their antigen at the nuclear space and the formation of intraluminal vesicles (ILVs), cell surface.39 which are vesicles containing cargo proteins that bud off of the Two recent studies employed a mutagenic strategy to engi- LE membrane inwardly into the LE lumen.24,25 Once ILVs are neer pH-dependent binding affinity to increase serum clearance formed, the proteins within are delivered to the lumen of lyso- half-life. The first study sought to modify tocilizumab (TCZ), somes where they are degraded. a humanized IgG antibody targeting the interleukin-6 recep- tor (IL-6R); TCZ is marketed for the treatment of moderate to . e Engineering Antibodies to Exploit severe rheumatoid arthritis.40 The IL-6R undergoes a high rate of t the Endo/Lysosomal System membrane turnover, which results in a high rate of TCZ cellular u clearance through lysosome mediated degradation and a conse- b The ideal tumor target for an ADC has the following features: (1) quential decrease in plasma elimination half-life. Consequently, ri the antigen is abundantly and exclusively expressed on the target the investigators of this study reduced the binding affinity of st cell; (2) it undergoes minimal secretion since secreted receptors TCZ to IL-6R at the endosomal pH 6.0 without affecting bind- i d can bind the antibody in the circulation, thus limiting exposure ing affinity at the cell surface pH of 7.4. This approach allowed to the target cell; (3) it possesses an appropriate rate of endocy- TCZ to dissociate from IL-6R in the endosome to become t o tosis; and (4) it undergoes an appropriate intracellular traffick- unlinked from the lysosomal degradation pathway that IL-6R n ing route for the desired outcome. Recent advances in antibody enters and bind FcRn to recycle back to the cell surface.41 To o engineering may allow investigators to design efficacious ADCs reengineer the binding affinity of TCZ at pH 6.0, the investi- D against targets that meet most of these criteria, but are other- gators utilized a histidine scanning approach.41 This involves wise unattractive because their particular intracellular trafficking mutating critical amino acids within the antigen binding inter- . e characteristics are not conducive to efficient ADC delivery to the face to histidine residues; histidine has a pK of 6.0 and thus its a c lysosome. Hence, there are now a few instances of antibodies have presence within a binding interface can have a dramatic effect on n been engineered to possess high antigen binding affinity at a pH the pH dependency of the binding affinity. One mutant, which e of 7.4 (extracellular pH) with an increased off-rate at a pH of 6.0 contained Y27H and S31H mutations on the heavy chain and i c (endosomal pH). This approach ensures increased dissociation Y32H and R53H mutations on the light chain of the antibody s of the antibody from the receptor within the endosome so that (clone PH2), reduced IL-6R binding affinity by ~4-fold at pH o it becomes an independent entity within the endosomal sorting 6.0 while actually increasing the affinity at pH 7.4.41 However, i B systems. While these particular studies were done to increase the increased dissociation from its target at pH 6.0 alone will not efficacy of naked antibodies, one may envisage that this mode of always save an antibody from lysosomal degradation, as retention s engineering could be utilized differently depending on the intra- within the fluid phase of the endosome will result in trafficking e cellular trafficking dynamics of the receptor being targeted and to the lysosome. Accordingly, when the pharmacokinetics of PH2 d the particular antibody- based therapeutic platform used. and TCZ were compared in mice that were administered a single n Receptors such as the transferrin receptor, LDL-receptor, the intravenous dose of 25 mg/kg, there was no significant increase in a metabotropic glutamate receptor 5, integrin receptors and HER2 plasma antibody concentration of PH2 over TCZ when tracked L continuously recycle back to the plasma membrane immediately over a 35 d period.41 As a result, PH2 was engineered to possess 2 after cellular internalization.26-30 When developing an ADC an increased binding affinity for FcRn at pH 6.0 (PH2-FcRn); 1 against receptors such as these, one could plausibly attempt this reduced lysosomal accumulation and successfully increased 0 to engineer antibody dissociation within the endosome in an the serum half-life 4 fold for the antibody in vivo (1 week for 2 attempt to reduce the likelihood that the ADC will cycle back PH2, 4 weeks for PH2-FcRn).41 © out of the cell with its receptor (Fig. 2). While this concept has In a different study, investigators sought to re-engineer an anti- not been demonstrated for ADCs, ligands such as tumor growth body directed against proprotein convertase subtilisin kexin type factor α and transferrin can indeed dissociate from their receptor 9 (PCSK9);42 PCSK9 promotes the degradation of low density within the endosome, avoid recycling out of the cell and traf- lipoprotein (LDL) receptor, thus increasing serum levels of LDL- fic to the lysosome for degradation.27,31-33 This mechanism would cholesterol. Accordingly, antibodies targeted to PCSK9, such as require the absence of the neonatal Fc receptor (FcRn) when J10 and the affinity matured J16, can be used to effectively lower using an ADC, as FcRn is a receptor present within the fluid LDL. Similar to the previous study discussed, the antibody stud- phase of endosomes which can transport IgG out of the endo- ied, J16, undergoes significant lysosome mediated degradation.42 somes to the cell surface.34 Therefore, these investigators also sought to decrease J16 binding Alternatively, receptors such as epidermal growth factor recep- affinity at the endosomal pH of 6.0 without affecting its binding tor 1, interleukin-2β, β2- adrenergic receptor and E-cadherin affinity at the plasma pH of 7.4. The investigators also utilized a are principally trafficked to the lysosome for degradation after histidine scanning approach to generate the J17 mutant antibody www.landesbioscience.com mAbs 15 of this characteristic relies on histi- dines placed both on the antibody and its receptor. Human prolactin (PRL), a member of the family of hematopoietic cytokines, binds to the PRL receptor (PRLR) in a pH- dependent manner; affinity of PRL for PRLR decreases 500-fold as the pH decreases from pH 8 to 6.43,44 To investigate the molecular mecha- nism by which this occurs, Kullkarni and colleagues sought to attribute . e pH dependence of PRL binding to t the effect of individual His residues u within the high affinity PRL-PRLR b interface.45 These histidines in PRL ri (His-27, His-30 and His-180), st which are located within the high i d affinity binding interface, appear to act cooperatively in creating pH- t o dependent binding characteristics, n as no single mutation drastically o changed the pH affinity profile of D PRL. This study revealed, however, Figure 2. Possible Intracellular trafficking routes of Antibody-Drug Conjugates. If an ADC binds to a that the pH dependent regulation of . receptor which undergoes continual recycling back to the plasma membrane, there is the potential e the PRL:PRLR interaction depends that the ADC may be recycled back to the plasma membrane without delivering the toxic payload, as c depicted on the right trafficking route. In certain cell types, the presence of the FcRn can complicate this critically on His-188 in the PRLR.45 n process, as FcRn binds IgGs and recycles them back to the plasma membrane; as depicted on the right Importantly, the binding of human e trafficking route. However, in the absence of FcRn, dissociation of the ADC from its receptor within the growth hormone (a disparate and i endosome may lead to lysosomal trafficking of the ADC, where it can release its toxic payload to the cell, c natural binding partner for PRLR) as depicted on the left trafficking route. s to the PRLR is pH-independent, o demonstrating specificity for pH- i B that contained histidine substitutions in complementarity deter- dependency within the same receptor.45 High resolution crystal- mining region (CDR)-1 (S30H) and CDR2 (S50H) of the light lographic structures of the binding interfaces of the PRLR in s chain and CDR2 (S52H) of the heavy chain.42 Surface plasmon complex with the WT and all possible single-site His mutants e resonance studies revealed that J17 possesses a 9.2-fold lower revealed the importance of electrostatic forces in regulating the d binding affinity at pH 6.0 compared with pH 7.4; most of this pH dependence rather than conformational effects.45 n pH-dependent effect comes from the difference in the rate of dis- a sociation of the complex.42 Balancing Receptor Kinetics, Binding Affinity, L When the pharmacokinetics of J16 and J17 were compared in Molecular Size and Tumor Penetration of Engineered 2 wild-type cynomolgus monkeys treated with a single intravenous Antibodies 1 injection of either J16 or J17 (1.5 mg/kg), there was a substantial 0 increase in antibody plasma elimination half-life of J17 (t : 7.4 d) Antibodies have also been reengineered to achieve increased anti- 2 1/2 over J16 (T : 0.9 d).42 Fur thermore, when either J10 or J17 were gen binding affinity at the cell surface. These particular modi- © 1/2 injected into FcRn –/– mice, there was no difference in the serum fications, in some circumstances, may prove to be obstructive half-life of either antibody, supporting the notion that FcRn plays because increasing binding affinity may adversely affect biology a pivotal role in increasing the serum half-life of J17; no pharma- and intracellular targeting. Accordingly, high affinity driven by a cokinetic comparisons between J16 and J17 in FcRn –/– mice fast on-rate and slow off rate may inhibit diffusion throughout the were reported in this study.42 Confocal microscopy and colocal- entire tumor mass after extravasation and lead to an accumula- ization analysis revealed that J17 co-localized with LAMP1 to an tion of antibodies at perivascular regions of the tumor where they ~15% lower extent than J10, supporting the hypothesis that the are internalized and catabolized by those cells.46 Furthermore, escape from lysosomal degradation of J17 helped increase serum the molecular size of the biological modality used and the inter- half-life; no direct comparison of the lysosomal colocalization nalization kinetics of the tumor antigen targeted can also drasti- characteristics of J16 and J17 was reported in this study.42 cally impact the degree of tumor penetration of an ADC. This Not surprisingly, histidine-regulated binding affinity is a con- concept is known as the “binding site barrier” effect and was pro- cept found in nature; however, one of the documented studies posed over two decades ago.47-49 The binding site barrier effect 16 mAbs Volume 5 Issue 1 suggested that: (A) greater antigen density; (B) higher antibody more than trastuzumab due to a diminished binding site barrier affinity; and (C) faster antibody internalization and metabolism effect. by cells would increase the “barrier” effect for antibodies binding To further explore how antibody binding affinity affects solid to antigens on the tumor, limiting the number of available dif- tumor accumulation and penetration, Rudnick and colleagues fusible molecules available for penetration deeper into the tumor tested the solid tumor uptake and penetration characteristics of mass.47,49,50 Thus, there is an appropriate range of antibody affin- C6.5-IgG and three affinity altered mutant IgGs (G98A, ML39, ity and molecular size required to balance specificity, selectivity, H3B1).46 These particular antibodies have intrinsic K values D pharmacokinetic properties, but also tumor accumulation and ranging from 10–7 M to 10–10 M.55 These affinity variants were penetration. generated by mutating the residues of C6.5 within the CDR Cell surface receptors exhibit a broad range of basal and anti- regions located within the center of the antibody:antigen bind- body-induced internalization rates and this can have a dramatic ing interface.55 The substantial variations in K values were pri- D effect on ADC efficacy. For example, when Ingle et al. compared marily driven by large changes in the k rate, as k rates for . off on e the efficacy of ADCs targeting CD19 and CD21, which are cell the variants generated were not significantly different from the t surface receptors present on normal and tumorigenic B-cells, they C6.5 wild-type antibody.56 To determine the differences in tumor u found that CD21 does not appreciably internalize upon antibody accumulation characteristics of these IgGs, 20 μg radioiodinated b binding, even when expressed at very high levels.51 Furthermore, IgG of each variant was injected intravenously into SCID mice ri while CD19 did undergo endocytosis and efficiently internal- carrying subcutaneous SK-OV-3 (HER2 overexpressing, human st ized anti-CD19 ADCs, it only did so in the absence of CD21.51 ovarian carcinoma) tumor xenografts.46 Twenty-four and 120 h i d Hence, CD21 and CD19 form a complex on the surface of B cells after IgG injection, the mice were euthanized and the amount of where CD21 prevents the internalization of CD19.51 The results radiolabeled IgG present within the tumor mass, the surround- t o of this study underscore the need to comprehensively understand ing tissue and in circulation was determined by a gamma coun- n the molecular mechanisms governing receptor endocytosis and ter.46 The results showed a correlation between binding affinity o intracellular trafficking properties of the respective target when and tumor accumulation with the highest affinity IgG (MH3B1, D designing an ADC. K 1 × 10–10 M) having the lowest tumor accumulation at The rate of receptor endocytosis can also affect ADC effi- boDth 24 and 120 h.46 In order to examine how binding affin- . e cacy through modifications in tumor penetration. Ackerman ity affects the distance to which an IgG can penetrate a tumor c et al. compared how a difference in the rate of receptor inter- after extravasation, 500 μg of unlabeled IgG was administered n nalization affects the distance to which the antibodies M85151a by intraperitoneal injection to SCID mice harboring established e and M111147 can penetrate a tumor spheroid.52 M85151a and SK-OV-3 tumors and the distance that each IgG variant traveled i c M111147 are antibodies which both bind to carcinoembryonic from the blood vessels through the tumor was assessed by stain- s antigen (CEA), however they possess an ~3-fold difference in ing for HER2, tumor vasculature (CD31) and human IgG after o internalization rates (M85151a t is 5 h and M111147 t is 13 the injection.46 The results from this experiment confirmed that i 1/2 1/2 B h) due to the fact that M85151a recognizes two epitopes and thus IgGs with a slower off rate failed to efficiently penetrate into the cross-links CEA, leading to more rapid rate of internalization.52,53 tumor.46 Subsequent analyses revealed that the IgGs with slow s When LS174T spheroids were incubated with each antibody, the off rates were internalized and catabolized by the perivascular e slower internalizing M111147 antibody penetrated much deeper tumor cells, and thus were eliminated before reaching other areas d within the spheroid tumor than did the more rapid internalizing of the tumor.46 n M85151a antibody.52 Thus, when developing an ADC, the target When Adams et al. studied the tumor penetration charac- a selection process must include a comprehensive analysis of recep- teristics of C6.5 and its affinity variants in single chain variable L tor internalization kinetics and how the receptor internalization fragment (scFv) format rather than a full-length IgG, they found 2 kinetics of the receptor will affect ADC delivery to the entire that increased binding affinity still correlated with a decrease 1 tumor mass. in tumor accumulation and the ability to penetrate a tumor, 0 Antibody affinity for the receptor also has an effect on tumor although this characteristic was less pronounced in scFv format.57 2 penetration. This notion is demonstrated by the differences in the To assess the magnitude that these scFvs can accumulate within a © tumor penetration efficacy of the anti-human epidermal growth tumor, 20 μg of radioiodinated scFv of each variant was injected factor receptor 2 (HER2) IgGs trastuzumab (Herceptin®) and into mice harboring SK-OV-3 human ovarian carcinoma xeno- C6.5 (in IgG format, C6.5-IgG). Although both C6.5 and trastu- grafts.57 Twenty four hours after scFv injection, the mice were zumab bind to HER2, they do so at distinct sites and do not sacrificed and the amount of radiolabeled scFv present in the compete for binding.54 Trastuzumab (K of 0.1 × 10–10 M) can tumors was determined by a gamma counter.57 A K of at least 1 D D penetrate SK-OV-3 tumors xenografts to a distance ~20% less × 10–7 M was required for any appreciable accumulation within than C6.5-IgG (K of 2.7 × 10–8 M),46 suggesting that C6.5-IgG the tumor.57 While overall scFv accumulation within the tumor D may be more effective in vivo, but published results of the direct positively correlated with lower K values in the range of 3 × 10–7 D comparison of the cancer cell toxicity between trastuzumab and M to 1 × 10–9 M, tumor accumulation reached a plateau and no C6.5-IgG were not found. Nevertheless, given that trastuzumab further scFv accumulation was observed with variants possessing has a k of 3 × 10–4 s–1 and C6.5 has a k of 6 × 10–3 s–1, it is pos- a K below 1 × 10–9 M.57 Additionally, 100 μg of low affinity (3 × off off D sible that a faster off rate allows C6.5 to penetrate solid tumors 10–7 M) or high affinity (1 × 10–11 M) scFv was injected into mice www.landesbioscience.com mAbs 17 bearing SK-OV-3 tumor xenografts and the distance that each a scFv, perhaps there is a critical molecular weight somewhere scFv variant traveled from the blood vessels through the tumor between 15kDa and 25/30 kDa in which binding affinity begins was assessed by staining for tumor vasculature (anti-CD31) and to influence tumor penetration. Another explanation for these scFv (a rabbit polyclonal antibody specific for scFv molecules results may be that DARpins have a different epitope on HER2 was used).57 Twenty-four hours after injection, the high affinity compared with C6.5 and trastuzumab, which may lead to a dif- scFv was present only in nearby blood vessels within the tumor ferent internalization rate and less catabolism. Future experi- mass while the low affinity scFv was dispersed throughout the ments aimed at addressing these questions may be revealing. vascularized regions of the tumor.57 Thus, while the smaller anti- To better understand how both molecular size and bind- HER2 scFv molecules exhibited affinity-based restrictions in ing affinity affect tumor accumulation, Schmidt and Wittrup tumor accumulation and penetration, it was to a lesser degree developed a mathematical model and utilized data available in than the larger IgG molecules.57 the literature to predict how binding affinity and molecular size Zahnd et al. came to a different conclusion when studying influence tumor accumulation.59 The model predicts that inter- . e the correlation between binding affinity and overall tumor accu- mediate-sized proteins (~25 kDa) accumulate within tumors the t mulation of the anti-HER2 designed ankyrin repeat proteins least, whereas both smaller and larger biological moieties accu- u (DARPins).58 DARPins are 14.5 kDa molecules (and therefore mulate to a greater degree within a tumor.59 IgGs are predicted b smaller than either the ~150 kDa IgG or ~25 kDa scFv) contain- to attain significantly higher levels of tumor accumulation due ri ing an NH -terminal capping repeat, two internal repeats carry- to both a slower serum clearance half-life and FcRn mediated st 2 ing the binding residues and a COOH-terminal capping repeat.58 retrieval from lysosomal degradation.59 The model also predicted i d In this study, anti-HER2 DARPins with affinities ranging from that smaller proteins require antigen binding affinities on the 9 × 10–11 M to 2 × 10–7 M were labeled with 99mTc(CO) on the order of 10–9 M to 10–12 M to achieve significant tumor accumu- t 3 o NH -terminal His-tag and were intravenously injected (single lation while larger proteins, such as an IgG can possess antigen n 2 dose of 8–10 μg DARPins) in mice bearing human ovarian car- binding affinity as low as 10–7 M and achieve substantial tumor o cinoma SK-OV-3 xenografts at the lateral flanks.58 The mice were accumulation.59 D sacrificed at various time points over the course of three days after DARPin injection and the level of radioactivity retained within Antibody Conjugation: Implications for Linker . e the tumor was measured in a gamma scintillation counter.58 Chemistry and the Conjugate c While these DARPins have a rapid serum elimination half-life (< n 3 min), tumor accumulation was proportional to DARPin affin- Another factor that affects the efficacy of an ADC is the effi- e ity for HER2, with the highest affinity DARPin (K 9 × 10–11 cient release of the drug from the antibody into the cell cytosol. D ci M) accumulating the most within the tumor mass.58 The tumor This is achieved through the disruption of the chemical linkage s accumulation of DARPins modified by the addition of PEG20 between the antibody and the toxic payload. Thus, the choice of o was also analyzed in mice harboring human ovarian carcinoma the linker used may have an impact on ADC efficacy and sev- i B SK-OV-3 xenografts at the lateral flanks.58 While the PEGylated eral different types have been synthesized. Acid-labile hydrazone DARPins had an increased serum half-life (~19 h), the addition linkers are designed to be stable within the neutral pH extra- s of PEG20 to the DARPins eliminated most of the correlation cellular environment, but become cleaved within the low pH e between binding affinity and tumor accumulation; with the environment of intracellular endosome and lysosome compart- d exception of the lowest affinity anti-HER2 PEGylated DARPin ments.60 Peptide linkers, such as citruline-valine, are designed to n (K : 2 × 10–7 M), which had negligible tumor accumulation, be selectively cleaved by lysosomal proteases (e.g., cathepsin or a theDre was little difference in the magnitude of accumulation of plasmin).61 Another linker that has been developed is disulfide- L any other PEGylated anti-HER2 DARPins.58 Thus, while there based which is selectively cleaved in the reductive environment 2 was a clear correlation between binding affinity and tumor accu- of the cell cytosol.61 Non-cleavable linkers, such as thioether or 1 mulation of the smaller, unmodified DARPins, increasing the amide bonds, have been utilized more recently and are intended 0 DARPIn hydrodynamic radius through PEGylation eliminated to retain stability throughout the plasma and most of the intracel- 2 this correlation. lular space. Thus, when using a non-cleavable linker, liberation of © While it is somewhat difficult to reconcile these seemingly the payload relies on the degradation of the antibody within the contradictory findings, given that all three studies utilized a bio- lysosome, however it is important to note that thioether linkers logical moiety of different molecular size, it is plausible that there can be less stable in the plasma due to thiol exchange reactions.62 is a complex interplay between molecular weight, hydrodynamic ADCs with cleavable disulfide linkers, such as the disulfide linker radius and binding affinity that contribute to tumor accumula- N-succinimydyl 4-(2-pyridyldithio)-pentanoate (SPP), can form tion and penetration characteristics. Thus, for much smaller mol- lipophilic drug metabolites within the lysosome which possess ecules such as DARPins, which diffuse through a tumor easily the ability to exit the target cell and re-enter neighboring cells by virtue of their size, an increased binding affinity may lead to which may lead to improved efficacy through bystander killing increased tumor accumulation. However, as molecules increase of neighboring tumor cells, but can also cause off-target toxic- in size and become inherently more difficult to diffuse through a ity.60,63 Alternatively, ADCs possessing a thioether linker, such as tumor, an increased binding affinity influences this penetration succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxyl- restriction. Thus, because DARPins are nearly half the size of ate (SMCC), form charged drug metabolites within the lysosome 18 mAbs Volume 5 Issue 1 that cannot freely diffuse across the plasma membrane of a neigh- a thioether linker, is undergoing evaluation in multiple Phase 3 boring cell. trials for HER2-positive metastatic breast cancer and a market- The efficacy of trastuzumab linked to the maytansinoid DM1 ing application for T-DM1 was submitted to the Food and Drug by a reducible disulfide linker (T-SPP-DM1) or a non-cleavable Administration in August 2012.70 Preliminary results from a thioether linker (T-MCC-DM1) has been compared by multi- Phase 3, randomized, multicenter, international, two-arm, open- ple groups. In the first study, mice bearing MMTV-HER2 Fo5 label clinical trial comparing the safety and efficacy of T-DM1 mammary tumor transplants were treated with a single intra- and the current standard of care capecitabine + lapatinib (XL) venous injection (10 mg/kg) of T-SPP-DM1, T-MCC-DM1 or in patients harboring HER2-positive advanced metastatic breast vehicle and tumor volume was monitored for 21 d.64 While both cancer was recently published.71 The primary analysis of the data T-SPP-DM1 and T-MCC-DM1 delayed tumor growth compared from the study showed that T-DM1 treatment is well tolerated with mice treated with vehicle, MCC-DM1 was significantly bet- and can provide a progression free survival of 9.4 mo compared ter at inhibiting the tumor growth; tumor volume reached a size with the 5.8 mo achieved with XL treatment.71 Gemtuzumab . e of ~800 mm3 by day 10 in vehicle treated mice, tumor volume ozogamicin (Mylotarg®) was approved by the FDA in 2000 for t reached a size of ~900 mm3 by day 21 in T-SPP-DM1 treated use in patients over 60 y of age suffering from relapsed acute u mice, and tumor volume reached a size of ~500 mm3 by day 21 myelocytic leukemia.72 Gemtuzumab ozogamicin consists b in T-MCC-DM1 treated mice.65 T-MCC-DMI was also better of a humanized anti-CD33 antibody linked to N-acetyl-γ- ri tolerated by female Sprague-Dawley rats in these studies; rats calicheamicin (an enediyne antibiotic that causes double-strand st treated with single doses of either 25 or 50 mg/kg T-MCC-DM1 breaks of DNA resulting in apoptosis) through a bifunctional, i d showed no appreciable weight loss over a 5 d period, while rats hydrazone-based linker which is conjugated to lysine residues on treated with a single dose of 25 mg/kg T-SPP-DM1 exhibited a the CD33 antibody.73 In June 2010, gemtuzumab ozogamicin t o 10% reduction in total body weight by day 5.65 The efficacy dif- was voluntarily withdrawn from the US market due to safety and n ferences observed between T-SPP-DM1 and T-MCC-DM1 may efficacy concerns that developed during a randomized Phase 3 o have been caused by either inefficient reduction of disulfide bond comparative clinical trial performed after accelerated approval.74 D linking SPP to the antibody and hence release of the payload to This outcome highlights the need for a better understanding of the cell or by the formation of lipophilic metabolites and the sub- all facets involved in ADC development. . e sequent exit of the linker/payload from the tumor cell. The lat- The efficacy of an ADC relies, in part, on the interactions of c ter explanation could be supported by evidence from the study the antibody component with elements of the extracellular envi- n showing weight loss in mice treated with T-SPP-DM1 which is ronment. It is clear that antigen binding affinity and molecular e indicative of off-target toxicity. size can affect tumor penetration, and a balance between molecu- i c A subsequent study by Erickson et al. reported no statisti- lar size and binding affinity is crucial to maximizing efficacy. s cal difference between the in vivo efficacy of T-SPP-DM1 and Furthermore, selection of a proper tumor antigen should involve o T-MCC-DM1 against BT-474EEI tumors.66 In this study, nude analyses of not only cell specificity and copy number, but also the i B XID mice harboring BT-474EEI xenografts on their mammary internalization kinetics of the receptor. However, we still lack the fat pads were treated with a single intravenous injection of 3–18 fundamental knowledge of how molecular size, binding affin- s mg/kg of T-SPP-DM1 or T-MCC-DM1. Over a 38 d period, ity and internalization kinetics of the receptor correlate to dif- e the tumor volume was reduced in similar proportions after either ferences in overall ADC cell kill potency. There is thus a need d T-SPP-DM1 or T-MCC-DM1 treatment.66 The reasons for the for additional functional readouts for correlation analyses such n disparate results of these two studies may lie within the inherent as tumor cell toxicity studies, analyses of changes in cell surface a biological differences that exist between the two tumors targeted. expression of the target receptor and analysis of how downstream L For example, differences in HER2 densities, endo/lysosomal signaling cascades of the target receptor are modulated by the 2 dynamics and other biological differences present within the two antibody. This information will provide a more comprehensive 1 tumors may have drastic effects on the efficacy of a given ADC. understanding of how to properly balance the selection of the 0 target antigen with the molecular size and binding affinity of the 2 Summary and Perspectives ADC. © The linker-payload choice is also a critical element in deter- Our current understanding of the nuances involved in ADC mining successful delivery of the payload to the cellular cytosol of design has led to some success within the clinic. As of early a target cell. We now know that different linker types can them- October 2012, there were at least 22 ADCs in clinical evalua- selves lead to differences in both the efficacy and the level of off- tion67 and one ADC, brentuximab vedotin is currently approved target toxicity observed for a given ADC. These facts highlight by the FDA and marketed for treatment in anaplastic large cell the importance of linker selection and provide the basis for future lymphoma and Hodgkin lymphoma.68,69 Brentuximab vedotin studies aimed at defining the precise behavior of these different is an anti-CD30 mAb conjugated to monomethyl auristatin E linkers in the intracellular and extracellular environment. Studies (MMAE, a synthetic analog of the tubulin polymerization inhib- aimed at assessing quantitatively the sub-cellular compartment in itor dolastatin 10) through a peptide valine-citrulline peptide which the linker is liberated from the antibody and the kinetics linker.64 Trastuzumab emtansine (T-DM1), composed of trastu- of that liberation will be critical to furthering our understanding zumab linked to the cytotoxin maytansinoid DM1 (DM1) via of how linker choice influences efficacy in different situations. www.landesbioscience.com mAbs 19 Quantitative data on how stable different linkers are in the extra- fullest extent for antibody-based therapy, we need to understand cellular space and how this stability impacts off-target toxicity is why antibodies follow a particular intracellular trafficking route. also needed. Therefore, identifying the precise molecular mechanisms mediat- Progress in the development of ADCs depends additionally ing endosomal sorting should remain a priority. Importantly, we on a better understanding of how the antibody component of need to resolve the precise molecular mechanisms mediating how an ADC interacts and traffics within the intracellular space of endosomes distinguish whether a receptor undergoes recycling vs. a target cell. Recent studies have shown that antibody engineer- endo-lysosomal degradation. This should also be assessed under ing can be used to create antibodies that exploit the endosomal different cellular contexts, including in different cell types, at dif- pathway and this may have a substantial impact in the field of ferent stages of the cell cycle and during differential ligand acti- ADCs in the future. Numerous cellular contexts will need to vation of the same receptor. As experimental assays improve and be considered, e.g., whether histidines within the binding inter- enable understanding of intracellular processes in greater detail, face of the receptor changes the antibody affinity in different the results may resolve current discrepancies and increase our abil- . e pH environments; whether the target is a recycling receptor or a ity to efficiently select novel and appropriate targets for ADCs. t lysosomal-targeted receptor; whether the cell expresses FcRn and u whether this will effect trafficking of an IgG back to the cell sur- Dislcosure of Potential Conflicts of Interest b face. To effectively exploit the endo/lysosomal pathways to the No potential conflicts of interest were disclosed. ri t s References 12. SNtuowcleelol tiMdeH-d,e pMenadrkens t B,c oWnfoigrgme atPi,o nMalc Mchahanogne sH Tin. 24. Rpeoreadtuerreer d Mep,e Bnodwensceer Rof, eMxpuorpsuhrye RoFf .e Kndinoectyitcos saendd m teamte-- di 1. Adams GP, Weiner LM. 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