Pharm Res DOI 10.1007/s11095-015-1729-8

EXPERT REVIEW

Internalization, Trafficking, Intracellular Processing and Actions of Antibody-Drug Conjugates Shi Xu 1

Received: 19 March 2014 / Accepted: 29 May 2015 # Springer Science+Business Media New York 2015

ABSTRACT Purpose This review discusses the molecular mechanism involved in the targeting and delivery of antibody-drug conjugates (ADCs), the new class of biopharmaceuticals mainly designed for targeted cancer therapy. Methods this review goes over major progress in preclinical and clinical studies of ADCs, in the past 5 years. Results The pharmacokinetics and pharmacodynamics of ADCs involve multiple mechanisms, including internalization of ADCs by target cells, intracellular trafficking, release of conjugated drugs, and payload. Conclusion These mechanisms actually jointly determine the efficacy of ADCs. Therefore, the optimization of ADCs should take them as necessary rationales.

KEY WORDS Antibody drug conjugate . Internalization . Recycling . Trafficking . Linker

ABBREVIATIONS ADC ADCC CDC DM1 EGFR FcRn MMAE MTD

Antibody-drug conjugates Antibody-dependent cellular cytotoxicity Complement-dependent cytotoxicity N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)maytansine Epidermal growth factor receptor Neonatal Fc receptor Monomethyl auristatin E Maximum tolerated doses

* Shi Xu [email protected] 1

Scientific Development Manager, Discovery Biology, GenScript USA Inc., 860 Centennial Ave., Piscataway, New Jersey 08854, USA

GENERAL PRINCIPLES OF ADC DEVELOPMENT The rationale for the development of ADCs is to combine the selectivity, favorable pharmacokinetics, and high potency of the conjugated drug molecule. An ideal ADC should retain the favorable pharmacokinetic and functional properties of antibodies; remain intact and nontoxic in the compartment of its systemic delivery (typically blood). The ADC will consequently accumulate in the tumor compartment, and release sufficient payload to kill tumor cells. An ideal ADC may even combine the cytotoxic activity of the drug molecule with the intrinsic anti-tumor activities of the antibody, e.g. antibodydependent cellular cytotoxicity (ADCC), complementdependent cytotoxicity (CDC), blocking receptor signal transduction, etc. (1). Desirable properties of the antibody backbone for ADCs include: high affinity; high specificity; appropriate half-life for exposure; immune effector functions such as CDC, ADCC and antibody-dependent cellular phagocytosis; tumorsuppressing modulation of antigen’s biological activity when possible; and minimal loss of the above-listed properties upon its conjugation to the drug molecule (1). Humanized IgG1 has been the most common isotype used for ADC antibodies in clinical development, due to its capability to trigger both ADCC and CDC (2).

MECHANISMS OF INTERNALIZATION OF ADCS BY TARGET CELLS Internalization of the ADCs is important for its effectiveness: many of the chemical-linking strategies used to construct ADCs rely on intracellular conditions, either in the cytoplasm or in the lysosome, to release the active drug molecule (3). Although the expression level of the target receptor (antigen) on cell surface appears to be an important factor

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determining the potency of ADCs, observations suggest that even antigen receptors with low copy numbers can be effectively targeted with a sufficiently potent ADC. For example, CD33-positive acute myelogenous leukemia tumors express relatively low levels of CD33 receptor (5000~10,000 copies per cell), but favorable clinical responses have been observed with gemtuzumab ozogamicin, an ADC that targets the CD33 (4,5). The binding of ADC to the membrane receptor antigen usually triggers rapid internalization of the ADC-receptor complex by receptor-mediated endocytosis (6–8). The internalization efficiency can vary between an ADC and the unconjugated antibody: in some cases both are internalized at the same rate, whereas in other cases the ADC internalization is more efficient (9,10). The rate and extent of internalization are important because it influences the uptake and release of drug molecules (11,12). Moreover, in some cases, antibody binding can alter the mechanism of internalization of the target receptor. For example, Epidermal growth factor receptor (EGFR) is typically internalized via the receptor-mediated endocytosis dependent on clathrin (13); however, the internalization of EGFR by C225 (Cetuximab) was found independent on clathrin and/ or dynamin, but dependent on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis causes internalization of large membrane areas, leading to highly efficient antibody-induced internalization of EGFR (14). The alteration of internalization mechanism means that the ADC-receptor compound may be internalized by target cells via pathways other than receptormediated endocytosis. The turn-over rate of receptor by endocytosis can further affect the tumor penetration capability of ADC. A study on antibody penetration showed that, an antibody that crosslinks carcinoembryonic antigen (CEA) led to a faster rate of internalization than the antibody without crosslinking function; consequently, the slower internalizing antibody penetrated much deeper within the spheroid tumor than did the faster internalizing counterpart (15). Therefore, the target selection for ADCs must include a comprehensive analysis of receptor internalization kinetics and its influence on ADC delivery to the entire tumor mass (7). Studies with anti-HER2 antibodies also suggest affinity’s influence on penetration capability: higher affinity antibodies were internalized and degraded faster than moderate affinity antibodies, thus limiting their tumor penetration (16). The inverse relationship between antigen density, antibody affinity, antibody internalization by cells and tumor penetration capability is termed as Bbinding site barrier^ effect: higher antigen density, higher affinity and faster antibody internalization will reduce diffusible antibodies available for deeper penetration into the tumor mass (7).

THE RECYCLING OF IGG AND ADCS BY NEONATAL FC RECEPTOR (FCRN) ADCs are designed for specific drug delivery into the tumor cells over-expressing the target receptor. However, because macropinocytosis without cargo specificity naturally occurs in vascular endothelial cells at all times (17,18), the uptake of systemically delivered ADCs by vascular endothelial cells is theoretically inevitable. Fortunately, internalized IgG can be rescued from endosomal/lysosomal pathway and recycled into the circulation as a consequence of their interaction with the neonatal Fc receptor (FcRn) (19). After being taken-up by vascular endothelial cells and other cells through macropinocytosis, IgG can interact with the FcRn in a pH-dependent manner: the binding occurs in endosomes at pH 6.0–6.5, followed by recycling and releasing at the cell surface at pH 7.0–7.4 (20). The interaction between the Fc region of IgG and FcRn plays a key role in IgG recycling and homeostasis, and is largely responsible for the long half-life of IgG (~21 days in humans for IgG1) (21). FcRn-knockout mice showed a 10~ 15 times higher IgG elimination rate than the control (22–24). Altering the half-life of IgG has been performed by mutating the Fc region at various amino acid residues. A mutant of tocilizumab (a humanized IgG targeting the interleukin-6 receptor) named PH2 was engineered to possess increased binding affinity for FcRn at pH 6.0 (PH2-FcRn); this reduced lysosomal accumulation and successfully increased the antibody’s serum half-life by 4 folds in-vivo (25): 1 week for PH2, and 4 weeks for PH2-FcRn. Interestingly, increased binding affinity between the Fc region and FcRn does not necessarily lead to proportional half-life extension. For example, a study on invivo half-life of antibodies involved 2 mutants of trastuzumab (Herceptin™): one with a 3-fold increase in FcRn binding at acidic pH, the other with a 12fold increased binding at acidic pH and also enhanced binding at neutral pH. The two mutants exhibited similar half-lives in a humanized FcRn transgenic mouse model (26). It has been hypothesized that increasing binding to FcRn at neutral pH prevents IgG release and may accelerate IgG degradation (26). Theoretically, Fc region can also be engineered to decrease the binding affinity with FcRn to decrease the half-life of ADC. For certain clinical applications, short half-life is desirable to avoid toxic adverse effects. For example, radiographic diagnostic imaging and toxin-conjugated products (i.e., for cancer treatment) would not require prolonged circulation in the bloodstream (27,28).

Internalization, Trafficking, Processing and Actions of ADCs

MECHANISMS OF INTRACELLULAR DRUG RELEASE FROM ADC The ADCs endocytosed by tumor cells are typically sorted into the late endosomal/lysosomal pathway. Many studies have clearly shown ADC accumulation in the lysosomes of tumor cells (29–33), where the drug molecule is released either by linker cleavage or degradation of the antibody backbone (29,32,34–36). The mechanism of release of drug molecule from ADCs depends on the linker design. The general principle of linker design is extreme stability in circulation, since release of the cytotoxic drug molecule before reaching the target would lead to toxicity. However, upon reaching the target cells, the linker must also be able to ensure the drug release to kill the target cells. There are currently four different classes of linkers in use that broadly fall under 2 categories: cleavable and noncleavable linkers (37). i. Cleavable linkers Cleavable linkers include acid-liable hydrazine linkers, disulfide linkers and peptide linkers. The first class of drug linker applied to ADCs is acid-labile hydrazine linkers, which are cleaved in lysosomes as a consequence of the lower pH within this compartment compared to the systemic circulation (38). The lysosomal compartment is both acidic (pH~5) and rich in proteolytic enzymes (39). Unfortunately, acid-labile hydrazine linkers have been associated with off-target release of drug molecules in clinical studies (40). The second class of drug linker undergoing clinical testing is disulfide linkers. This cleavage of disulfide linkers and release of drug molecules in cytosol are attributed to the more reductive intracellular environment (30,41,42): the concentration of glutathione, a thiol-containing tripeptide is in μM range in the blood, whereas its concentration in the cytoplasm is in the mM range (up to 1000 times higher) (3). However, the biochemical cleavage mechanism of disulfide bond also raises the concern of non-specific cytotoxicity: the ADCs internalized by vascular endothelial cells or other non-target cells are also subject to linker cleavage and drug release, because these non-target cells also have high intracellular glutathione concentration. The third class of drug linker is peptide linkers, with the potential for selective cleavage in the lysosomal compartment by lysosomal proteases such as cathepsin B (32,34,35). Compared to hydrazone linker compounds, peptide linkers are associated with increased serum stability and improved anti-tumor effects of ADCs. For example, dipeptide linkers Phe-Lys- and Val-Cit- are quite stable under physiological conditions, but undergo rapid hydrolysis in the presence of lysosomal extracts and purified human cathepsin B (36).

Evaluation of ADC from mouse circulation showed ∼144 h (6.0 days) half-life of a dipeptide linker, significantly greater than the half-lives of disulfide- or hydrazone-linked ADCs in mice or human trials (43). ii. Non- cleavable linkers Non-cleavable linkers have been developed more recently. The thioether bond of non-cleavable linkers is extremely stable in both extracellular and intracellular environments. The release of free, active drug from ADC with non-cleavable linkers is realized by catabolic degradation of internalized antibodies in lysosomes, releasing the active, cytotoxic drug component (32,42). Some recent studies have shown the superiority of noncleavable linkers over Btraditional^ cleavable linkers in in-vivo studies. In target-independent safety studies in rats, noncleavable linker ADCs showed reduced toxicity compared to cleavable linker ADCs, presumably due to the minimized systematic release of free drug or other toxic metabolites (44). In a study on anti-tumor activity of humanized anti-CD70 antibody h1F6 conjugated to the auristatin, 2 ADCs with noncleavable linkers both exhibited maximum tolerated doses (MTD) >150 mg/kg in single-dose experiments on mice, whereas the MTD of their counterpart with protease cleavable valine-citrulline peptide linker was only ~50 mg/kg as reported previously (45). More importantly, trastuzumab-DM1 (Kadcycla™), the most recently FDA approved ADC indicated for the treatment of human EGFR-2 (HER2) positive metastatic breast cancer, is based on non-cleavable linker: Derived from the HER2 targeting mAb trastuzumab (Herceptin™), trastuzumab-DM1 developed by Genentech consists of the antibody trastuzumab conjugated to the anti-mitotic maytasanoid N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)maytansine (DM1) via a non-cleavable N-[maleimidomethyl] cyclohexane-1 carboxylate (MCC) linker (46). An in-vivo study on a series of ADCs with various linkers showed that trastuzumab-DM with non-cleavable thioester linker was more effective than its counterparts with disulfide linkers in mouse breast cancer models (42). These studies all suggest that using non-cleavable linker for drug conjugation is a valuable strategy in ADC development.

DRUG-TO-ANTIBODY RATIO, A KEY FACTOR DETERMINING THE PHARMACOKINETICS OF ADC The number of drug molecules loaded per antibody is an important variable for ADC development, because the drugto-antibody ratio can alter the characteristics of the ADC. Efforts to conjugate more drug molecules onto the antibody

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have often failed because the resulting ADC suffers from increased aggregation, higher systemic clearance, or loss of affinity to the antigen (47). Therefore, most ADCs have 2~4 drug molecules per antibody (48). Conjugating more drug molecules to the antibody backbone does not necessarily lead to higher potency. The effects of conjugating 2, 4, and 8 molecules of the cytotoxin monomethyl auristatin E (MMAE) onto the anti-CD30 mAb (cAC10) were studied both in-vitro and in-vivo (49). MMAE conjugation did not affect binding of the ADC to CD30. Although in-vitro activity was directly correlated with the drug-to-antibody ratio, the in-vivo activities of the ADCs with 4 and 8 drug molecules per antibody were about equivalent. This was explained by the fact that clearance of the ADC was dependent on drug-to-antibody ratio: the exposure of the ADC with 8 drug molecules was only half of that of ADC with 4 drug molecules (49). This suggests that optimizing the drugto-antibody ratio helps to maintain favorable pharmacokinetics while maximizing payload in developing ADCs. The low achievable drug-to-antibody ratio (2~4 drug molecules per antibody) actually precludes the application of ADC strategy on most existing chemotherapeutic agents, and only a few extremely potent agents can be considered for the effective payload. The importance of drug potency to the efficacy of ADCs was shown in the clinical study of BR96-DOX (BMS182248), a rapidly internalized chimeric IgG1 anti-Lewisy monoclonal antibody conjugated to doxorubicin (DOX) via an acid-labile hydrazone linker (50,51). The ADC was evaluated in Phase I trials in patients with solid tumors expressing the Lewisy antigen. The results of this trial were disappointing, with objective responses seen in only 2 of 66 patients treated every 3 weeks at various dose levels of BR96-DOX (50). This study is quite typical to show the limitations of using a low potency drug such as DOX as the delivery payload for an ADC. In contrast, ADCs enable the clinical application of highly potent cytotoxic agents that cannot be used in conventional chemotherapy. For example, maytasine failed in the development as chemotherapeutic in 1980s (8) due to lack of therapeutic window, but its derivative DM1 has been successfully utilized in the FDA-approved ADC trastuzumab-DM1 (46,52). Conjugation with therapeutic antibodies is an effective method to increase the therapeutic index of highly potent cytotoxic agents. For application of the highly potent cytotoxic agents in ADCs, the agent used must have prolonged stability in aqueous formulations and plasma, in consistency with the relatively long half-life of ADCs (53).

DRUG MOLECULES, THE INTRACELLULAR EFFECTOR OF ADC Drugs are cleaved from the carrier antibody either lysosomes or cytosol (for disulfide linker) and take effect in the cytosol

(54,55), while therapeutic antibodies are thought to be eliminated predominantly by proteolytic degradation mechanism (56). Generally speaking, the highly potent cytotoxic agents that are commonly conjugated to antibodies belong to 2 categories: DNA Damaging drugs, and microtubule-targeting drugs. i. DNA damaging drugs An example of DNA damaging drugs applied in ADCs is calicheamicin, a highly cytotoxic enediyne antibiotic. It binds to the minor groove of DNA helix and therefore induces double-strand DNA breaks, which further result in cell death (1). N-acetyl-g-calicheamicin dimethyl hydrazide is a derivative of calicheamicin, and it is the payload of Mylotarg® gemtuzumab ozogamicin that targets CD33. This ADC was conditionally approved under the FDA’s acceleratedapproval program, but was finally withdrawn by Pfizer due to its failure to show improved efficacy in a Phase III combination trial. Another class of DNA damaging drugs applied in ADCs are analogs of duocarmycins, which are highly cytotoxic DNA minor-groove binding alkylating agents (57). For example, the ADC of the fully human anti-CD70 antibody and a duocarmycin analog, MDX-1203, has been developed at Bristol–Myers Squibb (58). ii. Microtubule-targeting drugs Microtubule-targeting drugs used in ADCs are represented by two major classes: maytansinoids and auristatins. ADCs based on both classes have entered clinical trials. Maytansinoids are derivatives of maytansine, a natural product originally isolated from the shrub Maytenus serrate. Maytansine has IC50 values in the range of 10 to 100 pM depending upon the cell type (59). It is significantly more cytotoxic in-vitro than microtubule-targeting chemotherapeutics like taxol. As mentioned in the previous section, the FDAapproved ADC trastuzumab-DM1 is a successful application of maytansine derivative DM1. The binding of trastuzumabDM1 to HER2 results in internalization of the ADC-HER2 complex and subsequent lysosomal degradation of trastuzumab-DM1 (30). Cytotoxic DM1 molecules are released into the cytoplasm, causing microtubule destabilization and tumor cell death (42). Auristatin is a potent antimitotic drug derived from peptides occurring in marine shell-less mollusk Dolabella auricularia called dolastatins (60). Brentuximab vedotin (SGN-35) developed by Seattle Genentics is an ADC targeting CD30. SGN35 uses MMAE as the conjugated payload. In preclinical studies, binding of brentuximab vedotin to cells was followed by internalization of the ADC–CD30 complex, and release of MMAE via proteolytic cleavage, leading to G2/M phase growth arrest and apoptotic cell death (34,35). In phase II

Internalization, Trafficking, Processing and Actions of ADCs

studies, SGN-35 induced overall response rates of 75% in relapsed or refractory Hodgkin lymphoma and 86% in anaplastic large cell lymphoma. The positive results led to the accelerated approval of the drug by the FDA. SGN-35 has overall manageable toxicity profile. However, cumulative peripheral neuropathy constitutes an important clinical consideration as it may limit prolonged administration of SGN-35 (34,35).

BYSTANDER EFFECTS Okeley et al. proposed that diffused drugs could enhance clinical outcomes if lesions consist of mixtures of cells with variable target antigen expression (31). Diffused drug molecules released from ADC kill not only antigen-positive tumor cells, but also proximally located antigen-negative tumor cells that are called Bbystander cells^. The killing mechanism induced by diffused drug molecules is termed Bbystander effect.^ For instance, auristatins are hydrophobic compounds that are membrane permeable. Once released from their antibody carriers, auristatins are free to diffuse to neighboring cells, regardless of whether they display the target antigen (54). Diffusion of MMAE released from SGN-35 in the tumor microenvironment and cytotoxicity on bystander cells may in part explain its activity, especially in Hodgkin lymphoma (60). Although the Bbystander effect^ has been noted in model tissue culture systems, it has not been very clearly characterized in tumors in-vivo (54). BBystander effect^ is obviously based on the permeability of the released payload. If the released drug or metabolic does not have membrane permeability, the Bbystander effect^ is avoided (61).

DISCUSSION In previous sections, we have discussed the important mechanisms of internalization, trafficking, intracellular processing and actions of ADCs. These mechanisms not only explain how ADCs take effect intracellularly, but also outline the rationale to further improve the efficacy and safety of ADCs. As the target of ADCs, the membrane receptor antigen sets the tone for the mechanism and dynamics of the internalization of ADC-receptor complex (6–8). However, target validation is a very complex process in the upstream drug discovery, thus the choice of target receptor of ADCs will not be discussed in detail within this article. Modifying the antibody Fc region of ADCs is a good way to set an appropriate half-life for the indicated uses of ADCs. Another factor relevant to ADC half-life is drug-to-antibody ratio, which may affect the clearance rate of ADCs (49). The drug-to-antibody ratio of ADCs should be optimized in the

proof-of-concept studies using animal models. However, due to the risk of significantly changing the antibody characteristics, the drug-to-antibody ratio cannot be increased in an unrestricted manner. This principle limits the amount of payload that ADC can deliver into the target cells, and further restricts the choices of conjugated drugs into a few extremely potent agents. A modern approach to significantly increase the payload is to use antibody-conjugated nanoparticles: Receptortargeted nanoparticles (10~400 nm) obviously have much more capacity to carry drug molecules than ADCs, and it is an emerging drug delivery system in development of cancer therapy (62). But it should also be noticed that receptortargeted nanoparticles may have different mechanisms of internalization and intracellular trafficking from ADCs (13). The design of linker between the antibody and the drug molecule has been extensively studied since ADCs were invented. Obviously, the early design of cleavable linkers aims to effectively release the drug molecule intracellularly. However, preclinical data has shown that ADCs with noncleavable linkers have better safety characteristics than their counterpart with cleavable linkers (42,44,45). Moreover, the FDA’s approval of trastuzumab-DM1 has proved the safety of non-cleavable linker in ADCs’ clinical application. These meaningful results have revised the rationale of ADC design: the release of drug molecule from ADCs is actually not a problem, because the antibody backbone will ultimately be degraded in lysosomes (31,32,42,63); however, the extreme stability of the linker before ADCs reach the target cells seems to be critical for minimizing the adverse effects and enhancing the tolerable doses of ADCs. Therefore, non-cleavable linkers do have preferable characteristics in the development of ADCs. In summary, the intracellular space is where ADCs take effect. The mechanisms of ADC internalization, trafficking, intracellular processing and actions provide helpful guidelines to optimize ADCs for favorable characteristics. Improving our understanding of these molecular mechanisms will pave the way for developing better ADCs in the future.

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Internalization, Trafficking, Intracellular Processing and Actions of Antibody-Drug Conjugates.

This review discusses the molecular mechanism involved in the targeting and delivery of antibody-drug conjugates (ADCs), the new class of biopharmaceu...
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