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`mAbs
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`Site-specific antibody drug conjugates for cancer
`therapy
`Siler Panowskia, Sunil Bhaktaa, Helga Raaba, Paul Polakisa & Jagath R Junutulaa
`a Genentech, Inc; South San Francisco, CA USA
`Published online: 01 Nov 2013.
`
`Click for updates
`
`To cite this article: Siler Panowski, Sunil Bhakta, Helga Raab, Paul Polakis & Jagath R Junutula (2014) Site-specific antibody
`drug conjugates for cancer therapy, mAbs, 6:1, 34-45, DOI: 10.4161/mabs.27022
`
`To link to this article: http://dx.doi.org/10.4161/mabs.27022
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` PAPer TyPe
`mAbs 6:1, 34–45; January/February 2014; © 2014 Landes Bioscience
`
`Site-specific antibody drug conjugates
`for cancer therapy
`
`Siler Panowski, Sunil Bhakta, Helga raab, Paul Polakis and Jagath r Junutula*
`
`Genentech, Inc; South San Francisco, CA USA
`
`Keywords: site-specific conjugation, antibody drug conjugate, THIOMAB, internalization, tumor antigen, linker, cytotoxic drug
`
`Antibody therapeutics have revolutionized the treatment of
`cancer over the past two decades. Antibodies that specifically
`bind tumor surface antigens can be effective therapeutics;
`however, many unmodified antibodies
`lack therapeutic
`activity. These antibodies can instead be applied successfully
`as guided missiles to deliver potent cytotoxic drugs in the
`form of antibody drug conjugates (ADCs). The success of ADCs
`is dependent on four factors—target antigen, antibody, linker,
`and payload. The field has made great progress in these areas,
`marked by the recent approval by the US Food and Drug
`Administration of two ADCs, brentuximab vedotin (Adcetris®)
`and ado-trastuzumab emtansine (Kadcyla®). However, the
`therapeutic window for many ADCs that are currently in pre-
`clinical or clinical development remains narrow and further
`improvements may be required to enhance the therapeutic
`potential of these ADCs. Production of ADCs is an area where
`improvement is needed because current methods yield
`heterogeneous mixtures that may include 0–8 drug species
`per antibody molecule. Site-specific conjugation has been
`recently shown to eliminate heterogeneity, improve conjugate
`stability, and increase the therapeutic window. Here, we review
`and describe various site-specific conjugation strategies
`that are currently used for the production of ADCs, including
`use of engineered cysteine residues, unnatural amino acids,
`and enzymatic conjugation through glycotransferases and
`transglutaminases. In addition, we also summarize differences
`among these methods and highlight critical considerations
`when building next-generation ADC therapeutics.
`
`Introduction
`
`Monoclonal antibodies (mAbs) have long been an integral
`tool in basic research due to their high specificity and affinity
`for target antigens. For the past two decades, therapeutic mAbs
`have had substantial effects on medical care for a wide range
`of diseases, including inflammatory diseases and cancers. A
`critical feature of mAbs is their high specificity and their ability
`to bind target antigens, marking them for removal by methods
`such as complement-dependent cytotoxicity (CDC) or antibody-
`dependent cell-mediated cytotoxicity (ADCC).1 Antibodies
`can also impart therapeutic benefit by binding and inhibiting
`
`*Correspondence to: Jagath R Junutula; Email: jagath@gene.com
`Submitted: 09/20/2013; Revised: 10/30/2013; Accepted: 10/31/2013
`http://dx.doi.org/10.4161/mabs.27022
`
`the function of target antigens, as in the case of trastuzumab
`(Herceptin®), bevacizumab
`(Avastin®),
`and
`cetuximab
`(Erbitux®).2 However, antibodies against tumor-specific antigens
`often lack therapeutic activity.3
`Conjugation to cytotoxic drugs or radionuclides can expand
`the utility of mAbs and improve their potency and effectiveness;
`the antibodies are thus used as a means to target and delivery
`a toxic payload to the selected diseased tissue. This approach is
`currently a major focus of therapeutic research. Antibodies have
`been conjugated to a number of cytotoxic drugs, though various
`linker chemistries and these antibody drug conjugates (ADCs)
`have the ability to selectively and potently kill antigen–expressing
`tumor cells in vitro and in xenograft studies.4-6 ADCs have
`demonstrated success in the clinic, and there are now two such
`drugs, ado-trastuzumab emtansine (Kadcyla®) and brentuximab
`vedotin (Adcetris®), marketed in the United States. With over 30
`ADCs currently undergoing clinical studies, it is likely that more
`conjugates will be approved in the future.
`ADC development has been an iterative learning process, with
`ADCs evolving from murine antibodies that were conjugated
`to standard chemotherapeutic drugs to fully human antibodies
`conjugated to highly potent cytotoxic drugs. Our understanding
`of ADCs has improved substantially over the past 10 years and
`we now understand many of the critical factors required for
`their successful development, including target antigen selection,
`antibody, linker, and payload. One area of research that has
`seen recent advancement is that of conjugation chemistry. The
`implementation of site-specific conjugation, in which conjugation
`occurs only at engineered cysteine residues or unnatural amino
`acids for example, has resulted in homogeneous ADC production
`and improved ADC pharmacokinetic (PK) properties. This
`review will focus on current methods of site-specific conjugation,
`as well as the history and our present understanding of ADCs.
`
`Antibody-Drug Conjugates
`
`The history of ADCs
`Historically, the use of drugs for the treatment of cancer
`has centered on chemotherapies that target rapidly dividing
`cancer cells. These chemotherapy drugs included the folate and
`purine analogs (methotrexate, 6-mercaptopurine), microtubule
`polymerization inhibitors/promoters (vinca alkaloids, taxanes)
`and DNA damaging agents (anthracyclines, nitrogen mustard).7
`These compounds target cancer cells but also other dividing cells
`in the body, and patients receiving treatment experience severe side
`
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`effects that greatly limit the administrable dose. The therapeutic
`index (maximum tolerated dose/minimum efficacious dose) for
`these drugs is small, resulting in a narrow therapeutic window
`(Fig. 1). To circumvent this obstacle in drug development and
`improve therapeutic index, researchers turned to ADCs. The
`promise of ADCs was that they could selectively deliver toxic
`compounds to diseased tissue, a concept first described by Paul
`Ehrlich as “Magic Bullets” in the early 1900s.8
`ADC development, however, was not straightforward and
`those studied in the 1980s and early 1990s faced a number of
`challenges. Several early attempts at ADC development included
`the KS1/4 antibody-methotrexate conjugate for non-small cell
`lung cancer and the BR96 antibody-doxorubicin conjugate for
`metastatic breast cancer.9,10 Both drugs were evaluated in the
`clinic, but despite localizing to tumors, the conjugates showed
`little or no therapeutic benefit.11,12 Poor target antigen selection
`was likely a primary reason for the failure of these early conjugates.
`The antigens targeted by KS1 and BR96 were initially selected
`because their expression was associated with cancer cells, but
`both antigens were also expressed in normal tissues, resulting
`in toxicity.11,13 Other factors that limited the success of these
`conjugates were the use of either chimeric or murine antibodies,
`which can elicit an immunogenic response, and the use of lower
`potency drugs.
`Wyeth and Celltech improved on these early ADCs with
`the development of gemtuzumab ozogamicin (Mylotarg®), an
`anti-CD33 conjugate for the treatment of acute myeloid leukemia
`(AML). Gemtuzumab ozogamicin
`incorporated a highly
`potent calicheamicin derivative to help improve efficacy and a
`humanized antibody to limit immunogenicity,14 but the mAb-
`drug linker was unstable and released 50% of bound drug in
`48 h. Although gemtuzumab ozogamicin demonstrated promising
`activity in the clinic and was granted accelerated approved by the
`US Food and Drug Administration (FDA) in 2000, the drug was
`later withdrawn from the market after subsequent clinical data
`raised concerns about safety and clinical benefit when combined
`with the frontline standard of care.15,16
`Lessons learned from the initial ADC programs mentioned
`above were incorporated into the development and design
`of second-generation ADCs, and two of these, brentuximab
`vedotin and ado-trastuzumab emtansine, showed impressive
`clinical efficacy and safety, and were recently approved by the
`FDA. Brentuximab vedotin, developed by Seattle Genetics
`in partnership with Millennium/Takeda for the treatment
`of anaplastic large cell lymphoma and Hodgkin lymphoma,
`chemically couples an anti-CD30 chimeric antibody with
`the highly potent antimitotic agent, monomethyl auristatin E
`(MMAE) through a protease cleavable linker.17 Ado-trastuzumab
`emtansine, developed by Genentech with ImmunoGen’s ADC
`linker-drug technology, targets human epidermal growth
`factor receptor 2 (Her2)-positive breast cancer and combines
`an anti-Her2 antibody (trastuzumab) with the cytotoxic agent
`maytansine (DM1) via a stable linker.18 Knowledge gained from
`the development of these and other ADCs has led to a better
`understanding of the ways in which ADCs function and their
`clinical performance.
`
`Figure 1. ADCs expand the therapeutic window. ADC therapeutics can
`increase efficacy and decrease toxicity in comparison to traditional che-
`motherapeutic cancer treatments. Select delivery of drugs to cancer
`cells increases the percent of dosed drug reaching the tumor, thus low-
`ering the minimum effective dose (MeD). The maximum tolerated dose
`(MTD) is increased, as less drug reaches normal, non-target tissue due to
`targeted delivery by the antibody. Taken together, the therapeutic win-
`dow is improved by the use of ADCs.
`
`ADC Function and Mechanism of Action
`
`ADCs are designed to kill cancer cells in a target-dependent
`manner and the first step in this process is binding of the antibody
`to its antigen. The tumor antigen must be localized to the cell-
`surface so it can be accessed by a circulating antibody. Upon
`ADC binding, the entire antigen-ADC complex is internalized
`through receptor-mediated endocytosis (Fig. 2). This process
`generally occurs when a ligand binds a cell-surface receptor and
`initiates a cascade of events, including recruitment of adaptins
`and clathrin, inward budding of the plasma membrane, formation
`of early endosomes, and lastly trafficking to late endosomes and
`lysosomes.19 Once inside lysosomes, ADCs are degraded and free
`cytotoxic drug is released into the cell, resulting in cell death. The
`mechanism of action of cell death can vary based on the class of
`cytotoxic drug used (e.g., disruption of cytokinesis by tubulin
`polymerization inhibitors such as maytansines and auristatins,
`DNA damage by DNA interacting agents such as calcheamicins
`and duocarmycins).20 Neighboring cancer cells may also be killed
`when free drug is released into the tumor environment by the dying
`cell in a process known as the bystander effect.21 For ADCs to
`work, a threshold level of free toxic drug must be reached inside and
`around tumor cells. Factors that influence whether this threshold is
`met, and thus determine the success of an ADC, include the target
`tumor antigen, antibody, linker and cytotoxic drug (Fig. 3).
`
`Anatomy of ADCs
`
`Importance of the tumor antigen
`As mentioned earlier, the ideal tumor antigen must be localized
`to the cell-surface to allow ADC binding. Preferably the antigen
`also displays differential expression between tumor and normal
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`if the antibody selected does not contain several
`crucial attributes. High specificity of the
`antibody for the tumor antigen is essential. An
`antibody that cross-reacts to other antigens or
`displays general non-specific binding can be
`taken up in normal tissues unpredictably and
`in high amounts, resulting in both toxicity and
`removal/elimination of the ADC before it can
`reach the tumor.5,11,13 The antibody must also
`bind the target antigen with high affinity (Kd
`< 10 nM) for efficient uptake into target cells
`and it should be minimally immunogenic. An
`immune response mounted against an ADC,
`such as human anti-mouse antibodies (HAMA)
`against a murine ADC, can prevent repeat
`cycles of therapy.23 It is also important to select
`an antibody with optimal PK properties (longer
`half-life with slower clearance in plasma).24
`Lastly, it should be noted that unknown factors
`related to the antibody appear to contribute to
`ADC activity, as demonstrated in a study where
`only two of seven antibody conjugates that bind
`CD22 were effective in vivo, a dramatic result
`not likely due to PK properties alone.25
`Linker selection and intracellular drug
`release
`tumor antigen
`step after
`The next
`identification and antibody development is
`selection of a suitable linker/cytotoxic drug.
`As might be expected, the drug plays a major
`role in ADC activity and characteristics.
`What might be less intuitive is that the linker
`between the antibody and drug also is very
`important. An ideal linker should be stable in
`circulating blood, but allow rapid release of
`active free drug inside tumor cells. If a linker
`is not stable in blood, drug will be lost and ADC activity will
`be decreased.15,26
`Current linker formats that are being evaluated can be
`broadly categorized into two groups: cleavable linkers (acid-
`labile linkers, protease cleavable linkers, and disulfide linkers)
`and non-cleavable linkers. Acid-labile linkers are designed to be
`stable at pH levels encountered in the blood, but become unstable
`and degrade when the low pH environment in lysosomes is
`encountered (e.g., gemtuzumab ozogamicin). Protease-cleavable
`linkers are also designed to be stable in blood/plasma, but rapidly
`release free drug inside lysosomes in cancer cells upon cleavage
`by lysosomal enzymes. They take advantage of the high levels of
`protease activity inside lysosomes and include a peptide sequence
`that is recognized and cleaved by these proteases, as occurs with a
`dipeptide Val-Cit linkage that is rapidly hydrolyzed by cathepsins
`(e.g., brentuximab vedotin).
`A third type of linker under consideration contains a disulfide
`linkage. This linker exploits the high level of intracellular
`reduced glutathione to release free drug inside the cell (e.g.,
`the anti-CD56-maytansine conjugate IMGN-901). Linkers in
`
`Figure  2. Delivery of cytotoxic drugs to cancer cells by ADCs. The monoclonal antibody
`component of an ADC selectively binds a cell-surface tumor antigen, resulting in internaliza-
`tion of the ADC-antigen complex through the process of receptor-mediated endocytosis.
`The ADC-antigen complex then traffics to lysosomal compartments and is degraded, releas-
`ing active cytotoxic drug inside the cell. Free drug causes cell death through either tubulin
`polymerization inhibition or DNA binding/damage depending on the drugs mechanism of
`action.
`
`tissue, with increased expression in cancer cells. Expression of an
`antigen in normal tissue could enhance uptake of conjugate by
`the tissue, resulting in toxicity and lowering the dose of conjugate
`available to the tumor. Another important characteristic of the
`tumor antigen is ability to internalize upon ADC binding. The
`internalization of an ADC-antigen complex through receptor-
`mediated endocytosis, followed by ADC degradation in the
`lysosome, results in optimal free drug release and effective cell
`killing. That endocytosis will occur is not guaranteed for all
`cell-surface antigens, and the rate of internalization can vary
`from rapid to zero. Minimal ADC recycling to the cell surface
`and enhanced delivery of an internalized antigen/ADC to the
`lysosome also needs to occur for the maximal release of toxic
`free drug into the cell. Therefore, the ideal tumor antigen should
`be cell-surface expressed, highly upregulated in cancer tissue,
`internalized upon ADC binding, and able to release the cytotoxic
`agent inside the cell.22
`Antibody specificity, affinity, and pharmacokinetics
`Another critical factor that influences ADC success is the
`antibody itself. Even the perfect tumor antigen cannot be targeted
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`the non-cleavable category provide
`high stability in the blood, but are
`solely dependent on internalization,
`lysosomal delivery, and degradation
`of the ADC complex to release active
`drug and kill cancer cells (e.g., ado-
`trastuzumab emtansine). They may
`not release drug in extracellular
`space and are incapable of killing
`neighboring tumor cells through
`the by-stander effect.27 Furthermore,
`optimal linker selection depends on
`the target antigen that is chosen. It
`was demonstrated that ADCs with
`cleavable linkers against seven B cell
`targets (CD19, CD20, CD21, CD22,
`CD79b, and CD180) showed in vivo
`efficacy. In contrast, only target
`antigens that were internalized and
`efficiently trafficked to lysosomes
`(CD22 and CD79b) displayed in
`vivo efficacy with non-cleavable
`linkers.28 The specificity of free drug
`release in cells is a main goal of all
`of the linkers, and it is important for
`controlling the toxicity of the highly
`potent drugs used to construct ADCs.
`However, the balancing act between
`efficacy and toxicity varies for the
`above-mentioned linkers and linker
`selection will ultimately depend
`on experimentally determining the
`optimal combination of the correct
`linker, the target antigen and desired
`payload.
`Cytotoxic drugs
`The success of an ADC also depends on the use of an optimal
`drug. The percent of an injected antibody that localizes to a
`solid tumor is very small (0.003–0.08% injected dose per gram
`of tumor); therefore, toxic compounds with sub-nanomolar
`potency are desirable.29 In addition, drugs must contain a suitable
`functional group for conjugation and need to be stable under
`physiological conditions. The drugs currently being used to
`construct ADCs generally fall into two categories: microtubule
`inhibitors and DNA-damaging agents. It should be noted that
`other drugs such as the polymerase II inhibitor, α-amanitin, are
`also under investigation.30
`Microtubule inhibitors bind tubulin, destabilize microtubules,
`and cause G2/M phase cell cycle arrest. Auristatins and
`maytansinoids are two classes of microtubule inhibitors currently
`used in ADC development. MMAE is a highly potent auristatin
`(free drug IC50: 10-11-10-9 M) developed by Seattle Genetics
`and used in brentuximab vedotin, and DM1 is a highly potent
`maytansinoid (free drug IC50: 10-11–10-9 M) developed by
`ImmunoGen and used in ado-trastuzumab emtansine.23,31-34
`
`Figure 3. Critical factors that influence ADC therapeutics. ADCs consist of a cytotoxic drug conjugated to
`a monoclonal antibody by means of a select linker. These components all affect ADC performance and
`their optimization is essential for development of successful conjugates.
`
`DNA-damaging agents include anthracyclines, calicheamicins,
`duocarmycins, and pyrrolobenzodiazepines (PBDs). All of these
`drugs function by binding the minor groove of DNA and causing
`DNA stand scission, alkylation, or cross-linking. The cytotoxins
`are highly potent, with free drug IC50 of <10-9 M, and ADCs
`that incorporate these agents have been explored in the clinic,
`including inotuzumab ozogamicin, an anti-CD22-calicheamicin
`conjugate developed by Pfizer, and MDX-1203, an anti-CD70-
`duocarmycin developed by Bristol-Myers Squib.14,20,35-38
`The evolution of ADCs from BR96-doxorubicin and KS1/4-
`methotrexate to the currently marketed brentuximab vedotin and
`ado-trastuzumab emtansine exemplifies the substantial efforts
`and innovation of many scientists in the ADC field, and required
`optimization of all components of ADCs, including antibodies,
`linkers, and payloads. Successful ADC development depends on
`optimization of the delicate balance between efficacy and toxicity
`(target dependent and independent). (Fig. 4). However, the work
`is far from over, and further development may be essential to
`the success of many future ADC products. One area of current
`research that will help us take the next step in ADC evolution is
`site-specific conjugation.
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`in ADC production
`consistency
`can be challenging and may require
`diligent manufacturing capabilities.
`Site-specific conjugation, in which
`a known number of linker-drugs are
`consistently conjugated to defined
`sites, is one way to overcome these
`challenges.43,44 Heterogeneity
`is
`minimized and ADC properties are
`more predictable, with consistent
`conjugate production from batch
`to batch. Drug-to-antibody ratio
`(DAR) is precisely controlled and
`can be tailored to various linker-
`drugs, producing either 2- or
`4-DAR site-specific ADCs (Table
`1). Thus, site-specific conjugation is
`a major improvement to ADC drug
`development and it is no surprise
`that researchers have focused on a
`number of methods to achieve site-
`specific conjugation.
`Site-specific conjugation through
`engineered cysteine residues
`The amino acid cysteine contains
`a reactive thiol group that serves
`essential
`roles
`in
`the
`structure
`and function of many proteins.
`Conjugation of thio-reactive probes
`to proteins through cysteine residues
`has long been a method for protein
`labeling, and
`it has also been
`applied to the generation of ADCs.
`As described above, this process
`involves partial reduction of inter-
`chain disulfide bonds and results in
`a heterogeneous mixture of ADCs
`that differ with respect to site of conjugation, number of drugs
`per antibody, and number of intact inter-chain disulfide bonds.42
`To avoid the problem of heterogeneity and to maintain
`disulfide bonds, cysteine residues can be engineered into proteins,
`but there are still many challenges to this approach. Engineered
`free cysteine residues on the surface of a protein can pair with
`cysteines on other molecules to form protein dimers.45 It is also
`possible that introduced cysteines can pair intra-molecularly
`with native cysteine residues to create improper disulfide bonds,
`resulting in disulfide bond shuffling and possibly protein
`inactivation.46 The success of using introduced cysteine residues
`for site-specific conjugation relies on the ability to select proper
`sites in which cysteine-substitution does not alter protein
`structure or function. To accomplish this, the Phage Elisa for
`Selection of Reactive Thiols (PHESELECTOR) was developed
`by introducing reactive cysteine residues into an antibody-Fab
`(trastuzumab-Fab 4D5) at various sites, displaying the Fab on
`phage, and screening to identify reactive cysteines that do not
`interfere with antigen binding.47
`
`Figure 4. ADC metabolism in vivo. The therapeutic window of an ADC depends on the optimization of
`the delicate balance between efficacy and toxicity. The desired effect of ADCs is the target-dependent
`killing of tumor cells expressing high levels of target antigen (A). Side effects can be caused by target-
`dependent toxicity and killing of normal cells expressing low levels of target antigen (B), or by target-
`independent toxicity caused by entry of free drug into normal cells (C). Free drug can be released by ADC
`catabolism or by unstable labile linkers in the plasma..
`
`Site-Specific Conjugation
`
`Conventional ADC conjugation processes
`Traditionally, conjugation of linker-drugs to an antibody
`takes place at solvent accessible reactive amino acids such as
`lysines or cysteines derived from the reduction of inter-chain
`disulfide bonds in the antibody. Lysine conjugation results in
`0–8 conjugated molecules per antibody (Fig. 5), and peptide
`mapping has determined that conjugation occurs on both the
`heavy and light chain at ~20 different lysine residues (40 lysines
`per mAb). Therefore, greater than one million different ADC
`species can be generated.6,39-41 Cysteine conjugation occurs after
`reduction of four inter-chain disulfide bonds, and the conjugation
`is thus limited to the eight exposed sulfhydryl groups. Linker-
`drugs per antibody can range from 0–8 (Fig. 5), generating
`more than one hundred different ADC species.42 The diversity
`in heterogeneity of an ADC mixture is 2-fold because these
`ADC species differ in drug load and conjugation site. Therefore,
`each species may have distinct properties, which may result in a
`wide range of in vivo PK properties. In addition, batch-to-batch
`
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`Figure 5. Conjugation methods for ADC development. ADC production using traditional conjugation through lysine residues or reduction of inter-chain
`disulfide bonds results in high heterogeneity in both drug to antibody ratio (DAr) and location of conjugation site. Site-specific conjugation greatly
`decreased this heterogeneity. (A) Lysine conjugation results in a DAr of 0–8 and potential conjugation at ~40 lysine residues/mAb. (B) Conjugation
`through reduced inter-chain disulfide bonds results in a DAr of 0–8 and potential conjugation at eight cysteine residues per mAb. (C) Site-specific con-
`jugation utilizing two engineered cysteine residues results in a DAr of 0–2 and conjugation at two sites/mAb. DAr can be doubled by engineering four
`sites if desired. Data displayed in graphs were re-plotted from previous publications.40,41,43
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`Amber
`stop codon
`substitution
`
`Cell line
`expressing
`orthogonal
`trNA/aarS
`
`Unnatural
`amino acids
`
`Addition of Sec
`insertion sequence
`
`None
`
`Sodium Selenite
`
`(Glutamine Tag,
`Glycoengineering, FGE)
`
`Addition of glutamine tag
`or aldehyde tag, none
`for glycoengineering
`or for pre-existing
`glutamine tag (Gln-295)
`Cell line overexpressing
`Formylglycine Generating
`enzyme (FGe) for FGe
`method, none for
`other methods
`None
`
`Antibody engineering
`required
`
`Cysteine substitution
`
`None
`
`None
`
`Cell line engineering
`required
`
`Additional reagents
`required at time of
`antibody expression
`
`Enzymes required
`for conjugation
`
`Table 1. Methods used for generation of site-specific ADCs
`Engineered
`Cysteine Residues
`
`Unnatural
`Amino Acids
`
`Selenocysteine
`
`Enzymatic Conjugation
`
`None
`
`None
`
`None
`
`Glycotransferase,
`Transglutaminase,
`
`Conjugation
`site location
`
`Any location
`
`Any location
`
`C-terminus (other
`locations unknown)
`
`Drug-to antibody
`ratio (DAR)
`
`2 or 4
`
`2 or 4
`
`2
`
`Conjugation chemistry
`
`Maleimide,
`Bromoacetamide
`
`Oxime, Click
`chemistry
`
`Maleimide
`
`Institutions exploring
`site-specific antibody
`conjugation methods
`
`Genentech,
`MedImmune,
`Seattle Genetics
`
`Allozyne,
`Ambrx, Sutro
`
`National Cancer
`Institute
`
`Asn-297 for
`glycoengineering,
`Pre-existing glutamine tag
`(Gln-295) or any location
`for other methods
`
`2 for glycoengineering, 2 or
`4 for glutamine tag and FGe
`
`Click chemistry,
`transamidation, hydrozino-
`Pictet-Spengler chemistry
`
`Innate Pharma, Glycos,
`Pfizer, redwood
`Bioscience, SynAffix
`
`To determine the generality and validity of this approach
`with full-length mAbs, conjugation of a cytotoxic drug to an
`anti-MUC16 mAb was investigated.43 Based on PHESELEC-
`TOR assay results, heavy chain alanine 114 (Kabat numbering)
`was selected as an optimal site for cysteine substitution. Unlike
`conventional cysteine conjugation, in which drug was conjugated
`to both the heavy and light chain of the antibody, conjugation
`using the engineered cysteine site occurred only on the heavy
`chain at engineered cysteine residues, with greater than 92% of
`the engineered thio antibody (THIOMAB) conjugates contain-
`ing two drugs (Fig. 5). Based on these results, conjugation to
`the engineered cysteine was both efficient and specific, especially
`compared with conventional cysteine conjugation. Importantly,
`substitution of cysteine at this position did not alter antigen bind-
`ing of the HC-A114C anti-MUC16 THIOMAB compared with
`the original anti-MUC16 antibody. These results are significant
`because they demonstrate that the optimal sites for cysteine
`
`conjugation found using an anti-HER2 Fab and the PHESE-
`LECTOR method can also be applied to full-length antibodies,
`and data now suggest that these sites work well for site-specific
`conjugation to other mAbs (trastuzumab THIOMAB, anti-
`CD22 THIOMAB, anti-Steap1 THIOMAB and anti-TenB2
`THIOMAB).48-50
`The
`importance of site-specific conjugation was next
`highlighted by comparing the therapeutic windows of traditional
`anti-MUC16 drug conjugates (ADCs) and HC-A114C engineered
`anti-MUC16 THIOMAB drug conjugates (TDCs). Efficacies of
`the two conjugates were compared and despite having a decreased
`drug load (~2 drugs per TDC vs ~3.5 drugs per ADC), the site-
`specific THIOMAB conjugates were as active and efficacious
`in both in vitro and in vivo studies, thus providing equivalent
`efficacy at half the drug dose. Interestingly, engineered site-
`specific TDC conjugates were also better tolerated in both rat and
`cynomolgus monkey toxicity models compared with traditional
`
`40
`
`mAbs
`
`Volume 6 Issue 1
`
`Downloaded by [208.184.134.166] at 07:08 01 December 2014
`
`IMMUNOGEN 2138, pg. 8
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`ADC conjugates.40,43 Animals
`administered
`anti-MUC16
`site-specific TDCs displayed
`reduced liver and bone marrow
`toxicity
`compared
`with
`conventional ADCs. Taken
`together, the above results
`demonstrate that site-specific
`TDCs displayed equivalent
`efficacy and greater safety
`than conventional ADCs and
`therefore have an improved
`therapeutic window, further
`highlighting the benefits of
`site-specific conjugation.43
`Unnatural amino acids
`and selenocysteine
`A second strategy for site-
`specific conjugation centers on
`the insertion of amino acids
`with bio-orthogonal reactive
`handles such as the twenty-first
`amino acid,
`selenocysteine,
`