Antibodies and associates: Partners in targeted drug delivery

    Antibodies and associates: Partners in targeted drug delivery Patrick J. Kennedy, Carla Oliveira, Pedro L. Granja, Bruno Sarmento PII: DOI: Reference:

S0163-7258(17)30084-0 doi:10.1016/j.pharmthera.2017.03.004 JPT 7055

To appear in:

Pharmacology and Therapeutics

Please cite this article as: Kennedy, P.J., Oliveira, C., Granja, P.L. & Sarmento, B., Antibodies and associates: Partners in targeted drug delivery, Pharmacology and Therapeutics (2017), doi:10.1016/j.pharmthera.2017.03.004

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ACCEPTED MANUSCRIPT P&T 23179

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Antibodies and associates: Partners in targeted drug delivery

i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 208,

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a

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Patrick J. Kennedya,b,c,d, Carla Oliveiraa,c, Pedro L. Granjaa,b,d,e, Bruno Sarmentoa,b,f *

b

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4200-393 Porto, Portugal

INEB – Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200-393

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Porto, Portugal

Ipatimup – Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Rua Alfredo Allen

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208, 4200-393 Porto, Portugal

ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo

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Ferreira 228, 4050-313 Porto, Portugal

FEUP – Faculdade de Engenharia da Universidade do Porto, Departmento de Engenharia Metalúrgica e

de Materiais, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal f

CESPU – Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde &

Instituto Universitário de Ciências da Saúde, Rua Central de Gandra, 1317, 4585-116 Gandra, Portugal ⁎

Corresponding author at: i3S – Instituto de Investigação e Inovação em Saúde, Rua Alfredo Allen, 208,

4200-135 Porto, Portugal. Tel.: +351 220 408 800. E-mail address: [email protected] (B. Sarmento). Tel: +351 226074949 | Fax: +351 226094567

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Abstract

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Monoclonal antibodies (mAbs) are well established in the clinic due to their specificity and

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affinity to a diverse array of biochemical targets. More recently, mAbs are being exploited as targeting agents in modern drug delivery systems, aiming to bypass normal host tissue and to accumulate a

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therapeutic agent to a specific tissue or cell for enhanced pharmacology. At sizes ranging from ~10-100 nm, antibody-based bioconjugates have opened up a whole new realm of clinical possibilities with several

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platforms emerging on the market. Antibody-drug conjugates combine the killing power of cytotoxic agents with mAb specificity and have great potential to treat cancer and beyond. Partnering a mAb with a

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biologic (protein/peptide, oligonucleotide (ON) or another mAb) is also gaining clinical traction. For

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example, many bispecific mAbs target and recruit immune effector cells to a tumor, while ON-based therapeutics against intracellular (regulatory) RNAs may be safely delivered into specific cells with mAb

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support. Finally, nanoparticles (NPs) offer significant drug delivery advantages including controlled release, large and diverse payloads, intracellular delivery and multi-functionality. Coupling mAbs to the surface of NPs can add further targeting capacity, and yet, therapeutic mAbs can also be encapsulated to take advantage of the above NP qualities. Here, we present an updated overview of the different aspects required for the successful development and engineering of antibody bioconjugates in current and emerging drug delivery technologies.

Keywords

Monoclonal antibody, antibody-drug conjugate, bispecific antibody, oligonucleotides, nanoparticle, intracellular antibody

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Table of contents

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1. Introduction

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2. Antibodies in nature and biotechnology

4. Antibody-biologic conjugates (ABCs)

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5. Antibody-nanoparticle conjugates (ANCs) 6. Conclusions and future directions Conflict of interest

Abbreviations

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References

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Acknowledgements

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3. Antibody-drug conjugates (ADCs)

ABC, antibody-biologic conjugate; ADC, antibody-drug conjugate; ADCC, antibody-dependent cellmediated cytotoxicity; ALL, acute lymphoblastic leukemia; ANC, antibody-nanoparticle conjugate; BBB, blood brain barrier; BiTE, bispecific T cell engager; CDC, complement-dependent cytotoxicity; CDR, complementary determining region; CDR H3, third CDR from the VH; CH, constant heavy chain; CL, constant light chain; DAR, drug-to-antibody ratio; DART®, dual-affinity re-targeting; dox, doxorubicin; DDS, drug delivery system; DM1, mertansine/emtansine; DM4, ravtansine/soravtansine; EGFR, epithelial growth factor; EpCAM, epithelial cell adhesion molecule; Fab, antigen-binding fragment; FAE, Fab arm exchange; Fc, crystallizable fragment; Fcab™, Fc domain with antigen binding; FcR, Fc receptor; FcRn, neonatal Fc receptor; HER2, human epidermal growth factor receptor 2; HIV, human immunodeficiency virus; Ig, immunoglobulin; IgG, immunoglobulin G; KiH, knobs-into-hole; mAb,

ACCEPTED MANUSCRIPT monoclonal antibody; MDR, multi-drug resistance; MMAE/F, monomethyl auristatin E/F; MS, mass spectrometry; ON, oligonucleotide; NP, nanoparticle; PBD, pyrrolobenzodiazepines; PEG, polyethylene

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glycol; scFv, single-chain variable fragment; sdAb, single-domain antibody; siRNA, small interfering RNA; SMCC, N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate; TCL, T cell lymphocyte;

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Tg, transglutaminase; VH, variable heavy chain; VEGF, vascular endothelial growth factor; VHH,

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variable domain of heavy chain antibody; VL, variable light chain

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1. Introduction

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Current drug delivery systems (DDSs) are designed to release effective drug doses over extended periods of time to improve patient adherence and convenience (Bae & Park, 2011). To this end, various

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DDSs administered by parenteral, mucosal (Sosnik et al., 2014) or transdermal routes have been

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developed. Yet, in most cases, the drug still acts systemically often leading to unwanted side effects. Reducing these side effects, and thus increasing the therapeutic index, can be achieved by avoiding healthy host tissues and accumulating the drug at the diseased site. Some modern DDSs actively target

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specific tissues/cells by exploiting defined biochemical interactions (e.g. ligand-receptor binding).

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Antibodies are nature’s molecular recognition machines designed to aid the immune system in

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destroying extracellular, as well as intracellular (Mallery et al., 2010), threats. Their affinity and specificity invokes Ehrlich’s “magic bullet” dream of targeting a select pathogen for therapeutic action while minimizing collateral damage to the host. Amazingly, almost anything can illicit an antibody response, and antibodies, as the first affinity reagent, have been heavily exploited by biotechnology in research, diagnostics and especially therapy. A monoclonal antibody (mAb) is a unique species of antibody with affinity to only one specific part of an antigen. mAbs in targeted therapy represent the fastest growing class of new medicines in the treatment of a wide range of human diseases (Rodgers & Chou, 2016). Early attempts at antibody therapy were problematic due to the animal origin from where the antibody was developed. Though, recombinant protein technologies are providing a wonderful alternative to animal-derived antibody sources and allowing for fully human constructs (Bradbury et al., 2011). Recombinant mAbs can also be constructed from their associated fragments/domains and (re-)engineered with more desirable pharmacological qualities.

ACCEPTED MANUSCRIPT This review aims to highlight the many ways in which the different antibody domains as bioconjugates are being exploited by modern DDSs ranging from 10 to >100 nm. First, the natural

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antibody structure and function is presented and followed up with a very general overview of novel mAb development. We then focus on the three main DDSs in which mAbs are used as targeting agents:

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antibody-drug conjugates (ADCs), antibody-biologic conjugates (ABCs) and antibody-nanoparticle

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conjugates (ANCs). The advantages of encapsulating therapeutic mAbs within a nanocarrier are also presented. Throughout, we provide some representative example studies and special considerations to be

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kept in mind during design and development of novel antibody bioconjugates, as well as insights into

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future advancements.

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2.1. Antibody biology

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2. Antibodies in nature and biotechnology

To better understand how mAbs can be utilized in the targeted DDSs detailed below, it is first necessary to introduce basic antibody biology. The core antibody, or immunoglobulin (Ig), structure is composed of a few domains/regions with their own unique role in the adaptive immunity process (Janeway et al., 2001). Ig contains two heavy (H) chains and two light (L) chains that are further subdivided into the constant (CH/CL) and variable (VH/VL) domains, and the overall structure is stabilized by interspersed disulfide bridges (Fig. 1a). Two fragment antigen-binding (Fab) regions are connected by a flexible hinge to one fragment crystallizable (Fc) region. One Fab expresses six complementarity determining regions (CDRs), three from each of the VH/VL domains, which are responsible for direct physical interaction with the target antigen (i.e. immune-stimulating molecule) and dictate binding affinity. The third CDR from the heavy chain (CDR H3) is particularly important. The glycosylated Fc, entirely composed of constant domains, is responsible for communication with the immune system through binding to various Fc receptors (FcRs). Fc is also necessary for the long circulation times of IgG,

ACCEPTED MANUSCRIPT the most common Ig isotype, due to lysosomal degradation escape mediated by the neonatal Fc receptor (FcRn). FcRn also aids in the transfer of maternal IgG across the placental and intestinal barriers to the

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fetus and neonate, respectively (Martins et al., 2016; Roopenian & Akilesh, 2007). Antibodies identify and destroy the vast array of pathogens (e.g. viruses, bacteria and diseased

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host cells) in several ways. Outside the cell, they can simply disrupt cellular entry or recruit the help of

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FcR-expressing immune effector cells mainly through antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) (Rogers et al., 2014). Inside the cell, antibody-

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coated pathogens are recognized by tripartite motif-containing protein 21 (Mallery et al., 2010), a high

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affinity cytosolic FcR (James et al., 2007), which recruits the proteasome through auto-ubiquitination for pathogen degradation to prevent fatal infection (Vaysburd et al., 2013). This immune mechanism is termed antibody-dependent intracellular neutralization (Foss et al., 2015). The plethora of

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pathogens/antigens one can encounter necessitates an on-demand, diverse antibody repertoire (~1012 possible unique antibody sequences) made possible by V(D)J recombination of only three Ig genes

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(Tonegawa, 1983).

2.2. Antibody fragments

Targeted DDSs with mAbs relies only on the antigen-binding variable domains and can dispense with the Fc, as long as FcR-dependent effector function is not a consideration. Many antibody fragmentbased formats exist that mainly revolve around the VH/VL domains (Holliger & Hudson, 2005). The most common ones are the single-chain variable fragment (scFv), Fab and F(ab’)2 (Fig. 1b). Interestingly, camelids (Hamers-Casterman et al., 1993) and some sharks (Greenberg et al., 1995) express heavy-chain only antibodies, making it possible to isolate the VH domain as VHH and VNAR, respectively. It is also possible to isolate the VH or VL domains from normal antibodies, and all these formats are considered as single-domain antibodies (sdAbs) (Fig. 1b). Antibody fragments constitute the basis for the bispecific

ACCEPTED MANUSCRIPT mAbs detailed in section 4 but can readily be used in ADCs (section 3) and ANCs (section 5) as well (Holliger & Hudson, 2005).

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2.3. Therapeutic monoclonal antibody (mAb) discovery and engineering

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Clinical utility of antibodies arose with the advent of hybridoma technology allowing for true

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monoclonality (Köhler & Milstein, 1975). Yet, it was only when an antibody repertoire from the B cells of an individual could be cloned (Orlandi et al., 1989) recombinantly expressed (Ward et al., 1989) and

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displayed in large combinatorial libraries (Huse et al., 1989; Lerner, 2016) that mAb technology really

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exploded. Although restricted to antibody fragments (scFv or Fab), this format linked genotype with phenotype and paved the way for in vitro mAb discovery and engineering as well as fully human mAbs. High affinity binders are then identified through process of selection and screening of a combinatorial

(Hoogenboom, 2005).

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library, often from an immunized animal, to a pre-determined target (e.g. cell surface receptor)

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Over the decades, in vitro display technologies have diversified into several different formats (Bradbury et al., 2011). Phage display was the first (Huse et al., 1989) and remains to be the most widely used display technology (Zhao et al., 2016). Other example technologies are based on yeast (Boder et al., 2012), mammalian cells (Ernest & Maurice, 2014) and cell-free (Stech & Kubick, 2015). These technologies rely on binding to identify target-specific mAbs, and function is only determined later in development. Recently, a mammalian cell system based on autocrine signaling was developed to identify mAbs with a pre-defined functional phenotype (Zhang et al., 2012) such as conversion of acute myeloblastic leukemia cells into natural killer cells (Yea et al., 2015). Pharmacological improvements of mAbs are possible through several antibody engineering strategies. For example, the early therapeutic mAbs derived from mouse hybridomas often invoked the human anti-murine antibody (HAMA) response, which would lead to varying levels of immune reactions. Chimerization and humanization of these mAbs reduced this immunogenicity by substituting most or all of the mAb support structure and retaining only the original mouse-derived CDRs to maintain target

ACCEPTED MANUSCRIPT specificity and affinity. During the early discovery phases, lead candidates often require higher binding affinity (i.e. sub nM). Thus, in vitro affinity maturation strategies, which mimic the in vivo process of

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somatic hypermutation, aim to mutate the CDRs until higher affinity clones are identified. Only a couple strategies have been presented above, but several more exist (e.g. increased circulation times and

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resistance to aggregation) that are relevant for the targeted DDSs detailed henceforth (Beck et al., 2010).

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3. Antibody-drug conjugates (ADCs)

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Targeted therapy of cancer with mAbs holds tremendous promise in the specific elimination of tumor cells without the systemic toxicity associated with conventional chemotherapeutic agents (e.g. rituximab and B cell lymphomas). Unfortunately, only modest success has been achieved in patients with

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solid tumors, as the immunological mechanisms associated with tumor cell elimination have not proven to be as effective as expected (Lambert, 2013).

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Antibody-drug conjugates (ADCs) combine the targeting ability of an antibody with the cell killing power of a cytotoxic agent (Casi & Neri, 2012). Importantly, coupling a cytotoxic agent with an antibody drastically reduces its liver toxicity while simultaneously enhancing the potency of the mAb. Most ADCs are designed to specifically target the cancer cell. Nevertheless, targeting the tumor vasculature (Gerber et al., 2009), akin to anti-vascular endothelial growth factor (VEGF) therapy (e.g. bevacizumab) to eliminate the blood supply to the tumor, may prove to be more clinically valid (Perrino et al., 2014). The research, development and clinical initiation of ADCs have been dominated by the pharmaceutical industry almost exclusively in cancer therapy. At present, there are >40 ADCs in clinical trials, and three have been approved: gemtuzumab ozogamicin for acute myelogenous leukemia, brentuximab vedotin for Hodgkin and systemic anaplastic large cell lymphoma and trastuzumab emtansine for human epidermal growth factor receptor 2 (HER2)+ metastatic breast cancer (Chudasama et al., 2016). Although gemtuzumab ozogamicin was withdrawn after 10 years in the market for safety

ACCEPTED MANUSCRIPT and efficacy reasons, brentuximab vedotin and trastuzumab emtansine are still clinically valid. These three ADCs are all quite different in their disease targets and the factors mentioned above. Brentuximab

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vedotin and, especially, trastuzumab emtansine are often used as benchmark ADCs during technology development and serve as good examples to illustrate the concepts outlined here.

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The basic ADC is composed of the antibody vehicle, the cytotoxic drug (i.e. “warhead”), and the

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linker that couples the warhead to the antibody. Several factors contribute to the efficacy of an ADC: a) qualities of the antibody vehicle, b) warhead potency/mode of action, c) number of drug molecules per

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antibody (i.e. drug-to-antibody ratio (DAR)), d) properties of the linker that attaches the warhead to the

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antibody, and e) location of the drug on the antibody (Fig. 2). Each of these factors is explained in more detail below.

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3.1. Antibody vehicle

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The antibody vehicle used to shuttle the cytotoxic agent to the target/site of action is of course important to the efficacy of the ADC, especially to minimize the toxicity of the warhead passenger (Fig. 2a). Briefly, most antibody vehicles are of human origin to minimize immunogenicity caused by the HAMA effect and are full-length IgG for possible effector function and to maximize circulation times via FcRn. As the vast majority of current ADCs are for cancer treatment, the antibody vehicle often targets tumor-specific, cell surface receptors. Using the approved ADCs as examples, gemtuzumab ozogamicin and brentuximab vedotin are chimeric antibodies against CD33 and CD30, respectively, while trastuzumab emtansine is a humanized IgG against HER2. Most ADCs are expected to be internalized; however, the ADC may have a combined or synergistic therapeutic effect of the antibody (by ADCC/CDC or signaling disruption) and the warhead (Adair et al., 2012).

3.2. Payloads/warheads

ACCEPTED MANUSCRIPT The warhead (Fig. 2b) is also very important in ADC efficacy often requiring nano- to pico-molar potency. The drug+linker combination is considered a “prodrug” and is often referred to as the payload.

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The warhead mainly comes in two classes: microtubule inhibitors or DNA-damaging agents. Interestingly, some types of warheads can be freed upon cell lysis to act upon neighboring cells in what is

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called the “bystander effect” (Diamantis & Banerji, 2016). As explained below, the ADC concept is not

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3.2.1. Microtubule inhibitors: Auristatins and maytansines

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restricted to just small molecule drugs.

Among the microtubule inhibitors, auristatins and maytansines are the most widely used (Gerber et al., 2009). Auristatins induce G2/M phase cell cycle arrest by inhibiting tubulin polymerase (Diamantis

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& Banerji, 2016). Monomethyl auristatin E (MMAE) has been used in several ADCs, including brentuximab vedotin (Senter & Sievers, 2012; Younes et al., 2010). The similar MMAF was used with a

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mAb against B-cell maturation antigen in the treatment of multiple myeloma (Tai et al., 2014) and with an anti-CD133 mAb for cancers of the liver and stomach (Smith et al., 2008). Maytansines and their derivatives (maytansinoids) function by binding to tubulin to inhibit microtubule assembly (Lopus et al., 2010). Mertansine/emtansine (DM1) and ravtansine/soravtansine (DM4) only differ according to their linker. DM1 is the warhead of trastuzumab (i.e trastuzumab emtansine) (Lewis Phillips et al., 2008) and others, while DM4 is currently in clinical trials combined with an anti-CD19 with relapsed or refractory acute lymphoblastic leukemia (ALL) (Kantarjian et al., 2016) or anti-folate receptor α for epithelial ovarian cancer (Lutz, 2015). One downside of tubulin inhibitors is that they only act on tumor cells when in a mitotic state.

3.2.2. DNA damage agents: Calicheamicins, duocarmycins and pyrrolobenzodiazepines (PBDs)

ACCEPTED MANUSCRIPT DNA damage-inducing agents, the other common class of warheads, are advantageous since they are active throughout the entire cell cycle, freeing the need to target actively proliferating cells (Diamantis

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& Banerji, 2016). Calicheamicins cleave DNA as a result of the Bergman cyclization process and induce catastrophic transcriptional events (Watanabe et al., 2002). They can be found coupled with an anti-CD22

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(inotuzumab ozogamicin) to treat non-Hodgkin lymphoma (NHL) or ALL (Kantarjian et al., 2013) and in

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gemtuzumab ozogamicin (Ricart, 2011).

Duocarmycins are DNA minor groove alkylating agents, and although relatively new to ADCs

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and the clinic (Dokter et al., 2014), duocarmycins exhibit superior efficacy in vitro and in patient-derived

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xenograft models when conjugated to trastuzumab than DM1 (van der Lee et al., 2015), and they may be effective in multi-drug resistance (MDR), a known problem with ADC therapy (Barok et al., 2014). Pyrrolobenzodiazepines (PBDs) are an extremely potent (low to mid picomolar) class of

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sequence-selective DNA minor-groove binding crosslinking agents (Hartley, 2011). Jeffrey et al. conjugated synthetic PBD dimers to an anti-CD70 antibody in a site-specific manner to yield ADCs with

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high uniformity in drug-loading and nominal aggregation (Jeffrey et al., 2013). This ADC showed superior antitumor activity in CD70-positive renal cell carcinomaand NHL cell lines in vitro and in vivo (xenograft models). PBD dimers combined with an anti-CD33 antibody demonstrated better efficacy than gemtuzumab ozogamicin in in vitro and in vivo models and, importantly, maintained activity in MDR models of acute myeloid leukemia (Kung Sutherland et al., 2013).

3.2.3. RNA polymerase II inhibitor: α-amanitin

The cyclic octapeptide α-amanitin is an inhibitor of RNA polymerase II (Pol II) and thus transcription, which ultimately leads to cell apoptosis. Similar to PBDs, α-amanitin acts on dividing and non-dividing cells and causes no drug resistance. ADCs harboring α-amanitin offer tremendous potential in cancer therapy as shown in preclinical studies of α-amanitin combined with an anti-epithelial cell adhesion molecule (EpCAM) antibody (Moldenhauer et al., 2012).

ACCEPTED MANUSCRIPT Recently, Liu et al. demonstrated that loss of TP53, the gene encoding for the tumor suppressor p53, directly leads to loss of POLR2A, the major subunit of Pol II, and that POLR2Alow cells are more

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sensitive to α-amanitin (Y. Liu et al., 2015). Furthermore, mouse models of human colorectal cancer with hemizygous deletion of POLR2A treated with low doses of the anti-EpCAM-α-amanitin ADC had

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complete tumor regression. Because p53 mutation/deletion, and thus decreased levels of Pol II, occurs in

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most human tumors, these studies suggest that ADCs harboring α-amanitin could be highly efficacious in

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general cancer therapy.

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3.2.4. Alternative payloads: Radionuclides, fluorescent molecules, photosensitizers and antibiotics

Alternative payloads (i.e. non-small molecule drugs and non-biologics (section 4)) can also be

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conjugated to mAbs; however, these conjugates may not be classified as classical ADCs. Conjugates harboring a radionuclide can be used in both cancer therapy (Steiner & Neri, 2011) and in vivo imaging

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(Barbet et al., 2012). In diagnostics, antibody fragments lacking Fc are more appropriate because whole IgG has long circulation times. As an example, a recombinant human Fab (Nilvebrant et al., 2012), and later a bivalent Fab (Haylock et al., 2016), against human CD44v6 radiolabeled with

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displayed high and specific tumor uptake in a squamous cell carcinoma xenograft mouse model (Haylock et al., 2014). Similarly, fluorescent/bioluminescent molecules can be conjugated to antibodies for in vivo imaging. In fact, infrared fluorescent conjugates with multiple antibodies targeting different receptors (HER2 and EpCAM) were used to visualize a single tumor (Sun et al., 2012). Photodynamic therapy (PDT) utilizes light at a specific wavelength to excite a photosensitizer to produce localized reactive oxygen species resulting in cell death. Although PDT is primarily used to treat skin-related disorders, often incorporated within semisolid formulations (Gonzalez-Delgado et al., 2016), it can also be used to treat internal cancers (Pereira et al., 2015). Construction of these photoimmunoconjugates utilizes similar chemistries as classic ADCs. As this technology is limited by the light penetration into the tissue, it is therefore mainly applicable to skin cancer or in a surgical setting.

ACCEPTED MANUSCRIPT Lehar et al. demonstrated that intracellular Staphylococcus aureus is protected from antibiotics and able to spread infection (Lehar et al., 2015). Thus, an antibody-antibiotic conjugate (AAC), composed

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of an anti-S. aureus antibody and a cleavable antibiotic, was developed to overcome this hurdle. Upon macrophage escape, S. aureus was coated with the AAC and when opsonized into another cell, the

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antibiotic was activated upon cleavage by phagolysosomal proteases, resulting in intracellular bacterial

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elimination. This same strategy has also been speculated to work for tuberculosis (TB) (Ekins, 2014).

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3.3. Drug-to-antibody ratio (DAR)

As the payload is significantly smaller than the large antibody vehicle, it is possible to attach multiple payloads per antibody without disturbing its binding and function. Therefore, the drug-to-

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antibody ratio (DAR) helps dictate the potency of a single ADC (Fig. 2c). Low DARs reduce the potential ADC potency; however high DARs may disrupt ADC pharmacokinetics.

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Given the inherent heterogeneity associated with conjugating drugs to mAbs (see section 3.5), it is difficult to accurately control and quantify the DAR. There are several biochemical methods for calculating the DAR of an ADC. UV/VIS spectroscopic analysis of the drug or drug-linker is the simplest method (Wakankar et al., 2011). Mass spectrometry (MS) and liquid chromatography are also commonly used. Chen et al. developed a native nanoelectrospray MS method (Chen et al., 2013), while Debaene et al. used native MS and native ion mobility MS to characterize ADCs (Debaene et al., 2014). DARs can also be assessed by exploiting the hydrophobicity common to most warheads through hydrophobic interaction chromatography, as well as reversed phase high-performance liquid chromatography (Ouyang, 2013). Importantly, some of these methods are able to be used for ADCs in plasma/serum (Xu et al., 2013). In general, ADCs with DARs greater than four tend to aggregate resulting in suboptimal clearance rates (Vankemmelbeke & Durrant, 2016). Therefore, investigators aim for DARs of two to four

ACCEPTED MANUSCRIPT to balance potency and aggregation, and data from clinical trials suggests that DARs averaging three to

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four seem to be optimal (Teicher & Chari, 2011).

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3.4. Linker properties

Given that antibodies have long circulation times and free drugs are quite toxic, stability of the

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linker in circulation is crucial to maintain ADC efficacy and safety. On the other hand, the linker should

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not interfere with the stability or targeting capability of the mAb. The properties of the linker used to attach the warhead to the mAb vehicle dictate the cytotoxin release profile and, ultimately, the selectivity, pharmacokinetics, and therapeutic index of the ADC. Most early ADCs were unsuccessful because of the

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linker properties and location. The importance of the linker is now fully recognized, and the lessons learnt are guiding current development protocols (Flygare et al., 2013).

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The linker portion can be divided into distinct components: a) connection of the linker to the antibody, b) polarity of the linker, c) trigger for linker cleavage (cleavable vs. non-cleavable) and d) selfimmolative spacer for drug release (Fig. 2d). The connection to the mAb depends on where the linker/payload will be located on the mAb. This is usually accomplished with a lysine or cysteine residue; however, other strategies can be exploited (see section 3.5). Because of the hydrophobicity of the commonly used linkers and warheads, the polarity of the linker dictates the DAR, among others. Use of a polar linker helps to reduce aggregation and ultimately allows for an increased DAR. Importantly, drug resistance can also be minimized with polar linkers due to reduced P-glycoprotein-mediated drug efflux. For example, DM1 conjugated with a maleimidyl-based hydrophilic linker containing polyethylene glycol (PEG(4)Mal) was more potent at killing

MDR1-expressing

cells/xenografts

than

with

the

nonpolar

(maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (Kovtun et al., 2010).

linker

N-succinimidyl-4-

ACCEPTED MANUSCRIPT The majority of linkers are designed to release the warhead by an intracellular trigger mechanism mediated by hydrolysis, reduction or proteases (Flygare et al., 2013). However, non-cleavable, thioether

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linkers may also release the warhead, upon catabolic degradation within the lysosome, but require that warhead activity can tolerate additional functional groups that may remain. For example, a lysine residue

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from the SMCC-based linker remains on DM1 of trastuzumab emtansine upon intracellular processing

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(Erickson et al., 2006). Non-cleavable linkers are advantageous due to their high stability in circulation. Among the cleavable linkers, hydrazone linkers release their payload upon exposure to the low pH found

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within the endosomes and lysosomes (Flygare et al., 2013). Both gemtuzumab ozogamicin

and

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inotuzumab ozogamicin connect calicheamicins to the antibody via a hydrazone linker. Linkers based on disulfide bridges exploit the intracellular reducing environment, presumably catalyzed by glutathione, but not in the oxygen-rich bloodstream for warhead release. Calicheamicins are reliant on disulfide reduction

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for activity, and the maytansinoids DM1 and DM4 have been extensively used with these linkers. Both the hydrazone and disulfide bridge strategies rely on chemical differences between the extra- and

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intracellular environments; however, exploiting lysosomal proteases for warhead release has gained strong favor as of late. Here, dipeptide linkers composed of cathepsin b cleavage motifs valine-alanine or valine-citrulline are commonly used (Ricart, 2011). The self-immolative spacer is the final part of the linker that liberates the free warhead upon activation of the trigger mechanism (Flygare et al., 2013). Using brentuximab vedotin as an example, this ADC contains a valine-citrulline site and a para-aminobenzylcarbamate spacer for MMAE release upon proteasomal degradation (Senter & Sievers, 2012; Younes et al., 2010). In conclusion, the linker needs to strike a balance between retaining the payload while in circulation versus liberating the payload while in the cell. The many linker types and modalities combined with the myriad antibody vehicles and warheads available necessitate extensive empiricism when developing novel ADCs.

3.5. Drug location: Site-specific strategies

ACCEPTED MANUSCRIPT The location of the linker/payload on the antibody is proving to be crucial for the overall stability and therapeutic efficacy of an ADC. Conversely, the antibody site may also affect conjugation efficiency.

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The initial strategies focused on conjugation to the lysine residues (e.g. gemtuzumab ozogamicin and trastuzumab emtansine). Depending on the antibody vehicle, lysine abundance and location will vary,

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thus making it difficult to control the DAR and, of course, location. More importantly, this approach

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results in highly heterogeneous ADC mixtures and thus narrow therapeutic windows and severe pharmacokinetic ramifications (Chudasama et al., 2016), such as disruption of antigen and Fc binding

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(Acchione et al., 2012). Later strategies to reduce heterogeneity focused on exploiting the natural

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cysteines. Here, the disulfide bridges are reduced and the resulting sulfhydryl groups are used with maleimide-based linkers (e.g. brentuximab vedotin). ADCs based on IgG1 tend towards the cysteines between the light and heavy chains, whereas IgG2 favors the hinge region (Wiggins et al., 2015).

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Targeting these cysteines results in drastically reduced heterogeneity; however, disruption of these structural regions may lead to antibody instability. This instability was addressed by inserting

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pyridazinediones (PDs) into native disulfide bonds. The bridges, and thus structure, are maintained, and the resulting PDs bear ‘clickable’ handles that are able to carry out two orthogonal transformations to yield versatile, multifunctionalized adducts (Maruani et al., 2015). Site-specific conjugation is paramount to achieving fully homogenous ADCs and has been under intense investigation of late (Fig. 2e). Site-specificity in (re-)engineered mAbs is mainly focused on enzymatic conjugation or targeted insertion of cysteines or unnatural amino acids with a functional group that can be chemoselectively reacted (Chudasama et al., 2016). The ultimate aim of these strategies is to impact the structure of the mAb as little as possible to maintain target specificity while maximizing stability, dispersity and therapeutic efficacy, and of course these strategies do not have to be restricted to antibodies and small molecule drugs. Below, we present a few examples of each strategy, but more details can be found elsewhere (Akkapeddi et al., 2016; Sochaj et al., 2015).

3.5.1. Natural glycans

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Exploiting glycans naturally-present on antibodies, an engineered glycotransferase was used to

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enzymatically add a reactive keto-galactose to an anti-HER2 antibody for further conjugation to MMAF (Zhu et al., 2014). The resulting ADC had comparable affinity to that of trastuzumab, but targeted a

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different epitope and, thus, was effective against trastuzumab-resistant breast cancer cells.

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Transglutaminase (TG) can form a covalent bond between a glutamine side chain and a primary amine. Although they do display some promiscuity, TGs do not recognize glutamines within the constant region

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of glycosylated antibodies. Thus, microbial TG was used to enzymatically conjugate diverse compounds

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to pre-defined glutamine residues in model antibodies, and drug location was shown to have an impact on ADC stability and pharmacokinetics (Strop et al., 2013).

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3.5.2. Engineered cysteines

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Conjugating payloads to cysteines is the method of choice for ADC generation (Akkapeddi et al., 2016). Cysteine substitutions that did not alter antigen binding for the loading of MMAE within an antiMUC16 antibody were identified by phage display (Junutula et al., 2008). These THIOMABS demonstrated higher tolerability and improved therapeutic efficacy in vivo compared to more conventional coupling strategies. Subsequent studies further confirmed that different drug sites on the antibody with similar DARs yielded different stabilities and therapeutic activities in vitro and in vivo. More specifically, sites with high solvent accessibility lead to increased instability from the maleimide exchange process and, thus, higher liver toxicity due to free drug in serum. In contrast, sites with low accessibility and surrounded by positively-charged residues resulted in increased stability and efficacy (Shen et al., 2012).

3.5.3. Unnatural amino acids

ACCEPTED MANUSCRIPT Incorporation of unnatural amino acids (uAAs) within an antibody (Noren et al., 1989) has also proven to be an effective way to generate specific conjugation sites for use with unique orthogonal

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coupling strategies (Hallam et al., 2015). Axup et al. started by generating an E. coli-produced Fab and a mammalian cell-produced full-length IgG against human HER2 engineered to express p-

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acetylphenylalanine (pAcPhe) at defined antibody positions with high surface accessibility and

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conjugation efficiency (Axup et al., 2012). Auristatin was conjugated to the pAcPhe residues through a stable oxime linkage. The resulting ADCs demonstrated potent cytotoxicity to HER2+ cells and complete

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tumor regression in xenograft animal models. The mammalian platform was later improved with the

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development of Chinese hamster ovary ells that stably express uAA-containing antibodies with high titers (>1 g/L) (Tian et al., 2014). A cell-free system has also been developed that incorporates a uAA into an antibody (Zimmerman et al., 2014). As a proof-of-principle, para-azidomethyl-l-phenylalanine (pAMF)

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was used to conjugate an auristatin derivative to trastuzumab using strain-promoted azide-alkyne cycloaddition copper-free click chemistry with highly potent cell cytotoxicity. It should be noted though

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that uAAs may illicit a strong immune response (Grunewald et al., 2009) against the resulting ADC, and manufacturing may be problematic in cell-based, but possibly not in cell-free, production systems (Hallam et al., 2015).

3.5.4. Multiple warheads

Arming ADCs with two different types of warheads, to help overcome/minimize drug resistance, also requires site-directed conjugation. However, screening all the different combinations of drug locations and DARs for the most efficient/potent is logistically problematic. In this sense, a protein A/Lbased solid-phase (bead), site-specific conjugation and purification method in 96-well format was developed as a way to screen, in a high-throughput manner, multiple linker payloads on multiple sites of an antibody (Puthenveetil et al., 2016). This versatile platform is compatible with various conjugation functional handles such as maleimides, haloacetamides, copper-free click substrates and transglutaminase

ACCEPTED MANUSCRIPT substrates. Li et al. incorporated selenocysteine, the 21st natural amino acid, at the C-terminus of an scFv THIOMAB derivative of trastuzumab, already containing an engineered cysteine, to generate a THIO-

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SELENOMAB. Model molecules and linker technologies provided a proof-of-concept of the dual labeled ADCs. These two technologies have thus paved the way for ADCs with multiple drug types or fluorescent

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(theranostic) ADCs for in vivo tracking (Li et al., 2015).

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The different aspects that need to be addressed for successful ADC development have been previously presented. Importantly, regulatory approval of novel ADCs may be hastened by using

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established anti-cancer mAbs. ADCs may also provide a way to repurpose tumor-specific mAbs with

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lackluster therapeutic efficacy or, in the same vain, chemotherapeutic agents that are too toxic when administered alone. Lastly, ADCs may help to overcome resistance to chemotherapeutic drugs (Kovtun et al., 2010) or therapeutic mAbs, as seen in the EMILIA trial with trastuzumab emtansine and trastuzumab-

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resistant breast cancer patients with persistent HER2 overexpression (Verma et al., 2012). The technologies now exist to generate highly specific, highly potent and highly homogenous

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ADCs to ensure their future as therapeutic (and theranostic?) agents in the treatment of various cancers (Lambert, 2013) and beyond (Lehar et al., 2015). Though, it must be noted that the interactions of the drug-linker, serum stability and therapeutic index of ADCs in humans may not reflect those observed in pre-clinical rodent models (Vankemmelbeke & Durrant, 2016). In the future, ADC technology might utilize smaller antibody formats for improvements in tumor penetration, homogeneity or production or to harbor non-classical drugs (e.g. to modulate specific intracellular signaling pathways).

4. Antibody-biologic conjugates (ABCs)

Combining one antibody with one or more other separate biologics (e.g. other mAbs, proteins or nucleic acids) confers many clinical advantages. Although this combined therapy is still a viable option (Raju & Strohl, 2013), a considerable obstacle to the clinical implementation of this strategy resides in the high costs associated with regulatory approval, manufacturing and clinical studies of the individual

ACCEPTED MANUSCRIPT biologics (Kontermann, 2012b). Thus, engaging multiple targets (i.e. antigens or epitopes) with a single antibody-biologic conjugate (ABC) can overcome this hurdle. Depending on their function, these ABCs

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could be considered as ADCs; yet, often times, it is difficult to differentiate between who is the targeting ligand and who is driving the therapeutic mode of action (i.e. drug). This becomes especially confusing

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when one of the partners recruits the immune system. Aside from hitting multiple targets, another

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advantage to bispecificity is the possibility for increased avidity. For example, low antigen density on cells or low affinity of single binders together may generate more specificity and allow for higher dosing.

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Furthermore, as signaling pathways typically have redundancies and escape mechanisms, resistance to

generated by targeting multiple antigens.

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single target mAb therapy precludes long-term efficacy. Hence, a more robust clinical response may be

ABCs, similar to normal antibodies, function in two ways: direct action or redirection. Direct

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action ABCs bind to extracellular ligands and/or their receptors to block the ligand/receptor interaction and/or to (ant)agonize the receptor to alter signaling. ABCs that function by redirection either: a) engage

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the immune system for ADCC/CDC and/or to recruit and activate cytotoxic T cell lymphocytes (TCLs), b) behave like an ADC by harboring an immunotoxin, cytokine, or prodrug-converting enzyme, or c) target a drug-loaded nanoparticle (expanded in section 5.1) (Kontermann, 2012b).

4.1. Other antibodies

Many “flavors” of bispecific mAbs have been developed over the years with much of the focus on recruitment of TCLs for immunotherapy of hematological or solid tumors (Kontermann, 2012b). Here, a few selected formats, as whole IgG or as fragments, are highlighted that help to illustrate the power of bispecifics and their clinical uses and potentials (Fig. 3).

4.1.1. Whole IgG bispecifics

ACCEPTED MANUSCRIPT Quadromas were the first generation bispecific mAbs (full IgG; Fig. 3a) and are produced through fusion of two hybridomas (Staerz & Bevan, 1986). Catumaxomab is a rat/murine quadroma used

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to treat malignant ascites. One arm targets CD3 and the other targets EpCAM while the Fc region binds to FcRs. This “trifunctional” TriomAb® aims to recruit TCLs (via CD3) and FcR+ effector cells to EpCAM-

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positive carcinomas for action through TCL-mediated lysis and ADCC. The predominance of EpCAM in

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ovarian, lung and gastric cancer has warranted further study in the clinic, and the same format is being applied to HER2 (ertumaxomab), and CD20 (FBTA05) as well (Linke et al., 2010).

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Interestingly, bispecifics naturally occur in IgG4 molecules through the process of Fab arm

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exchange (FAE) (van der Neut Kolfschoten et al., 2007); however, FAE can occur between therapeutic and endogenous IgG4 with some serious consequences (Labrijn et al., 2009). Using controlled FAE, stable bispecific IgG1 molecules termed DuoBodies were developed, and HER2-CD3 and HER2-HER2

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constructs were superior to parental counterparts in proof-of-concept studies (Labrijn et al., 2013).

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The “knobs-into-hole” (KiH) technology allows for efficient heterodimerization of the antibody

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heavy chains by incorporating a “hole” in the Fc region of one heavy chain and a “knob” in the other (Merchant et al., 1998). As an example, a pre-clinical bispecific was designed to shuttle an anti-βsecretase 1 antibody across the blood brain barrier (BBB) by partnering with an anti-transferrin receptor (TfR) antibody for the treatment of Alzheimer’s disease (Yu et al., 2011). Importantly, the bispecific had better brain uptake only when the anti-TfR had moderate affinity, further lending credence to the notion that high affinity is not always ideal. Unfortunately, when using this technology, the light chains can still pair indiscriminately with either heavy chain making isolation and purification of the intended construct difficult. CrossMAb technology resolved the light chain mispairing in the original KiH antibodies by exchanging the non-Fc domains of the heavy and light chains from one of the antibody halves in three different formats: VH-VL, CH1-CL, or Fab (Schaefer et al., 2011). Among others, CrossMAb bispecifics targeting VEGF-A and angiopoietin-2 were developed with antitumor, antiangiogenic and antimetastatic

ACCEPTED MANUSCRIPT properties (Kienast et al., 2013). Cell-free systems have also been extended to KiH-based bispecific

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generation (Xu et al., 2015).

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4.1.2. Fragment-based bispecifics

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The bispecific T cell engager (BiTE) and dual-affinity re-targeting (DART®) technologies are the most successful bispecifics derived from antibody fragments (Fig. 3b). BiTEs, are based on scFvs with

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one targeting CD3 and the other targeting a tumor antigen (Baeuerle & Reinhardt, 2009). Blinatumomab

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recruits TCLs to CD19+ cells (Zimmerman et al., 2015) for the treatment of non-Hodgkin’s lymphoma and ALL (Topp et al., 2015; Topp et al., 2011). This was the first bispecific to be approved by the American Food and Drug Administration (FDA) (Mullard, 2015); however, some patients developed

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cytokine release syndrome caused by abnormal macrophage activation (Teachey et al., 2013). BiTEs have a five residue, non-immunogenic linker with rotational flexibility, and, similar to ADCs, the size and

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shape of the linker can drastically dictate its therapeutic efficacy (i.e. T cell activation) (Nagorsen et al., 2012). Solitomab is a BiTE directed towards EpCAM that is currently in clinical trials for the treatment of gastrointestinal and lung cancers. Intracellular antigens, elusive to standard small molecules and mAbs, have also been targeted by BiTEs to treat leukemias and solid tumors (Dao et al., 2015). DARTs® are structurally quite similar to BiTEs, except that BiTEs are a single polypeptide chain and DARTs® consist of two polypeptide chains connected by an interchain disulfide bridge. One study demonstrated that an anti-CD19xCD3 DART® was more potent than the corresponding BiTE at eliminating CD19-positive cells in a pre-clinical mouse model (Moore et al., 2011). DARTs® are also used in receptor signaling, checkpoint modulation and pathogen neutralization/clearance and are currently being evaluated in a handful of clinical trials. Several other fragment-based bispecific formats have been developed (Kontermann, 2012b), especially exploiting the scFv as the base (Holliger & Hudson, 2005). Bis-scFvs and diabodies are just two scFvs conjugated together to form a bivalent mAb, either as a monospecific or bispecific. Taking it

ACCEPTED MANUSCRIPT further, two diabodies have been used to form the tetravalent dimer TandAb®. Two TandAbs®, AFM13 (CD30 + CD16) and AFM11 (CD19 + CD3), are currently under trial for the treatment of Hodgkin’s

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disease and non-Hodgkin’s lymphoma, respectively (Kontermann & Brinkmann, 2015). Fc domain with antigen binding (Fcab™) technology constitutes an interesting alternative on

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exploiting the antibody scaffold (Lobner et al., 2016). Here, the Fc region of human IgG1 is engineered to

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express antigen binding sites in the CH3 domain and still maintain FcR and FcRn-mediated effector function and half-life, respectively. Proof-of-concept studies with an anti-HER2 Fcab™ demonstrated

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superior efficacy to trastuzumab in patient-derived xenograft tumor models (Leung et al., 2015). The technology was taken further with the development of the mAb2™, a full-length mAb with the normal Fc

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substituted with an Fcab™.

With all the possible ways to engineer antibodies into bispecifics that are available, the field is

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rife with different technologies and where companies try to one-up each other with their molecules often

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focusing on established “safe” targets (e.g. HER2 and programmed cell death protein 1), making us

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wonder if all these different formats really make a difference in the clinic (Moran, 2011). Regardless, most clinical bispecifics (even as ADCs (Metz et al., 2011)) were developed for the treatment of cancer, although several other diseases are also being addressed, such as macular degeneration, inflammation/arthritis, hemophilia and Alzheimer’s disease (Kontermann & Brinkmann, 2015).

4.2. Proteins

Proteins have been used as therapeutics for decades and represent a significant financial market with around half coming from antibodies (Dimitrov, 2012). They can be divided into several different molecular types and act in many ways and on many diseases. In most cases, they do not need specific targeting; however, some would be too toxic, or not potent enough, if not directed towards a more specific site of action. A few examples of where a protein has benefitted from conjugation to an antibody are presented below.

ACCEPTED MANUSCRIPT Toxins derived from plants (e.g. ricin) or bacteria (e.g. Diptheria toxin) were the first tumoricidal agents to be conjugated to antibodies (Teicher & Chari, 2011). Problems associated with murine

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antibodies and large sizes were overcome by recombinant expression of the toxin with only the antigenbinding region of the antibody. For example, an anti-CD22-Pseudomonas exotoxin conjugate has been

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clinically evaluated for the treatment of B-cell malignancies (Kreitman & Pastan, 2011).

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Cytokines are frequently used in the clinic to treat a range of diseases (e.g. hepatitis and cancer). Yet, similar to classical chemotherapy, the required therapeutic doses may be too high, potentially

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causing severe adverse effects due to systemic action. Cytokine-antibody conjugates help focus the

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cytokine activity to reduce side effects and improve pharmacokinetics. Cytokines can be fused to either a whole IgG, Fc fragment or antigen-binding domain, and this format dictates the function and multimeric and multivalent nature of the resulting fusion. Currently, there are ~10 cytokine-antibody conjugates in

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clinical trials. For example, a fusion of an antibody against the disialoganglioside GD2 with interleukin-2 is under trial to treat advanced melanoma and neuroblastoma (Kontermann, 2012a).

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Rapid renal clearance is an unfortunate consequence of the small size of protein/antibody fragment-based therapeutics. Conjugation of PEG or PEG-mimetic peptides is one strategy to increase circulation times (Kontermann, 2016); however, several strategies exploiting FcRn have also been developed. The engineering of the Fc region of therapeutic mAbs was previously presented as one strategy. Of course, though, conjugating an (engineered) Fc to a (protein-based) therapeutic has also been extensively, and successfully, used to increase half-life (Martins et al., 2016). Alternatively, fusing Fc may also function to engage with other FcRs to activate or inhibit the immune system depending on the structure of the Fc (Levin et al., 2015). An elegant system of therapeutic protein display on an antibody scaffold was recently developed. Bovine antibodies have exceptionally long CDR H3s (Vadnais & Smider, 2016) that presumably neutralize the many pathogens associated with grazing. These extra-long CDR H3s (up to 67 residues) consist of many cysteines that help to form a “knob and stalk” structure (Wang et al., 2013). This minidomain has thus been exploited to present therapeutic proteins such as granulocyte colony-stimulating

ACCEPTED MANUSCRIPT factor (Zhang et al., 2013), erythropoietin (Zhang et al., 2015), a C-X-C chemokine receptor type 4targeting peptide (Liu et al., 2014), growth hormone or leptin (T. Liu et al., 2015) with enhanced

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pharmacological properties.

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4.3. Oligonucleotides (ONs)

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Therapeutic oligonucleotides (ONs) present an interesting alternative to established

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biotherapeutics. Current antibody drugs are restricted to cell surface and circulating antigens, and most drugs in general act as inhibitors. However, ONs are advantageous in that every gene product is “druggable” and intracellular molecules can be regulated, thus drastically increasing the overall target

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space (Wahlestedt, 2013). Antisense ONs were the first to be investigated and in fact comprise the only two approved therapeutic ONs, fomivirsen and mipomersen (Moreno & Pêgo, 2014). As recent studies

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have uncovered the myriad regulatory non-coding RNAs (ncRNAs) (Esteller, 2011), steric-blocking ONs (e.g. splice-switching ONs) and antagomirs that regulate the function of ncRNAs have been generating interest as possible therapeutic strategies (Wahlestedt, 2013). All these nucleic acid-based strategies are not without their delivery hurdles (i.e. biological barriers (Juliano et al., 2009)); yet, ONs can certainly benefit from the targeting afforded by mAbs (Ming & Laing, 2015). Interference RNA technology, mainly as small interfering RNAs (siRNAs), offer a real clinical viability (Wittrup & Lieberman, 2015), and conjugating siRNA to a ligand (e.g. mAb) for targeted delivery is critical for successful, and safe, therapy (Ikeda & Taira, 2006). Attaching a negatively-charged siRNA to a mAb has mainly been achieved through non-covalent charge interaction with a string of cationic amino acids. Antibody-mediated siRNA delivery was initiated with a conjugate comprised of a siRNA against the human immunodeficiency virus (HIV) capsid gene gag captured by protamine, a small arginine-rich nucleoprotein that was fused to an anti-HIV envelope Fab. This conjugate only targeted HIV

ACCEPTED MANUSCRIPT envelope-expressing B16 melanoma cells in mouse models, and the concept was taken further with antiHER2 scFv-delivered siRNAs into HER2-expressing cancer cells (Song et al., 2005).

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Kumar et al (2008) captured a different anti-HIV siRNA with an oligo-9-arginine peptide that was fused to a T cell-specific, anti-CD7 scFv to suppress HIV infection in vivo (P. Kumar et al., 2008).

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Covalent conjugation of an siRNA to an antibody was achieved by exploiting the clinically-established

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THIOMAB (Junutula et al., 2008) technology (Cuellar et al., 2015). Seven targets with different internalization routes were evaluated in vitro and in a prostate carcinoma mouse model. Targeted

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silencing was limited by endocytic entrapment independent of the internalization route (P. Kumar et al.,

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2008).

Similar to ADCs, the location of the ON/siRNA on the antibody can also have a dramatic impact on the efficacy of the final conjugate. Conjugation sites on the antibody will be similar for ONs as for

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small molecule drugs (e.g. amines and thiols) with associated heterogenous mixture problems (Winkler, 2013). Lu and colleagues (2013) expanded their work on ADCs (Axup et al., 2012) to antibody-siRNA

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conjugates (Lu et al., 2013). Here, they exploited their uAA-expressing antibody technology (section 3.5) to conjugate an aminooxy-derivatized cationic block copolymer for siRNA capture on three different sites of trastuzumab, as a Fab or whole IgG. Although all three conjugates had high binding affinity to HER2, one of them had nominal gene silencing in HER2+ cells presumably due to poor antigen-mediated internalization. It should be noted that investigations into other antibody-ON conjugates for therapeutic purposes have been scarce so far (Walker et al., 1995; Winkler, 2013). ONs directly conjugated to antibodies are still prone to instability and innate immune response (Lu et al., 2013). Recent technologies have addressed these issues to help make them a viable therapeutic platform. Incorporation of locked nucleic acids provides ONs with increased thermal and enzymatic stability and target (mainly DNA/RNA) specificity, while still maintaining Watson-Crick base pairing (Koshkin et al., 1998). For example, miravirsen is an antisense RNA, partly composed of locked nucleic acids, that targets the liver microRNA miR-122 to treat hepatitis C virus infection (Janssen et al., 2013). Nucleotides can also be chemically modified by replacing the 2’ position with fluoro, O-methyl or O-

ACCEPTED MANUSCRIPT methoxyethyl groups (Gomes-da-Silva et al., 2014). Though, protection by nanoencapsulation could significantly impact the delivery of ONs to areas such as the central nervous system (Gomes et al., 2015)

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or tumors (Moreno & Pêgo, 2014).

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5. Antibody-nanoparticle conjugates (ANCs)

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Nanotechnology applications in medicine are proving their worth. Nanoparticles (NPs) are extremely versatile and can be engineered to display a multitude of pharmacological properties. In the context of drug delivery, nanotechnology is used to encapsulate the drug, or payload, for enhanced

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pharmacokinetics, bioavailability, and drug release to increase the therapeutic index by increasing efficacy and/or reducing side effects, similar to ADCs (section 4). As an example, Doxil is an approved

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nanomedicine that encapsulates doxorubicin (dox) (Barenholz, 2012). Doxil is not more effective in reducing tumor volume; however, the nanoformulation helps to greatly reduce the cardiotoxicity common with free dox (Singal & Iliskovic 1998). Nanomedicine can impact a large spectrum of human diseases, including cancer (Fernandes et al., 2015), HIV (das Neves et al., 2010) and TB (Costa et al., 2016), and can be engineered with many nanomaterials to harbor many payload types (e.g. small to big (bio-)molecules). Small molecule drug candidates with high therapeutic potential often have poor solubility leading to poor bioavailability (Kalepu & Nekkanti, 2015), and several formulation strategies exist to increase solubility (e.g. solid dispersion (Vasconcelos et al., 2016) and nanoencapsulation (Kalepu & Nekkanti, 2015)). Though biologics usually do not have solubility issues, they do face other problems associated with biology. Nanovaccines have also been developed for immunotherapy of cancer and beyond (Shao et al., 2015). Administration of photosensitizers (Gonzalez-Delgado et al., 2016) and multiple therapeutics (Kemp et al., 2016) is also possible with NPs. Beyond drug delivery, NPs can be used in in vivo diagnostics either

ACCEPTED MANUSCRIPT as contrast agents themselves (e.g. quantum dots or gold NPs) or harboring them as payloads (Sharma et al., 2006).

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The lifespan of a biopharmaceutical is severely dependent on the mode of administration. For example, insulin is mainly administered by painful injection. Patients prefer to take drugs by less invasive

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means (e.g. mucosal); however, most biopharmaceuticals do not bode well in these environments.

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Nanotechnology is currently being exploited for systemic drug delivery by non-parenteral routes (Sosnik et al., 2014). Our own lab has extensive experience in the nanoencapsulation of macromolecular

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biotherapeutics based on proteins (e.g. insulin (Sarmento et al., 2007)), peptides (glucagon-like peptide (Araujo et al., 2015)) and nucleic acids (e.g. siRNA (Gomes et al., 2016)).

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The early NP-based cancer therapeutics (e.g. Doxil) focused on “passive” targeting to tumors by avoiding the reticuloendothelial system and exploiting the enhanced permeability and retention effect, a

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consequence of leaky tumor vasculature (Bertrand et al., 2014; Brannon-Peppas & Blanchette, 2004). However, a major goal for nanomedicine is to drive the therapeutic directly to the site of action (e.g.

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receptor-overexpressing tumor cell) through decoration with ligands that bind to a specific target molecule (e.g. receptor) (Bertrand et al., 2014). Although no targeted NPs have been approved yet, close to 20 are in clinical trials. BIND-014, one example that completed phase II trials for the treatment of prostate and lung cancer, is a PEGylated polymeric NP harboring docetaxol and decorated with a small molecule specific to prostate-specific membrane antigen (Hrkach et al., 2012). Given the vast multitude of targeting ligands, drugs and nanomaterials, the possible combinations of the three are boundless. NPs partnered with mAbs, as targeting ligands or as therapeutic payloads, are presented below.

5.1. Antibodies as targeting agents

Many factors contribute to the overall success or failure of targeted NPs including: a) route of administration, b) biological barriers (Blanco et al., 2015), c) adsorption of host proteins to the NP surface (Lundqvist et al., 2008), d) conjugation chemistry, e) hydrophobicity of the NP, f) composition, size,

ACCEPTED MANUSCRIPT shape and charge of both the ligand and NP, g) density and orientation of the conjugated ligand (Bertrand et al., 2014) and h) ligand affinity/avidity (Fig. 4). Additionally, the properties of the drug to be

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incorporated will dictate the method of nanoencapsulation. Antibodies serve as ideal targeting ligands for their affinity, specificity and engineering malleability. This section presents the strategies to consider

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when developing antibody-decorated NPs.

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Active targeting of nanocarriers is possible through decoration with ligands, and antibodies or associated fragments have been proven to be viable for this purpose (Bertrand et al., 2014). These systems

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are often designed to target cell surface receptors and degrade and release their payload once the NPs

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enter the cell by a number of means. Fortunately, it seems that P-glycoprotein does not recognize the NPs, thus resulting in enhanced intracellular accumulation and nominal drug resistance (Arruebo et al., 2009). Early studies with targeted NPs focused on encapsulating classic chemotherapy agents to increase

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therapeutic index, mainly by lowering systemic toxicity. Park and colleagues demonstrated that immunoliposomes harboring dox and decorated with anti-HER2 (Fab or scFv) had more tumor targeting

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efficacy and lower toxicity than all the other control treatments (Park et al., 2002). The conjugation strategies and challenges associated with ADCs also apply to nanocarriers. As seen before with ADCs and bispecifics, the choice of linker may impact the therapeutic efficacy of the NP conjugate (Clark & Davis, 2015). Here, an acid-cleavable linker demonstrated more brain uptake than a non-cleavable linker in a TfR-mediated BBB model. Bispecific antibodies have also been coupled to NPs (Schneider et al., 2012). In this example, siRNA-digoxigenin conjugates were used during production of dynamic polyconjugates or lipid-based NPs. Bispecific antibodies, with one arm against a tumor antigen (e.g. HER2), were captured onto the NPs by the other antibody arm against digoxigenin. The NP strategy was able to show cell-specific gene downregulation, while the non-NP, bispecific-siRNA-digoxigenin conjugate could not. Different antibodies conjugated to NPs can also be used to target disparate antigens on the same cell or each antibody may serve different functions. Zhang et al (2002 & 2004) designed an “artificial virus” to cross different biological barriers to deliver a plasmid encoding antisense mRNA against the

ACCEPTED MANUSCRIPT epithelial growth factor (EGFR) gene in a human glioma mouse model. An anti-TfR receptor mAb shuttled the NPs across the BBB while an anti-insulin receptor mAb aimed to cross the plasma and

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nuclear membranes. This nanosystem ultimately resulted in 100% lifespan increase compared to controls (Zhang et al., 2004; Zhang et al., 2002).

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Unlike ADCs and ABCs, the antibody (~10-20 nm) in this case is usually much smaller than the

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conjugated NP “cargo” (~100 nm), opening the possibility to attach many antibodies to a single NP. Similar to natural and bispecific antibodies, avidity may be exploited to enhance overall binding and/or

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specificity to the target antigen(s). A site-specific conjugation strategy using expressed protein ligation

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and copper-free click chemistry was used to decorate anti-HER2 affibodies to superparamagnetic iron oxide NPs at controlled ligand densities (Elias et al., 2013). In fact, intermediate ligand density resulted in the highest cell binding, and this was confirmed with an alternative (non-antibody) ligand. Thus, optimal

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ligand density must be empirically determined for highest efficacy. Ligand orientation on the NP can also affect antigen binding. Conventional chemistries do not

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afford control of where on the mAb (e.g. amines) the conjugation bridge is formed. This leads to nonideal conditions where many of the antibodies may be oriented on the NP such that the antigen-binding domains are unavailable for direct antigen contact. Engineering and recombinant strategies can overcome this disorientation and maximize antibody functionality. Whole IgG can be coupled to gold NPs through the Fc domain with the heterobifunctional linker, hydrazide-polyethylene glycol-dithiol (S. Kumar et al., 2008). Another example is to engineer a Fab with a C-terminal cysteine and use a maleimide-based linker (e.g. SMCC) to couple to amine groups commonly found on various nanomaterials (e.g. chitosan (Antunes et al., 2012)). The numerous ways in which (directional) conjugation can be achieved are described in detail elsewhere (Sapsford et al., 2013). As previously mentioned, ONs can be viable therapeutic agents against a wide variety of targets and diseases, but their fragile nature must be preserved before introduction to the cytoplasmic site of action. Encapsulation within NPs represents a proven option for protection and intracellular delivery of ONs (e.g. siRNA). In fact, interference RNA in humans was confirmed by encapsulating siRNA into

ACCEPTED MANUSCRIPT targeted NPs (Davis et al., 2010). As an example, EGFR-targeted, chitosan-based NPs were developed to encapsulate siRNA against Mad2, essential for the mitotic checkpoint, in non-small cell lung cancer

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models. This system effectively silenced Mad2 causing apoptotic cell death only in EGFR-expressing cells in vitro (Nascimento et al., 2014) and had preferential tumor targeting in vivo compared to non-

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targeted NPs (Nascimento et al., 2016).

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Aside from using the antigen-binding regions of the antibody, Fc-decorated NPs have been developed for drug delivery across the intestinal (Pridgen et al., 2013) and lung (Vllasaliu et al., 2012)

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epithelial layers. Both systems exploit FcRn for receptor-mediated transcytosis to reach systemic

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circulation. For example, oral delivery of insulin to diabetic mice with this strategy resulted in levels high enough for therapeutic efficacy (Pridgen et al., 2013).

In vivo diagnostics can also benefit from targeted NPs. Anti-EGFR-coated gold NPs were

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developed to aid in endoscopic tumor imaging (Sokolov et al., 2003). Furthermore, combining a diagnostic agent along with the therapeutic payload (i.e. theranostic) offers the advantage of monitoring a

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treatment regimen during NP administration (Yao et al., 2016). It should be noted though that more complexity added to a nanocarrier imposes greater cost (e.g. regulatory); thus, the trade-off should be considered before embarking on (pre-)clinical studies (Cheng et al., 2012). Although the overall strategy of “active targeting” has been criticized, highlighting the dearth of clinical evidence (van der Meel et al., 2013), the current literature points to the extreme validity of this strategy. Yet, clearly more research efforts must be dedicated into ways to identify and overcome the biological barriers inhibiting the targeting (Blanco et al., 2015). For example, the protein corona (Pearson et al., 2014) is a well-characterized phenomenon known to disrupt targeting (Salvati et al., 2013). When in biological fluids (e.g. serum), biomolecules adsorb as a “hard” or “soft” layer onto the NP surface. The composition of this corona is dependent on the biological environment but also on the NP qualities (size, shape, charge, hydrophobicity, etc) (Lundqvist et al., 2008). These proteins can thus mask the ligand overriding any targeting capabilities, as seen in transferrin-coated NPs (Salvati et al., 2013). One way to

ACCEPTED MANUSCRIPT overcome the inhibitory effects of the protein corona is through backfilling with PEG once the ligand has been conjugated to the NP (Dai et al., 2014).

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The infinite combinations of drugs, ligands/antibodies, nanomaterials, encapsulation and conjugation methods and specific disease indications can frustrate the initial planning stages. On one

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hand, making decisions about the ideal system is quite challenging, but, on the other hand, these

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technologies can bring unprecedented possibilities. Regardless, the antibody-decorated nanocarrier

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platform is emerging as a highly promising tool in targeted drug delivery.

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5.2. Antibodies as payload

Encapsulating antibodies within nanocarriers offers several promising applications from

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intracellular delivery to enhanced residence times and delivery by non-parenteral routes (Fig. 5). The encapsulation into nanoparticles is also powerful methodology to deliver antibodies in a sustained manner

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and to preserve its structural stability. As the storage of antibodies in conventional formulations is a limiting issue in their market chain, nanoencapsulation may play an important role towards the improvement of stability and long-term use. Yet, these nanosystems are still quite new, and each application has its own set of hurdles.

A major issue with antibody encapsulation is that they may not survive the harsh nanoformulation process encountered with some polymers, because the use of solvents, sonication, and heat leads to structural instability. Gdowski et al optimized a method to have moderate encapsulation efficiency of whole IgG in poly(lactic-co-glycolic acid) NPs and still maintain antigen binding (Gdowski et al., 2015). The use of stabilizers like albumin (Canton et al., 2013) and mannitol (Son et al., 2009) has helped to protect the antibody structure from poly(lactic-co-glycolic acid) entrapment stress. Self-assembling, polymeric nanovesicles were able to deliver antibodies against common proteins (e.g. NF-κB) inside cells with low toxicity (Canton et al., 2013). Kim et al. recently designed block/homo polyion complex micelles to deliver transiently charge-converted whole IgGs into the cytosol (Kim et al., 2016). Similarly,

ACCEPTED MANUSCRIPT fusing a supercharged anionic protein to an antibody could allow its intracellular delivery using any common cationic lipid-based transfection reagent designed for nucleic acids (Zuris et al., 2015). The large

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size of whole IgG further hinders drug loading and entrapment efficiencies. Of course, the smaller size of antibody fragments should help improve their survival and allow for more efficient encapsulation.

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Many diseases are caused by abnormal protein functions and the protein-protein interaction

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consequences inside the cell (Zeng et al., 2013). Thus, modulation of intracellular signaling holds tremendous therapeutic potential. This is currently accomplished by stimulating surface receptors to

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perturb downstream pathways. The endless molecules that antibodies can recognize make them ideal for

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inhibiting intracellular protein-protein interaction s and beyond. The use of intracellular antibodies for basic science or as a therapeutic strategy is still very much in its infancy (Perez-Martinez et al., 2010), and nanotechnology offers a good strategy for the intracellular delivery of antibodies.

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One dogma of intracellular antibodies is that they do not function in the reductive environment of the cytosol since disulfide bridges can be reduced leading to disrupted antibody structure (Sousa et al.,

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2016). However, antibody-dependent intracellular neutralization clearly demonstrates that intracellular antibodies can in fact function properly in a natural setting (section 2 (Mallery et al., 2010)). Also, recombinant antibody fragments (e.g. scFvs (Son et al., 2009) or sdAbs (Zeng et al., 2013)) are more stable and thus should engage with intracellular target antigens more readily. Or, at the very least, they can be selected for in a reductive environment during the discovery process (Zeng et al., 2013). Rabbitts and colleagues have developed a method for expressing and capturing intracellular antibodies against a specific target antigen (Tanaka & Rabbitts, 2012). This technology was initially used with VH and VL domain libraries, but has been further extended to llama VHHs (Newnham et al., 2015) and human sdAbs (Zeng et al., 2015). Prolonging residence times to decrease the number of doses is extremely helpful in many therapeutic regimens (Uhrich et al., 1999). Whole IgG certainly has long circulation due lysosomal escape (Martins et al., 2016). Yet, this does not preclude the need for repeated administrations every month or so causing reduced patient adherence. Nanoencapsulation is proven to be highly effective at controlled drug

ACCEPTED MANUSCRIPT release (Uhrich et al., 1999), but its use with antibodies is severely lacking. In one example though, encapsulated anti-VEGF (bevacizumab) in nanoliposomes resulted in 5-fold greater concentrations of the

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antibody, versus free mAb, 42 days after retinal injection (Abrishami et al., 2009). Administration of most antibodies is made by intravenous infusion or subcutaneous injection,

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raising costs and pain levels. A time when antibodies can be delivered by non-parenteral means with NPs

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is on the horizon (Sosnik et al., 2014) with oral delivery as the holy grail (Reilly et al., 1997). It must be possible as newborns are able to acquire passive immunity from their mother’s milk, a process mediated

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by intestinal FcRn that is retained in adulthood (Martins et al., 2016). Of course, the principles guiding

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oral delivery of proteins in general will apply to antibodies as well (des Rieux et al., 2006; Morishita & Peppas, 2006). The journey from the mouth to the circulation is a perilous one fraught with low pH, degrading enzymes, the mucous layer and the epithelial barrier. Passage beyond the stomach and

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temporary disruption of proteases is possible with well-established pharmaceutical formulations. Penetrating the mucous layer is possible with specific nanomaterials (e.g. chitosan), surfactants or

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functional groups displaying amenable surface qualities (e.g. charge or PEG) (Sosnik et al., 2014). Though, surpassing the epithelial layer remains the most challenging with exploitation of receptormediated transcytosis having potential (Pridgen et al., 2013). .

6. Conclusions and future directions

This review is intended to constitute a general reference for all those interested in using any one of the myriad applications of antibody domains as therapeutic, diagnostic or targeting agents with an emphasis on drug delivery. Conjugating a mAb to a therapeutic clearly has many pharmacologic advantages from enhanced activity to lowered systemic toxicity and dosage regimens. Though cancer was the main disease indication discussed here, the wide range of targets accessible to antibodies suggests that many acute and chronic diseases can be treated using the platforms presented here. Additionally, the intracellular delivery of nucleic acids or mAbs vastly increases the druggable target space by, for

ACCEPTED MANUSCRIPT example, modulating mRNA/ncRNA or protein-protein interactions, respectively. Upon reflection, several questions and thoughts about the future come to mind.

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First, have all the ways in which to exploit all the antibody regions/domains been exhausted? In particular, the Fc domain and the corresponding Fc receptors deserve more interest for drug delivery and

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enhanced pharmacology. Targeting FcRn with Fc conjugates has proven clinically successful in

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prolonging drug circulation times (Martins et al., 2016) and is emerging as a viable strategy in oral nanoDDSs (Pridgen et al., 2013). Thus, exploiting the Fc-decorated nanosystem to encapsulate and orally

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deliver mAbs is a definite possibility. With the clinical success of llama VHHs (i.e. nanobodies)

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(Peyvandi et al., 2016) and the potential of bovine CDR H3s (Wang et al., 2013), we encourage the exploration of immune repertoires from non-traditional sources (e.g. scavengers (Ohishi et al., 1979) and insects (Dong et al., 2012)). Alternatively, nucleic acid-based affinity reagents (e.g. aptamers) should be

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exploited more thoroughly as therapeutics (Keefe et al., 2010) and/or targeting agents (Farokhzad et al., 2006; Sun & Zu, 2015; Zhu et al., 2015). Most of the principles and technologies outlined here can, of

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course, be translated to other affinity binders. Several of the biotechnologies presented here aim to recruit immune effector cells (e.g. CrossMAbs and DARTs®) to a specific cell (usually cancerous). Immune checkpoint blockade, usually mediated with mAb therapy, results in durable responses, but unfortunately only works in the small fraction of patients with high amounts of T cells already residing in the tumor (Gubin et al., 2014; Pardoll, 2012). Thus, therapeutic efficacy may be improved by combining an immune checkpoint blockade antibody with: a) a genomically targeted one (Fig. 6a) (Sharma & Allison, 2015), b) a bispecific mAb to help recruit T cells, c) another checkpoint blockade antibody (Boutros et al., 2016; Mahoney et al., 2015) or d) an inhibitor of myeloid suppressor cells (De Henau et al., 2016) possibly delivered by ADCs or ANCs. This concept can be also extended to ADCs harboring either a stimulator of effector T cell recruitment or dendritic cell activation and maturation (Fig. 6b) (Gerber et al., 2016; Müller et al., 2015). The fusion concept of each of these bioconjugate platforms could also be further expanded. For example, bispecific ADCs (Sellmann et al., 2016), ABCs coupled to a NP to form a bispecific ANC (Wu

ACCEPTED MANUSCRIPT et al., 2016), or ADCs/ABCs encapsulated within a NP to exploit the enhanced permeability and retention effect (Bilodeau et al., 2015) are all under development. Alternatively, these platforms could be

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incorporated within biomaterials for other delivery modalities. Hydrogels and films offer localized and sustained release of mAbs (Tian et al., 2005) or drugs within (targeted) NPs (e.g. vaginal delivery for HIV

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prevention (Cunha-Reis et al., 2016; Gu et al., 2015)). Translating these strategies to mAb conjugates is a

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logical direction.

With significant interest of late in aging, regeneration, microbiomes and genome editing, will

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antibodies and associates play a therapeutic role? As a possible example, encapsulating guide RNAs and

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endonucleases (e.g. Cas9, Cpf1 (Zetsche et al., 2015) or C2c2 (Abudayyeh et al., 2016)) within an antibody-decorated nanocarrier (Zuris et al., 2015) and shuttled to a specific cell/tissue for directed genome or transcriptome editing is envisioned.

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The success of targeted therapy is evident. The specificity and binding kinetics of antibodies coupled with powerful drug delivery technologies can be exploited to tailor disease treatment with greater

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precision. Will one of the three different approaches to drug delivery presented here ultimately become victorious, or will all of them prove their worth? We hope the reader will apply the principles outlined here to their own research and/or (pre-)clinical agent and to seek out partnerships to narrow the “innovation gap” and advance human health (Gehr & Garner, 2016).

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operacional Programme for Competitiveness and Internationalisation

ACCEPTED MANUSCRIPT (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação in the framework of the project "Institute for

“Applied

Biomolecular

Sciences

Unit”

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Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274) and of the project (POCI-01-0145-FEDER-007728

and

PT2020

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UID/MULTI/04378/2013). This work was also supported by the FCT PhD Programmes and by Programa

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Operacional Potencial Humano (POCH), specifically by the BiotechHealth Programme (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences). Patrick J. Kennedy

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gratefully acknowledges the BiotechHealth Doctoral Programme and FCT for financial support

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(SFRH/BD/99036/2013) and beyond.

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References

Abrishami, M., Zarei-Ghanavati, S., Soroush, D., Rouhbakhsh, M., Jaafari, M. R., & Malaekeh-Nikouei,

AC CE P

B. (2009). Preparation, characterization, and in vivo evaluation of nanoliposomes-encapsulated bevacizumab (avastin) for intravitreal administration. Retina, 29, 699-703. Abudayyeh, O. O., Gootenberg, J. S., Konermann, S., Joung, J., Slaymaker, I. M., Cox, D. B., et al. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science, 353, aaf5573. Acchione, M., Kwon, H., Jochheim, C. M., & Atkins, W. M. (2012). Impact of linker and conjugation chemistry on antigen binding, Fc receptor binding and thermal stability of model antibody-drug conjugates. MAbs, 4, 362-372. Adair, J. R., Howard, P. W., Hartley, J. A., Williams, D. G., & Chester, K. A. (2012). Antibody–drug conjugates – a perfect synergy. Expert Opin Biol Ther, 12, 1191-1206. Akkapeddi, P., Azizi, S.-A., Freedy, A. M., Cal, P. M. S. D., Gois, P. M. P., & Bernardes, G. J. L. (2016). Construction of homogeneous antibody-drug conjugates using site-selective protein chemistry. Chem Sci, 7, 2954-2963.

ACCEPTED MANUSCRIPT Antunes, F., Andrade, F., & Sarmento, B. (2012). Chitosan-based nanoparticulates for oral delivery of biopharmaceuticals. In

Chitosan-based systems for biopharmaceuticals (pp. 225-241): John

PT

Wiley & Sons, Ltd. Araujo, F., Shrestha, N., Shahbazi, M. A., Liu, D., Herranz-Blanco, B., Makila, E. M., et al. (2015).

RI

Microfluidic assembly of a multifunctional tailorable composite system designed for site specific

SC

combined oral delivery of peptide drugs. ACS Nano, 9, 8291-8302.

Arruebo, M., Valladares, M., & González-Fernández, Á. (2009). Antibody-conjugated nanoparticles for

NU

biomedical applications. J Nanomater, 2009, 1-24.

MA

Axup, J. Y., Bajjuri, K. M., Ritland, M., Hutchins, B. M., Kim, C. H., Kazane, S. A., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A, 109, 16101-16106.

Release, 153, 198-205.

TE

D

Bae, Y. H., & Park, K. (2011). Targeted drug delivery to tumors: Myths, reality and possibility. J Control

AC CE P

Baeuerle, P. A., & Reinhardt, C. (2009). Bispecific T-cell engaging antibodies for cancer therapy. Cancer Res, 69, 4941-4944.

Barbet, J., Bardies, M., Bourgeois, M., Chatal, J. F., Cherel, M., Davodeau, F., et al. (2012). Radiolabeled antibodies for cancer imaging and therapy. Methods Mol Biol, 907, 681-697. Barenholz, Y. (2012). Doxil® — The first FDA-approved nano-drug: Lessons learned. J Control Release, 160, 117-134. Barok, M., Joensuu, H., & Isola, J. (2014). Trastuzumab emtansine: Mechanisms of action and drug resistance. Breast Cancer Res, 16, 209. Beck, A., Wurch, T., Bailly, C., & Corvaia, N. (2010). Strategies and challenges for the next generation of therapeutic antibodies. Nat Rev Immunol, 10, 345-352. Bertrand, N., Wu, J., Xu, X., Kamaly, N., & Farokhzad, O. C. (2014). Cancer nanotechnology: The impact of passive and active targeting in the era of modern cancer biology. Adv Drug Deliv Rev, 66, 2-25.

ACCEPTED MANUSCRIPT Bilodeau, M. T., Shinde, R., White, B., Bazinet, P., Whalen, K., Dupont, M., et al. (2015). Abstract 3674: Pentarins: Improved tumor targeting through nanoparticle encapsulation of miniaturized biologic

PT

drug conjugates. Cancer Res, 75, 3674-3674.

barriers to drug delivery. Nat Biotechnol, 33, 941-951.

RI

Blanco, E., Shen, H., & Ferrari, M. (2015). Principles of nanoparticle design for overcoming biological

SC

Boder, E. T., Raeeszadeh-Sarmazdeh, M., & Price, J. V. (2012). Engineering antibodies by yeast display. Arch Biochem Biophys, 526, 99-106.

NU

Boutros, C., Tarhini, A., Routier, E., Lambotte, O., Ladurie, F. L., Carbonnel, F., et al. (2016). Safety

MA

profiles of anti-CTLA-4 and anti-PD-1 antibodies alone and in combination. Nat Rev Clin Oncol, 13, 473-486.

Bradbury, A. R., Sidhu, S., Dübel, S., & McCafferty, J. (2011). Beyond natural antibodies: The power of

TE

D

in vitro display technologies. Nat Biotechnol, 29, 245-254. Brannon-Peppas, L., & Blanchette, J. O. (2004). Nanoparticle and targeted systems for cancer therapy.

AC CE P

Adv Drug Deliv Rev, 56, 1649-1659. Canton, I., Massignani, M., Patikarnmonthon, N., Chierico, L., Robertson, J., Renshaw, S. A., et al. (2013). Fully synthetic polymer vesicles for intracellular delivery of antibodies in live cells. FASEB J, 27, 98-108.

Casi, G., & Neri, D. (2012). Antibody–drug conjugates: Basic concepts, examples and future perspectives. J Control Release, 161, 422-428. Chen, J., Yin, S., Wu, Y., & Ouyang, J. (2013). Development of a native nanoelectrospray mass spectrometry method for determination of the drug-to-antibody ratio of antibody–drug conjugates. Anal Chem, 85, 1699-1704. Cheng, Z., Al Zaki, A., Hui, J. Z., Muzykantov, V. R., & Tsourkas, A. (2012). Multifunctional nanoparticles: Cost versus benefit of adding targeting and imaging capabilities. Science, 338, 903-910.

ACCEPTED MANUSCRIPT Chudasama, V., Maruani, A., & Caddick, S. (2016). Recent advances in the construction of antibody-drug conjugates. Nat Chem, 8, 114-119.

PT

Clark, A. J., & Davis, M. E. (2015). Increased brain uptake of targeted nanoparticles by adding an acidcleavable linkage between transferrin and the nanoparticle core. Proc Natl Acad Sci U S A, 112,

RI

12486-12491.

SC

Costa, A., Pinheiro, M., Magalhaes, J., Ribeiro, R., Seabra, V., Reis, S., et al. (2016). The formulation of nanomedicines for treating tuberculosis. Adv Drug Deliv Rev, 102, 102-115.

NU

Cuellar, T. L., Barnes, D., Nelson, C., Tanguay, J., Yu, S. F., Wen, X., et al. (2015). Systematic

MA

evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMABsiRNA conjugates. Nucleic Acids Res, 43, 1189-1203. Cunha-Reis, C., Machado, A., Barreiros, L., Araújo, F., Nunes, R., Seabra, V., et al. (2016).

Release, 243, 43-53.

TE

D

Nanoparticles-in-film for the combined vaginal delivery of anti-HIV microbicide drugs. J Control

AC CE P

Dai, Q., Walkey, C., & Chan, W. C. W. (2014). Polyethylene glycol backfilling mitigates the negative impact of the protein corona on nanoparticle cell targeting. Angew Chem Int Ed Engl, 53, 50935096.

Dao, T., Pankov, D., Scott, A., Korontsvit, T., Zakhaleva, V., Xu, Y., et al. (2015). Therapeutic bispecific T-cell engager antibody targeting the intracellular oncoprotein WT1. Nat Biotechnol, 33, 10791086. das Neves, J., Amiji, M. M., Bahia, M. F., & Sarmento, B. (2010). Nanotechnology-based systems for the treatment and prevention of HIV/AIDS. Adv Drug Deliv Rev, 62, 458-477. Davis, M. E., Zuckerman, J. E., Choi, C. H. J., Seligson, D., Tolcher, A., Alabi, C. A., et al. (2010). Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature, 464, 1067-1070.

ACCEPTED MANUSCRIPT De Henau, O., Rausch, M., Winkler, D., Campesato, L. F., Liu, C., Cymerman, D. H., et al. (2016). Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells.

PT

Nature, 539, 443-447. Debaene, F., Boeuf, A., Wagner-Rousset, E., Colas, O., Ayoub, D., Corvaia, N., et al. (2014). Innovative

RI

native MS methodologies for antibody drug conjugate characterization: High resolution native

SC

MS and IM-MS for average DAR and DAR distribution assessment. Anal Chem, 86, 1067410683.

NU

des Rieux, A., Fievez, V., Garinot, M., Schneider, Y.-J., & Préat, V. (2006). Nanoparticles as potential

MA

oral delivery systems of proteins and vaccines: A mechanistic approach. J Control Release, 116, 1-27.

TE

Br J Cancer, 114, 362-367.

D

Diamantis, N., & Banerji, U. (2016). Antibody-drug conjugates--An emerging class of cancer treatment.

Dimitrov, D. S. (2012). Therapeutic proteins. Methods Mol Biol, 899, 1-26.

AC CE P

Dokter, W. H. A., Lee, M. v. d., Groothuis, P., Achterberg, T. v., Loosveld, E., Jacobs, D., et al. (2014). Abstract 2652: In vitro and in vivo antitumor activity of SYD985, a novel HER2-targeting ADC: a comparison with T-DM1. Cancer Res, 74, 2652. Dong, Y., Cirimotich, C. M., Pike, A., Chandra, R., & Dimopoulos, G. (2012). Anopheles NF-kappaBregulated splicing factors direct pathogen-specific repertoires of the hypervariable pattern recognition receptor AgDscam. Cell Host Microbe, 12, 521-530. Ekins, S. (2014). Hacking into the granuloma: Could antibody antibiotic conjugates be developed for TB? Tuberculosis (Edinb), 94, 715-716. Elias, D. R., Poloukhtine, A., Popik, V., & Tsourkas, A. (2013). Effect of ligand density, receptor density, and nanoparticle size on cell targeting. Nanomedicine, 9, 194-201. Erickson, H. K., Park, P. U., Widdison, W. C., Kovtun, Y. V., Garrett, L. M., Hoffman, K., et al. (2006). Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing. Cancer Res, 66, 4426-4433.

ACCEPTED MANUSCRIPT Ernest, S. S., & Maurice, Z. (2014). Antibody library display on a mammalian virus vector: combining the advantages of both phage and yeast display into one technology. Curr Drug Discov Technol,

PT

11, 48-55. Esteller, M. (2011). Non-coding RNAs in human disease. Nat Rev Genet, 12, 861-874.

RI

Farokhzad, O. C., Cheng, J., Teply, B. A., Sherifi, I., Jon, S., Kantoff, P. W., et al. (2006). Targeted

SC

nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. Proc Natl Acad Sci U S A, 103, 6315-6320.

NU

Fernandes, E., Ferreira, J. A., Andreia, P., Luís, L., Barroso, S., Sarmento, B., et al. (2015). New trends in

MA

guided nanotherapies for digestive cancers: A systematic review. J Control Release, 209, 288307.

Flygare, J. A., Pillow, T. H., & Aristoff, P. (2013). Antibody-drug conjugates for the treatment of cancer.

TE

D

Chem Biol Drug Des, 81, 113-121.

Foss, S., Watkinson, R., Sandlie, I., James, L. C., & Andersen, J. T. (2015). TRIM21: A cytosolic Fc

AC CE P

receptor with broad antibody isotype specificity. Immunol Rev, 268, 328-339. Gdowski, A., Ranjan, A., Mukerjee, A., & Vishwanatha, J. (2015). Development of biodegradable nanocarriers loaded with a monoclonal antibody. Int J Mol Sci, 16, 3990-3995. Gehr, S., & Garner, C. C. (2016). Rescuing the lost in translation. Cell, 165, 765-770. Gerber, H.-P., Sapra, P., Loganzo, F., & May, C. (2016). Combining antibody–drug conjugates and immune-mediated cancer therapy: What to expect? Biochem Pharmacol, 102, 1-6. Gerber, H.-P., Senter, P. D., & Grewal, I. S. (2009). Antibody drug-conjugates targeting the tumor vasculature: Current and future developments. MAbs, 1, 247-253. Gomes-da-Silva, L. C., Simões, S., & Moreira, J. N. (2014). Challenging the future of siRNA therapeutics against cancer: The crucial role of nanotechnology. Cell Mol Life Sci, 71, 1417-1438. Gomes, M. J., Fernandes, C., Martins, S., Borges, F., & Sarmento, B. (2016). Tailoring lipid and polymeric nanoparticles as siRNA carriers towards the blood-brain barrier – From targeting to safe administration. J Neuroimmune Pharmacol, 1-13.

ACCEPTED MANUSCRIPT Gomes, M. J., Martins, S., & Sarmento, B. (2015). siRNA as a tool to improve the treatment of brain diseases: Mechanism, targets and delivery. Ageing Res Rev, 21, 43-54.

PT

Gonzalez-Delgado, J. A., Kennedy, P. J., Ferreira, M., Tome, J. P., & Sarmento, B. (2016). Use of photosensitizers in semisolid formulations for microbial photodynamic inactivation. J Med Chem,

RI

59, 4428-4442.

SC

Greenberg, A. S., Avila, D., Hughes, M., Hughes, A., McKinney, E. C., & Flajnik, M. F. (1995). A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification

NU

in sharks. Nature, 374, 168-173.

MA

Grunewald, J., Hunt, G. S., Dong, L., Niessen, F., Wen, B. G., Tsao, M. L., et al. (2009). Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids. Proc Natl Acad Sci U S A, 106, 4337-4342.

TE

D

Gu, J., Yang, S., & Ho, E. A. (2015). Biodegradable film for the targeted delivery of siRNA-loaded nanoparticles to vaginal immune cells. Mol Pharm, 12, 2889-2903.

AC CE P

Gubin, M. M., Zhang, X., Schuster, H., Caron, E., Ward, J. P., Noguchi, T., et al. (2014). Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature, 515, 577-581. Hallam, T. J., Wold, E., Wahl, A., & Smider, V. V. (2015). Antibody conjugates with unnatural amino acids. Mol Pharm, 12, 1848-1862. Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Songa, E. B., et al. (1993). Naturally occurring antibodies devoid of light chains. Nature, 363, 446-448. Hartley, J. A. (2011). The development of pyrrolobenzodiazepines as antitumour agents. Expert Opin Investig Drugs, 20, 733-744. Haylock, A. K., Spiegelberg, D., Mortensen, A. C., Selvaraju, R. K., Nilvebrant, J., Eriksson, O., et al. (2016). Evaluation of a novel type of imaging probe based on a recombinant bivalent miniantibody construct for detection of CD44v6-expressing squamous cell carcinoma. Int J Oncol, 48, 461-470.

ACCEPTED MANUSCRIPT Haylock, A. K., Spiegelberg, D., Nilvebrant, J., Sandstrom, K., & Nestor, M. (2014). In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging

PT

of squamous cell carcinoma: A dual-isotope study. EJNMMI Res, 4, 11. Holliger, P., & Hudson, P. J. (2005). Engineered antibody fragments and the rise of single domains. Nat

RI

Biotechnol, 23, 1126-1136.

SC

Hoogenboom, H. R. (2005). Selecting and screening recombinant antibody libraries. Nat Biotechnol, 23, 1105-1116.

NU

Hrkach, J., Von Hoff, D., Ali, M. M., Andrianova, E., Auer, J., Campbell, T., et al. (2012). Preclinical

MA

development and clinical translation of a PSMA-targeted docetaxel nanoparticle with a differentiated pharmacological profile. Sci Transl Med, 4, 128ra139. Huse, W. D., Sastry, L., Iverson, S. A., Kang, A. S., Alting-Mees, M., Burton, D. R., et al. (1989).

TE

D

Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science, 246, 1275-1281.

AC CE P

Ikeda, Y., & Taira, K. (2006). Ligand-targeted delivery of therapeutic siRNA. Pharm Res, 23, 1631-1640. James, L. C., Keeble, A. H., Khan, Z., Rhodes, D. A., & Trowsdale, J. (2007). Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function. Proc Natl Acad Sci U S A, 104, 6200-6205.

Janeway, C. A., Travers, P., Walport, M., & Shlomchik, M. J. (2001). Immunobiology: The immune system in health and disease (Vol. 2): Garland New York. Janssen, H. L., Reesink, H. W., Lawitz, E. J., Zeuzem, S., Rodriguez-Torres, M., Patel, K., et al. (2013). Treatment of HCV infection by targeting microRNA. N Engl J Med, 368, 1685-1694. Jeffrey, S. C., Burke, P. J., Lyon, R. P., Meyer, D. W., Sussman, D., Anderson, M., et al. (2013). A potent anti-CD70 antibody–drug conjugate combining a dimeric pyrrolobenzodiazepine drug with sitespecific conjugation technology. Bioconjug Chem, 24, 1256-1263. Juliano, R., Bauman, J., Kang, H., & Ming, X. (2009). Biological barriers to therapy with antisense and siRNA oligonucleotides. Mol Pharm, 6, 686-695.

ACCEPTED MANUSCRIPT Junutula, J. R., Raab, H., Clark, S., Bhakta, S., Leipold, D. D., Weir, S., et al. (2008). Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol,

PT

26, 925-932. Kalepu, S., & Nekkanti, V. (2015). Insoluble drug delivery strategies: Review of recent advances and

RI

business prospects. Acta Pharm Sin B, 5, 442-453.

SC

Kantarjian, H. M., Lioure, B., Kim, S. K., Atallah, E., Leguay, T., Kelly, K., et al. (2016). A phase II study of coltuximab ravtansine (SAR3419) monotherapy in patients with relapsed or refractory

NU

acute lymphoblastic leukemia. Clinical Lymphoma, Myeloma and Leukemia, 16, 139-145.

MA

Kantarjian, H. M., Thomas, D., Jorgensen, J., Kebriaei, P., Jabbour, E., Rytting, M., et al. (2013). Results of inotuzumab ozogamicin, a CD22 monoclonal antibody, in refractory and relapsed acute lymphocytic leukemia. Cancer, 119, 2728-2736.

D

Keefe, A. D., Pai, S., & Ellington, A. (2010). Aptamers as therapeutics. Nat Rev Drug Discov, 9, 537-550.

TE

Kemp, J. A., Shim, M. S., Heo, C. Y., & Kwon, Y. J. (2016). “Combo” nanomedicine: Co-delivery of

3-18.

AC CE P

multi-modal therapeutics for efficient, targeted, and safe cancer therapy. Adv Drug Deliv Rev, 98,

Kienast, Y., Klein, C., Scheuer, W., Raemsch, R., Lorenzon, E., Bernicke, D., et al. (2013). Ang-2VEGF-A CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2 functions simultaneously, mediates potent antitumor, antiangiogenic, and antimetastatic efficacy. Clin Cancer Res, 19, 6730-6740. Kim, A., Miura, Y., Ishii, T., Mutaf, O. F., Nishiyama, N., Cabral, H., et al. (2016). Intracellular delivery of charge-converted monoclonal antibodies by combinatorial design of block/homo polyion complex micelles. Biomacromolecules, 17, 446-453. Köhler, G., & Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, 495-497. Kontermann, R. E. (2012a). Antibody–cytokine fusion proteins. Arch Biochem Biophys, 526, 194-205. Kontermann, R. E. (2012b). Dual targeting strategies with bispecific antibodies. MAbs, 4, 182-197.

ACCEPTED MANUSCRIPT Kontermann, R. E. (2016). Half-life extended biotherapeutics. Expert Opin Biol Ther, 1-13. Kontermann, R. E., & Brinkmann, U. (2015). Bispecific antibodies. Drug Discov Today, 20, 838-847.

PT

Koshkin, A. A., Singh, S. K., Nielsen, P., Rajwanshi, V. K., Kumar, R., Meldgaard, M., et al. (1998). LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine,

RI

thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic

SC

acid recognition. Tetrahedron, 54, 3607-3630.

Kovtun, Y. V., Audette, C. A., Mayo, M. F., Jones, G. E., Doherty, H., Maloney, E. K., et al. (2010).

NU

Antibody-maytansinoid conjugates designed to bypass multidrug resistance. Cancer Res, 70,

MA

2528-2537.

Kreitman, R. J., & Pastan, I. (2011). Antibody fusion proteins: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox. Clin Cancer Res, 17, 6398-6405.

TE

D

Kumar, P., Ban, H. S., Kim, S. S., Wu, H., Pearson, T., Greiner, D. L., et al. (2008). T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell, 134, 577-586.

AC CE P

Kumar, S., Aaron, J., & Sokolov, K. (2008). Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties. Nat Protoc, 3, 314-320.

Kung Sutherland, M. S., Walter, R. B., Jeffrey, S. C., Burke, P. J., Yu, C., Kostner, H., et al. (2013). SGN-CD33A: A novel CD33-targeting antibody–drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML. Blood, 122, 1455-1463. Labrijn, A. F., Buijsse, A. O., van den Bremer, E. T., Verwilligen, A. Y., Bleeker, W. K., Thorpe, S. J., et al. (2009). Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo. Nat Biotechnol, 27, 767-771. Labrijn, A. F., Meesters, J. I., de Goeij, B. E., van den Bremer, E. T., Neijssen, J., van Kampen, M. D., et al. (2013). Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. Proc Natl Acad Sci U S A, 110, 5145-5150.

ACCEPTED MANUSCRIPT Lambert, J. M. (2013). Drug-conjugated antibodies for the treatment of cancer. British Journal of Clinical Pharmacology, 76, 248-262.

PT

Lehar, S. M., Pillow, T., Xu, M., Staben, L., Kajihara, K. K., Vandlen, R., et al. (2015). Novel antibodyantibiotic conjugate eliminates intracellular S. aureus. Nature, 527, 323-328.

RI

Lerner, R. A. (2016). Combinatorial antibody libraries: New advances, new immunological insights. Nat

SC

Rev Immunol.

Leung, K.-M., Batey, S., Rowlands, R., Isaac, S. J., Jones, P., Drewett, V., et al. (2015). A HER2-specific

MA

apoptosis. Mol Ther, 23, 1722-1733.

NU

modified Fc fragment (Fcab) induces antitumor effects through degradation of HER2 and

Levin, D., Golding, B., Strome, S. E., & Sauna, Z. E. (2015). Fc fusion as a platform technology: Potential for modulating immunogenicity. Trends Biotechnol, 33, 27-34.

TE

D

Lewis Phillips, G. D., Li, G., Dugger, D. L., Crocker, L. M., Parsons, K. L., Mai, E., et al. (2008). Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody–cytotoxic drug

AC CE P

conjugate. Cancer Res, 68, 9280-9290. Li, X., Patterson, J. T., Sarkar, M., Pedzisa, L., Kodadek, T., Roush, W. R., et al. (2015). Site-specific dual antibody conjugation via engineered cysteine and selenocysteine residues. Bioconjug Chem, 26, 2243-2248.

Linke, R., Klein, A., & Seimetz, D. (2010). Catumaxomab: Clinical development and future directions. MAbs, 2, 129-136. Liu, T., Liu, Y., Wang, Y., Hull, M., Schultz, P. G., & Wang, F. (2014). Rational design of CXCR4 specific antibodies with elongated CDRs. J Am Chem Soc, 136, 10557-10560. Liu, T., Zhang, Y., Liu, Y., Wang, Y., Jia, H., Kang, M., et al. (2015). Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists. Proc Natl Acad Sci U S A, 112, 1356-1361. Liu, Y., Zhang, X., Han, C., Wan, G., Huang, X., Ivan, C., et al. (2015). TP53 loss creates therapeutic vulnerability in colorectal cancer. Nature, 520, 697-701.

ACCEPTED MANUSCRIPT Lobner, E., Traxlmayr, M. W., Obinger, C., & Hasenhindl, C. (2016). Engineered IgG1-Fc – one fragment to bind them all. Immunol Rev, 270, 113-131.

PT

Lopus, M., Oroudjev, E., Wilson, L., Wilhelm, S., Widdison, W., Chari, R., et al. (2010). Maytansine and cellular metabolites of antibody-maytansinoid conjugates strongly suppress microtubule

RI

dynamics by binding to microtubules. Mol Cancer Ther, 9, 2689-2699.

SC

Lu, H., Wang, D., Kazane, S., Javahishvili, T., Tian, F., Song, F., et al. (2013). Site-specific antibody– polymer conjugates for siRNA delivery. J Am Chem Soc, 135, 13885-13891.

NU

Lundqvist, M., Stigler, J., Elia, G., Lynch, I., Cedervall, T., & Dawson, K. A. (2008). Nanoparticle size

MA

and surface properties determine the protein corona with possible implications for biological impacts. Proc Natl Acad Sci U S A, 105, 14265-14270. Lutz, R. J. (2015). Targeting the folate receptor for the treatment of ovarian cancer. Transl Cancer Res, 4,

TE

D

118-126.

Mahoney, K. M., Rennert, P. D., & Freeman, G. J. (2015). Combination cancer immunotherapy and new

AC CE P

immunomodulatory targets. Nat Rev Drug Discov, 14, 561-584. Mallery, D. L., McEwan, W. A., Bidgood, S. R., Towers, G. J., Johnson, C. M., & James, L. C. (2010). Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21). Proc Natl Acad Sci U S A, 107, 19985-19990. Martins, J. P., Kennedy, P. J., Santos, H. A., Barrias, C., & Sarmento, B. (2016). A comprehensive review of the neonatal Fc receptor and its application in drug delivery. Pharmacol Ther, 161, 22-39. Maruani, A., Smith, M. E. B., Miranda, E., Chester, K. A., Chudasama, V., & Caddick, S. (2015). A plugand-play approach to antibody-based therapeutics via a chemoselective dual click strategy. Nat Commun, 6, 6645. Merchant, A. M., Zhu, Z., Yuan, J. Q., Goddard, A., Adams, C. W., Presta, L. G., et al. (1998). An efficient route to human bispecific IgG. Nat Biotechnol, 16, 677-681. Metz, S., Haas, A. K., Daub, K., Croasdale, R., Stracke, J., Lau, W., et al. (2011). Bispecific digoxigeninbinding antibodies for targeted payload delivery. Proc Natl Acad Sci U S A, 108, 8194-8199.

ACCEPTED MANUSCRIPT Ming, X., & Laing, B. (2015). Bioconjugates for targeted delivery of therapeutic oligonucleotides. Adv Drug Deliv Rev, 87, 81-89.

PT

Moldenhauer, G., Salnikov, A. V., Luttgau, S., Herr, I., Anderl, J., & Faulstich, H. (2012). Therapeutic potential of amanitin-conjugated anti-epithelial cell adhesion molecule monoclonal antibody

RI

against pancreatic carcinoma. J Natl Cancer Inst, 104, 622-634.

SC

Moore, P. A., Zhang, W., Rainey, G. J., Burke, S., Li, H., Huang, L., et al. (2011). Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma.

NU

Blood, 117, 4542-4551.

MA

Moran, N. (2011). Boehringer splashes out on bispecific antibody platforms. Nat Biotechnol, 29, 5-6. Moreno, P. M. D., & Pêgo, A. P. (2014). Therapeutic antisense oligonucleotides against cancer: Hurdling to the clinic. Front Chem, 2, 87.

TE

D

Morishita, M., & Peppas, N. A. (2006). Is the oral route possible for peptide and protein drug delivery? Drug Discov Today, 11, 905-910.

AC CE P

Mullard, A. (2015). 2014 FDA drug approvals. Nat Rev Drug Discov, 14, 77-81. Müller, P., Kreuzaler, M., Khan, T., Thommen, D. S., Martin, K., Glatz, K., et al. (2015). Trastuzumab emtansine (T-DM1) renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade. Sci Transl Med, 7, 315ra188.

Nagorsen, D., Kufer, P., Baeuerle, P. A., & Bargou, R. (2012). Blinatumomab: A historical perspective. Pharmacol Ther, 136, 334-342. Nascimento, A. V., Gattacceca, F., Singh, A., Bousbaa, H., Ferreira, D., Sarmento, B., et al. (2016). Biodistribution and pharmacokinetics of Mad2 siRNA-loaded EGFR-targeted chitosan nanoparticles in cisplatin sensitive and resistant lung cancer models. Nanomedicine (Lond), 11, 767-781. Nascimento, A. V., Singh, A., Bousbaa, H., Ferreira, D., Sarmento, B., & Amiji, M. M. (2014). Mad2 checkpoint gene silencing using epidermal growth factor receptor-targeted chitosan nanoparticles in non-small cell lung cancer model. Mol Pharm, 11, 3515-3527.

ACCEPTED MANUSCRIPT Newnham, L. E., Wright, M. J., Holdsworth, G., Kostarelos, K., Robinson, M. K., Rabbitts, T. H., et al. (2015). Functional inhibition of beta-catenin-mediated Wnt signaling by intracellular VHH

PT

antibodies. MAbs, 7, 180-191. Nilvebrant, J., Kuku, G., Bjorkelund, H., & Nestor, M. (2012). Selection and in vitro characterization of

RI

human CD44v6-binding antibody fragments. Biotechnol Appl Biochem, 59, 367-380.

SC

Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., & Schultz, P. G. (1989). A general method for sitespecific incorporation of unnatural amino acids into proteins. Science, 244, 182-188.

NU

Ohishi, I., Sakaguchi, G., Riemann, H., Behymer, D., & Hurvell, B. (1979). Antibodies to Clostridium

MA

botulinum toxins in free-living birds and mammals. J Wildl Dis, 15, 3-9. Orlandi, R., Güssow, D. H., Jones, P. T., & Winter, G. (1989). Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc Natl Acad Sci U S A, 86, 3833-3837.

TE

D

Ouyang, J. (2013). Drug-to-antibody ratio (DAR) and drug load distribution by hydrophobic interaction chromatography and reversed phase high-performance liquid chromatography. Methods Mol Biol,

AC CE P

1045, 275-283.

Pardoll, D. M. (2012). The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 12, 252-264.

Park, J. W., Hong, K., Kirpotin, D. B., Colbern, G., Shalaby, R., Baselga, J., et al. (2002). Anti-HER2 immunoliposomes: Enhanced efficacy attributable to targeted delivery. Clin Cancer Res, 8, 11721181. Pearson, R. M., Juettner, V., & Hong, S. (2014). Biomolecular corona on nanoparticles: A survey of recent literature and its implications in targeted drug delivery. Front Chem, 2, 108. Pereira, P. M., Korsak, B., Sarmento, B., Schneider, R. J., Fernandes, R., & Tome, J. P. (2015). Antibodies armed with photosensitizers: From chemical synthesis to photobiological applications. Org Biomol Chem, 13, 2518-2529. Perez-Martinez, D., Tanaka, T., & Rabbitts, T. H. (2010). Intracellular antibodies and cancer: New technologies offer therapeutic opportunities. Bioessays, 32, 589-598.

ACCEPTED MANUSCRIPT Perrino, E., Steiner, M., Krall, N., Bernardes, G. J. L., Pretto, F., Casi, G., et al. (2014). Curative Properties of Noninternalizing Antibody–Drug Conjugates Based on Maytansinoids. Cancer Res,

PT

74, 2569-2578. Peyvandi, F., Scully, M., Kremer Hovinga, J. A., Cataland, S., Knöbl, P., Wu, H., et al. (2016).

RI

Caplacizumab for acquired thrombotic thrombocytopenic purpura. N Engl J Med, 374, 511-522.

SC

Pridgen, E. M., Alexis, F., Kuo, T. T., Levy-Nissenbaum, E., Karnik, R., Blumberg, R. S., et al. (2013). Transepithelial transport of Fc-targeted nanoparticles by the neonatal fc receptor for oral delivery.

NU

Sci Transl Med, 5, 213ra167.

MA

Puthenveetil, S., Musto, S., Loganzo, F., Tumey, L. N., O'Donnell, C. J., & Graziani, E. I. (2016). Development of solid-phase site-specific conjugation and its application towards generation of dual labeled antibody and Fab drug conjugates. Bioconjug Chem, 27, 1030-1039.

Ther, 13, 1347-1352.

TE

D

Raju, T. S., & Strohl, W. R. (2013). Potential therapeutic roles for antibody mixtures. Expert Opin Biol

AC CE P

Reilly, R. M., Domingo, R., & Sandhu, J. (1997). Oral delivery of antibodies. Future pharmacokinetic trends. Clin Pharmacokinet, 32, 313-323. Ricart, A. D. (2011). Antibody-drug conjugates of calicheamicin derivative: Gemtuzumab ozogamicin and inotuzumab ozogamicin. Clin Cancer Res, 17, 6417-6427. Rodgers, K. R., & Chou, R. C. (2016). Therapeutic monoclonal antibodies and derivatives: Historical perspectives and future directions. Biotechnol Adv, 34, 1149-1158. Rogers, L. M., Veeramani, S., & Weiner, G. J. (2014). Complement in monoclonal antibody therapy of cancer. Immunol Res, 59, 203-210. Roopenian, D. C., & Akilesh, S. (2007). FcRn: The neonatal Fc receptor comes of age. Nat Rev Immunol, 7, 715-725. Salvati, A., Pitek, A. S., Monopoli, M. P., Prapainop, K., Bombelli, F. B., Hristov, D. R., et al. (2013). Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nat Nanotechnol, 8, 137-143.

ACCEPTED MANUSCRIPT Sapsford, K. E., Algar, W. R., Berti, L., Gemmill, K. B., Casey, B. J., Oh, E., et al. (2013). Functionalizing nanoparticles with biological molecules: Developing chemistries that facilitate

PT

nanotechnology. Chem Rev, 113, 1904-2074. Sarmento, B., Ribeiro, A., Veiga, F., Sampaio, P., Neufeld, R., & Ferreira, D. (2007). Alginate/chitosan

RI

nanoparticles are effective for oral insulin delivery. Pharm Res, 24, 2198-2206.

SC

Schaefer, W., Regula, J. T., Bahner, M., Schanzer, J., Croasdale, R., Durr, H., et al. (2011). Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG

NU

antibodies. Proc Natl Acad Sci U S A, 108, 11187-11192.

MA

Schneider, B., Grote, M., John, M., Haas, A., Bramlage, B., lckenstein, L. M., et al. (2012). Targeted siRNA delivery and mRNA knockdown mediated by bispecific digoxigenin-binding antibodies. Mol Ther Nucleic Acids, 1, e46.

TE

D

Sellmann, C., Doerner, A., Knuehl, C., Rasche, N., Sood, V., Krah, S., et al. (2016). Balancing selectivity and efficacy of bispecific EGFR x c-MET antibodies and antibody-drug conjugates. J Biol Chem.

AC CE P

Senter, P. D., & Sievers, E. L. (2012). The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat Biotechnol, 30, 631-637.

Shao, K., Singha, S., Clemente-Casares, X., Tsai, S., Yang, Y., & Santamaria, P. (2015). Nanoparticlebased immunotherapy for cancer. ACS Nano, 9, 16-30. Sharma, P., & Allison, J. P. (2015). Immune checkpoint targeting in cancer therapy: Toward combination strategies with curative potential. Cell, 161, 205-214. Sharma, P., Brown, S., Walter, G., Santra, S., & Moudgil, B. (2006). Nanoparticles for bioimaging. Adv Colloid Interface Sci, 123–126, 471-485. Shen, B. Q., Xu, K., Liu, L., Raab, H., Bhakta, S., Kenrick, M., et al. (2012). Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol, 30, 184189.

ACCEPTED MANUSCRIPT Singal , P. K., & Iliskovic , N. (1998). Doxorubicin-induced cardiomyopathy. N Engl J Med, 339, 900905.

PT

Smith, L. M., Nesterova, A., Ryan, M. C., Duniho, S., Jonas, M., Anderson, M., et al. (2008). CD133/prominin-1 is a potential therapeutic target for antibody-drug conjugates in hepatocellular

RI

and gastric cancers. Br J Cancer, 99, 100-109.

SC

Sochaj, A. M., Świderska, K. W., & Otlewski, J. (2015). Current methods for the synthesis of homogeneous antibody–drug conjugates. Biotechnol Adv, 33, 775-784.

NU

Sokolov, K., Follen, M., Aaron, J., Pavlova, I., Malpica, A., Lotan, R., et al. (2003). Real-time vital

MA

optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles. Cancer Res, 63, 1999-2004.

Son, S., Lee, W. R., Joung, Y. K., Kwon, M. H., Kim, Y. S., & Park, K. D. (2009). Optimized stability

TE

D

retention of a monoclonal antibody in the PLGA nanoparticles. Int J Pharm, 368, 178-185. Song, E., Zhu, P., Lee, S. K., Chowdhury, D., Kussman, S., Dykxhoorn, D. M., et al. (2005). Antibody

AC CE P

mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat Biotechnol, 23, 709-717.

Sosnik, A., das Neves, J., & Sarmento, B. (2014). Mucoadhesive polymers in the design of nano-drug delivery systems for administration by non-parenteral routes: A review. Prog Polym Sci, 39, 2030-2075.

Sousa, F., Castro, P., Fonte, P., Kennedy, P. J., Neves-Petersen, M. T., & Sarmento, B. (2016). Nanoparticles for the delivery of therapeutic antibodies: Dogma or promising strategy? Expert Opin Drug Deliv, 1-14. Staerz, U. D., & Bevan, M. J. (1986). Hybrid hybridoma producing a bispecific monoclonal antibody that can focus effector T-cell activity. Proc Natl Acad Sci U S A, 83, 1453-1457. Stech, M., & Kubick, S. (2015). Cell-free synthesis meets antibody production: A review. Antibodies (Basel), 4, 12.

ACCEPTED MANUSCRIPT Steiner, M., & Neri, D. (2011). Antibody-radionuclide conjugates for cancer therapy: Historical considerations and new trends. Clin Cancer Res, 17, 6406-6416.

PT

Strop, P., Liu, S. H., Dorywalska, M., Delaria, K., Dushin, R. G., Tran, T. T., et al. (2013). Location matters: Site of conjugation modulates stability and pharmacokinetics of antibody drug

RI

conjugates. Chem Biol, 20, 161-167.

SC

Sun, H., & Zu, Y. (2015). Aptamers and their applications in nanomedicine. Small, 11, 2352-2364. Sun, Y., Shukla, G., Pero, S. C., Currier, E., Sholler, G., & Krag, D. (2012). Single tumor imaging with

NU

multiple antibodies targeting different antigens. Biotechniques, 52.

MA

Tai, Y. T., Mayes, P. A., Acharya, C., Zhong, M. Y., Cea, M., Cagnetta, A., et al. (2014). Novel anti-Bcell maturation antigen antibody-drug conjugate (GSK2857916) selectively induces killing of multiple myeloma. Blood, 123, 3128-3138.

TE

D

Tanaka, T., & Rabbitts, T. H. (2012). Intracellular antibody capture (IAC) methods for single domain antibodies. Methods Mol Biol, 911, 151-173.

AC CE P

Teachey, D. T., Rheingold, S. R., Maude, S. L., Zugmaier, G., Barrett, D. M., Seif, A. E., et al. (2013). Cytokine release syndrome after blinatumomab treatment related to abnormal macrophage activation and ameliorated with cytokine-directed therapy. Blood, 121, 5154-5157. Teicher, B. A., & Chari, R. V. J. (2011). Antibody conjugate therapeutics: Challenges and potential. Clin Cancer Res, 17, 6389-6397.

Tian, F., Lu, Y., Manibusan, A., Sellers, A., Tran, H., Sun, Y., et al. (2014). A general approach to sitespecific antibody drug conjugates. Proc Natl Acad Sci U S A, 111, 1766-1771. Tian, W. M., Zhang, C. L., Hou, S. P., Yu, X., Cui, F. Z., Xu, Q. Y., et al. (2005). Hyaluronic acid hydrogel as Nogo-66 receptor antibody delivery system for the repairing of injured rat brain: in vitro. J Control Release, 102, 13-22. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature, 302, 575-581.

ACCEPTED MANUSCRIPT Topp, M. S., Gokbuget, N., Stein, A. S., Zugmaier, G., O'Brien, S., Bargou, R. C., et al. (2015). Safety and activity of blinatumomab for adult patients with relapsed or refractory B-precursor acute

PT

lymphoblastic leukaemia: A multicentre, single-arm, phase 2 study. Lancet Oncol, 16, 57-66. Topp, M. S., Kufer, P., Gokbuget, N., Goebeler, M., Klinger, M., Neumann, S., et al. (2011). Targeted

RI

therapy with the T-cell-engaging antibody blinatumomab of chemotherapy-refractory minimal

SC

residual disease in B-lineage acute lymphoblastic leukemia patients results in high response rate and prolonged leukemia-free survival. J Clin Oncol, 29, 2493-2498.

NU

Uhrich, K. E., Cannizzaro, S. M., Langer, R. S., & Shakesheff, K. M. (1999). Polymeric systems for

MA

controlled drug release. Chem Rev, 99, 3181-3198.

Vadnais, M. L., & Smider, V. V. (2016). Bos taurus ultralong CDR H3 antibodies. Curr Opin Struct Biol, 38, 62-67.

TE

D

van der Lee, M. M., Groothuis, P. G., Ubink, R., van der Vleuten, M. A., van Achterberg, T. A., Loosveld, E. M., et al. (2015). The preclinical profile of the duocarmycin-based HER2-targeting

AC CE P

ADC SYD985 predicts for clinical benefit in low HER2-expressing breast cancers. Mol Cancer Ther, 14, 692-703.

van der Meel, R., Vehmeijer, L. J., Kok, R. J., Storm, G., & van Gaal, E. V. (2013). Ligand-targeted particulate nanomedicines undergoing clinical evaluation: Current status. Adv Drug Deliv Rev, 65, 1284-1298.

van der Neut Kolfschoten, M., Schuurman, J., Losen, M., Bleeker, W. K., Martinez-Martinez, P., Vermeulen, E., et al. (2007). Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange. Science, 317, 1554-1557. Vankemmelbeke, M., & Durrant, L. (2016). Third-generation antibody drug conjugates for cancer therapy – a balancing act. Ther Deliv, 7, 141-144. Vasconcelos, T., Marques, S., das Neves, J., & Sarmento, B. (2016). Amorphous solid dispersions: Rational selection of a manufacturing process. Adv Drug Deliv Rev, 100, 85-101.

ACCEPTED MANUSCRIPT Vaysburd, M., Watkinson, R. E., Cooper, H., Reed, M., O'Connell, K., Smith, J., et al. (2013). Intracellular antibody receptor TRIM21 prevents fatal viral infection. Proc Natl Acad Sci U S A,

PT

110, 12397-12401. Verma, S., Miles, D., Gianni, L., Krop, I. E., Welslau, M., Baselga, J., et al. (2012). Trastuzumab

RI

emtansine for HER2-positive advanced breast cancer. N Engl J Med, 367, 1783-1791.

SC

Vllasaliu, D., Alexander, C., Garnett, M., Eaton, M., & Stolnik, S. (2012). Fc-mediated transport of nanoparticles across airway epithelial cell layers. J Control Release, 158, 479-486.

NU

Wahlestedt, C. (2013). Targeting long non-coding RNA to therapeutically upregulate gene expression.

MA

Nat Rev Drug Discov, 12, 433-446.

Wakankar, A., Chen, Y., Gokarn, Y., & Jacobson, F. S. (2011). Analytical methods for physicochemical characterization of antibody drug conjugates. MAbs, 3, 161-172.

TE

D

Walker, I., Irwin, W. J., & Akhtar, S. (1995). Improved cellular delivery of antisense oligonucleotides using transferrin receptor antibody-oligonucleotide conjugates. Pharm Res, 12, 1548-1553.

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Wang, F., Ekiert, D. C., Ahmad, I., Yu, W., Zhang, Y., Bazirgan, O., et al. (2013). Reshaping antibody diversity. Cell, 153, 1379-1393. Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T., & Winter, G. (1989). Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature, 341, 544-546.

Watanabe, C. M., Supekova, L., & Schultz, P. G. (2002). Transcriptional effects of the potent enediyne anti-cancer agent Calicheamicin gamma(I)(1). Chem Biol, 9, 245-251. Wiggins, B., Liu-Shin, L., Yamaguchi, H., & Ratnaswamy, G. (2015). Characterization of cysteine-linked conjugation profiles of immunoglobulin G1 and immunoglobulin G2 antibody-drug conjugates. J Pharm Sci, 104, 1362-1372. Winkler, J. (2013). Oligonucleotide conjugates for therapeutic applications. Ther Deliv, 4, 791-809. Wittrup, A., & Lieberman, J. (2015). Knocking down disease: A progress report on siRNA therapeutics. Nat Rev Genet, 16, 543-552.

ACCEPTED MANUSCRIPT Wu, S.-C., Chen, Y.-J., Wang, H.-C., Chou, M.-Y., Chang, T.-Y., Yuan, S.-S., et al. (2016). Bispecific antibody conjugated manganese-based magnetic engineered iron oxide for imaging of HER2/neu-

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and EGFR-expressing tumors. Theranostics, 6, 118-130. Xu, K., Liu, L., Dere, R., Mai, E., Erickson, R., Hendricks, A., et al. (2013). Characterization of the drug-

RI

to-antibody ratio distribution for antibody–drug conjugates in plasma/serum. Bioanalysis, 5,

SC

1057-1071.

Xu, Y., Lee, J., Tran, C., Heibeck, T. H., Wang, W. D., Yang, J., et al. (2015). Production of bispecific

NU

antibodies in "knobs-into-holes" using a cell-free expression system. MAbs, 7, 231-242.

MA

Yao, V. J., D'Angelo, S., Butler, K. S., Theron, C., Smith, T. L., Marchio, S., et al. (2016). Ligandtargeted theranostic nanomedicines against cancer. J Control Release. Yea, K., Zhang, H., Xie, J., Jones, T. M., Lin, C. W., Francesconi, W., et al. (2015). Agonist antibody that

TE

D

induces human malignant cells to kill one another. Proc Natl Acad Sci U S A, 112, E6158-6165. Younes , A., Bartlett , N. L., Leonard , J. P., Kennedy , D. A., Lynch , C. M., Sievers , E. L., et al. (2010).

AC CE P

Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N Engl J Med, 363, 1812-1821.

Yu, Y. J., Zhang, Y., Kenrick, M., Hoyte, K., Luk, W., Lu, Y., et al. (2011). Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target. Sci Transl Med, 3, 84ra44. Zeng, J., Li, H. C., Tanaka, T., & Rabbitts, T. H. (2015). Selection of human single domain antibodies recognizing the CMYC protein using enhanced intracellular antibody capture. J Immunol Methods, 426, 140-143. Zeng, J., Zhang, J., Tanaka, T., & Rabbitts, T. (2013). Single domain antibody fragments as drug surrogates targeting protein–protein interactions inside cells. Antibodies (Basel), 2, 306-320. Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., et al. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163, 759-771.

ACCEPTED MANUSCRIPT Zhang, H., Wilson, I. A., & Lerner, R. A. (2012). Selection of antibodies that regulate phenotype from intracellular combinatorial antibody libraries. Proc Natl Acad Sci U S A, 109, 15728-15733.

PT

Zhang, Y., Liu, Y., Wang, Y., Schultz, P. G., & Wang, F. (2015). Rational design of humanized dualagonist antibodies. J Am Chem Soc, 137, 38-41.

RI

Zhang, Y., Wang, D., de Lichtervelde, L., Sun, S. B., Smider, V. V., Schultz, P. G., et al. (2013).

SC

Functional antibody CDR3 fusion proteins with enhanced pharmacological properties. Angew Chem Int Ed Engl, 52, 8295-8298.

NU

Zhang, Y., Zhang, Y. F., Bryant, J., Charles, A., Boado, R. J., & Pardridge, W. M. (2004). Intravenous

MA

RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer. Clin Cancer Res, 10, 3667-3677. Zhang, Y., Zhu, C., & Pardridge, W. M. (2002). Antisense gene therapy of brain cancer with an artificial

TE

D

virus gene delivery system. Mol Ther, 6, 67-72. Zhao, A., Tohidkia, M. R., Siegel, D. L., Coukos, G., & Omidi, Y. (2016). Phage antibody display

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libraries: a powerful antibody discovery platform for immunotherapy. Crit Rev Biotechnol, 36, 276-289.

Zhu, G., Niu, G., & Chen, X. (2015). Aptamer–Drug Conjugates. Bioconjug Chem, 26, 2186-2197. Zhu, Z., Ramakrishnan, B., Li, J., Wang, Y., Feng, Y., Prabakaran, P., et al. (2014). Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar. MAbs, 6, 1190-1200. Zimmerman, E. S., Heibeck, T. H., Gill, A., Li, X., Murray, C. J., Madlansacay, M. R., et al. (2014). Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjug Chem, 25, 351-361. Zimmerman, Z., Maniar, T., & Nagorsen, D. (2015). Unleashing the clinical power of T cells: CD19/CD3 bi-specific T cell engager (BiTE(R)) antibody construct blinatumomab as a potential therapy. Int Immunol, 27, 31-37.

ACCEPTED MANUSCRIPT Zuris, J. A., Thompson, D. B., Shu, Y., Guilinger, J. P., Bessen, J. L., Hu, J. H., et al. (2015). Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in

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vivo. Nat Biotechnol, 33, 73-80.

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Fig. 1. The structural components of (a) a whole monoclonal antibody (mAb)/IgG include the fragment antigen-binding (Fab) and fragment crystallizable (Fc) regions that are composed of the constant (CH and

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CL) and variable (VH and VL) domains each containing a disulfide bridge (yellow curves). Direct antigen

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binding to the target antigen is made through the complementarity determining regions (CDRs) of the Fab fragment and immune effector function is mediated by the Fc fragment. (b) The most common antibody

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fragment derivatives are: F(ab’)2, Fab, single chain variable fragment (scFv) and single-domain antibodies

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(sdAbs) from llamas (VHH), sharks (VNAR) or other species.

Fig. 2. There are several characteristics that need to be addressed when developing novel antibody-drug

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conjugates (ADCs) with maximum therapeutic efficacy. (a) The antibody vehicle qualities to address are: high affinity and specificity, human scaffolding to minimize immunogenicity and Fc presence for

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possibly effector function and long circulation times. (b) The mode of action of the warhead passenger. Common microtubule inhibitors are the auristatins and maytansines, while the common DNA damage agents are the calicheamicins, duocarmycins and pyrrolobenzodiazepines (PBDs). The α-amanitin peptide is highly potent RNA polymerase II inhibitor. Alternative payloads include radionuclides, fluorescent molecules, photosensitizers and antibiotics. (c) The drug-to-antibody ratio (DAR) needs to strike a balance between potency and aggregation. (d) The linker qualities include the direct connection to the antibody, the linker polarity and trigger mechanism and the self-immolative spacer. Adapted with permission from (Flygare et al., 2013). (e) The location of the payload on the mAb can greatly influence ADC potency. Thus, site specific strategies have been developed by exploiting naturally-present glycans or by engineering the mAb with cysteines or unnatural amino acids (uAAs) in specific locations.

Fig. 3. A handful of bispecific mAb formats as whole IgG (a) or fragment-based (b) antibodies are presented here. In the context of cancer, these bispecific mAbs often target CD3 with the aim of recruiting

ACCEPTED MANUSCRIPT cytotoxic T cells to tumors overexpressing a particular receptor. Whole IgGs have the added advantage of recruiting Fc receptor (FcR) expressing immune effector cells. The “knobs-into-hole” (KiH) technology

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allows for heavy chain heterodimerization, and the CrossMAb technology exchanges the CH1 and CL domains of one of the antibody halves. The bispecific T cell engager (BiTE) and dual-affinity re-targeting

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(DART®) formats are structurally similar but with minor differences in the flexible connection between

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the variable domains. Fc domain with antigen binding (Fcab™) technology expresses antigen binding sites in the Fc region but is still able to retain binding to FcR and FcRn for effector function and escape from

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lysosomal degradation. TandAbs® attempt to exploit avidity through their tetravalent nature.

Fig. 4. Many factors dictate the in vivo behavior of ligand-decorated nanoparticles (NPs) including those associated with the nanoparticle (NP; left) as well as the ligand (right). The specific NP qualities to keep

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in mind are size, shape, charge and hydrophobicity, and these properties can also dictate the amount and composition of the protein corona. The specific ligand (i.e. mAb) qualities to consider are size and

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affinity, as well as density and orientation when coupled to the NP. Inspired by (Bertrand et al., 2014).

Fig. 5. Encapsulating mAbs inside nanoparticles (NPs) have several advantages such as sustained release, intracellular delivery, non-parenteral administration and enhanced storage capacity.

Fig. 6. Kaplan-Meier survival curves illustrating the potential of combining immune checkpoint blockade with (a) a genomically targeted therapy (reprinted with permission from (Sharma & Allison, 2015)) or (b) an antibody-drug conjugate (ADC) that stimulates T cell recruitment or dendritic cell activation (reprinted with permission from (Gerber et al., 2016)).

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