Antibody Therapy

Antibody Therapy Brandon G Smaglo, Dalal Aldeghaither, and Louis M Weiner, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA Ó 2016 Elsevier Ltd. All rights reserved.

Abstract Antibody therapy has emerged as an important weapon in the anticancer armament. These therapies take advantage of their ability to target proteins uniquely expressed on the surface of cancer cells. A number of monoclonal antibodies, which target a specific tumor cell surface protein and in doing so result in cellular destruction, have been approved for clinical use, and many more continue to be developed. Expanding upon monoclonal antibody therapy are immunoconjugates, which deliver a therapeutic entity to the cancer cell with high precision by linking it to the antibody. Cancer antibody therapy may also target the immune system, preventing its natural downregulation and thus augmenting the body’s natural immune response against the cancer. An important component of all of these therapies is the design of antibodies that specifically target specific cancer epitopes, and much of the ongoing research into cancer antibody therapy is focused on the engineering antibodies that bind with high specificity.

Antibodies Targeting Cancer Antibodies Already Approved Antibodies are proteins secreted by B cells in response to antigens. The basic building blocks of the antibody structure are the heavy and light chains that together shape two functional units: the constant region (Fc) and the fragment of antigen-binding region (Fab) (Weiner et al., 2010). The Fab domain is composed of three hypervariable complementarity-determining regions (CDRs) which serve as determinants of antibody specificity and antigen recognition (Weiner et al., 2010). The Fc domain acts as a link between the Fab’s capacity to bind antigens and immune system activation and recruit effector cells by engaging their Fcg. Antibodies have five structural categories: IgM, IgG, IgA, IgE, and IgD, based on their heavy chain compositions and Fc regions. IgG is the most commonly used antibody structure for monoclonal antibody therapy.

Monoclonal Antibodies Monoclonal antibodies were first produced in 1975 using the then newly developed hybridoma technology (Köhler and Milstein, 1975). Their murine origin resulted in immunogenicity in humans and accompanying inability to induce robust human immune effector responses against the targeted tumor antigens and cancer cells, limiting their clinical application (Shawler et al., 1985; Khazaeli et al., 1994). Scientific and technical advancements have enabled the design and development of chimeric antibodies, composed of murine variable regions transferred onto human Fc regions, humanized antibodies, with murine CDRs grafted onto a human antibody scaffold, and fully human antibodies derived from xenogeneic mice or by recombinant human antibody display technologies (Robinson et al., 2004). The modified structures constitute improvements in target binding, effector potency, and reduced immunogenicity. These modified antibody structures now dominate the human antibody therapy landscape. To date, 14 conjugated and unconjugated mAbs have been FDA approved for the treatment of cancer (Table 1).

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The chimeric monoclonal antibody rituximab targets and destroys B cells by binding the surface protein CD20. One of the first monoclonal antibodies to enter into clinical practice, rituximab is generally well-tolerated and routinely used as a part of systemic treatment for a number of B cell leukemias and lymphomas, in both first and refractory lines of therapy (Marcus et al., 2005; Coiffier et al., 1998; Coiffier et al., 2002; Tam et al., 2008; Pfreundschuh et al., 2011). Ofatumumab, a humanized monoclonal antibody, also targets and binds CD20 and, in doing so, appears to inhibit early-stage activation of the B cell lymphocytes. In the treatment of chemotherapyrefractory CLL, ofatumumab was found to be active and welltolerated (Wierda et al., 2010). Ofatumumab is being explored in the first line treatment of CLL and also in other B cell malignancies, although questions of toxicity may limit its development in all clinical settings, and it is generally reserved for the treatment of rituximab-refractory B cell malignancies (Shanafelt et al., 2013; Czuczman et al., 2012; Ujjani et al., 2015). Alemtuzumab is a humanized monoclonal antibody that targets CD52; this surface protein is highly expressed on lymphocytes. A 42% reduction in risk of disease progression or death was reported when alemtuzumab was used in firstline therapy for patients with CLL, compared to standard therapy with chlorambucil (Hillmen et al., 2007). Presently, alemtuzumab is not available for clinical use, though it may be developed for future use in other diseases. Cetuximab, a chimeric monoclonal antibody, and panitumumab, a fully humanized monoclonal antibody, both target the epidermal growth factor receptor (EGFR). EGFR is overexpressed on the surface of some cancer cells. When ligands bind to EGFR, several intracellular protein pathways are stimulated that ultimately result in cellular growth and proliferation. In metastatic colorectal cancer, cetuximab and panitumumab have demonstrated benefit when added to standard chemotherapy regimens (Van Cutsem et al., 2009; Bokemeyer et al., 2009). Importantly, in colorectal cancer, these antibodies are only effective in patients with tumors that do not possess mutations in the Ras protein; Ras mutations result in

Encyclopedia of Immunobiology, Volume 4

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Table 1 Monoclonal antibodies approved for use, as identified by target and cancer type, with the studies that support their use in these diseases Monoclonal antibody

Target

Disease type

References

Rituximab

CD20

B cell lymphoma, CLL

Ofatumumab

CD20

B cell lymphoma, CLL

Alemtuzumab Cetuximab

CD52 EGFR

Panitumumab Trastuzumab

EGFR HER2

Ziv-aflibercept Bevacizumab

VEGF VEGF

Ramucirumab

VEGF-R2

B cell malignancies Colorectal cancer Head/neck squamous cell cancer Nonsmall cell lung cancer Colorectal cancer Breast cancer Gastric cancer Colorectal cancer Colorectal cancer Nonsmall cell lung cancer Glioblastoma Gastric cancer Colon cancer, lung cancer

Marcus et al. (2005), Coiffier et al. (1998), Coiffier et al. (2002), Tam et al. (2008), and Pfreundschuh et al. (2011) Wierda et al. (2010), Shanafelt et al. (2013), Czuczman et al. (2012), and Ujjani et al. (2015) Hillmen et al. (2007) Van Cutsem et al. (2009), Bonner et al. (2010), Vermorken et al. (2008), and Pirker et al. (2009)

constitutively activated proteins, regardless of upstream influence from EGFR. Cetuximab also has benefit in the treatment of head and neck squamous cell cancers, both as primary therapy in conjunction with definitive radiation (Bonner et al., 2010) and in conjunction with chemotherapy for unresectable disease (Vermorken et al., 2008). Finally, when added to chemotherapy, cetuximab leads to a modest increase in survival over chemotherapy alone in metastatic nonsmall cell lung cancer (Pirker et al., 2009). In the same family as EGFR, HER2 receptors are responsible for stimulation of cellular proliferation; overexpression of this protein by certain cancers results in uncontrollable cancer cell growth and reproduction (Hudis, 2007). This overexpression has been taken advantage of clinically, with the design of monoclonal antibodies targeting this protein. Trastuzumab, a recombinant monoclonal antibody-targeting HER2, was first demonstrated to have clinical benefit for the treatment of patients with metastatic breast cancer whose tumors overexpressed HER2 in 2001 (Slamon et al., 2001). For the management of women with a HER2 overexpressing breast cancer, this benefit has since been demonstrated to extend into the adjuvant setting as well (Leyland-Jones et al., 2005; Romond et al., 2005). Overexpression of HER2 can also be seen in patients with gastric cancer. The results of the ToGA trial (Bang et al., 2010) demonstrated an improvement in overall survival when trastuzumab was added to standard chemotherapy for the management of patients with metastatic gastric cancer whose tumors were found to overexpress HER2. Use of this monoclonal antibody is now standard for the treatment of these patients. An important therapeutic strategy in cancer treatment is targeting angiogenesis, the process of new blood vessel formation necessary for tumor growth. This process is primarily driven by ligand-binding vascular endothelial growth factor, or VEGF, which consists of a family of six different proteins delineated as VEGF A through E, and PIGF (Smaglo and Hwang, 2013). These proteins have proven to be important targets for

Bokemeyer et al. (2009) Slamon et al. (2001), Leyland-Jones et al. (2005), Romond et al. (2005), and Bang et al. (2010) Van Cutsem et al. (2012) Fuchs et al. (2007), Hochster et al. (2008), Giantonio et al. (2007), Bennouna et al. (2013), Sandler et al. (2006), and Vredenburgh et al. (2007) Fuchs et al. (2014), Wilke et al. (2014), Garon et al. (2014), and Tabernero et al. (2015)

monoclonal antibodies. The most thoroughly explored antiVEGF antibody is bevacizumab, which binds to VEGF-A and prevents its subsequent receptor binding. In the management of metastatic colorectal cancer, bevacizumab has demonstrated survival benefit when added to both conventional oxaliplatin and irinotecan chemotherapy regimens (Fuchs et al., 2007; Hochster et al., 2008). This benefit holds true regardless of line of therapy or previous bevacizumab exposure (Giantonio et al., 2007; Bennouna et al., 2013). Bevacizumab is also employed in the treatment of certain lung cancers and in glioblastoma multiforme (Sandler et al., 2006; Vredenburgh et al., 2007). A second monoclonal antibody-like structure targeting the VEGF system, ziv-aflibercept, works by binding several of the VEGF ligand proteins together and thus preventing their receptor binding and activation. The Velour study demonstrated the benefit of this newer antibody, which presently has only the single indication for treatment in second line metastatic colon cancer in combination with irinotecan-based chemotherapy (Van Cutsem et al., 2012).

Immunoconjugates Antibodies have also been utilized against tumors by delivering cytotoxic payloads using immunoconjugates. Immunoconjugates are complex therapeutic molecules that make use of antibodies’ cancer-targeting specificities, linking therapeutic agents to the antibody in order to deliver toxic payloads to the tumor target. Three components are essential to the construction of an effective immunoconjugate: the antibody, the therapeutic payload, and the linker that joins the two components. Immunoconjugates are divided based upon the mechanism of action of the therapeutic agent conjugated to the antibody, which may be a pharmacologic agent, a radionucleotide, or a catalytic toxin. Of these therapeutic entities, immunoconjugates employing pharmacologic and radionucleotide agents have obtained FDA approval. Pharmacologic agents that block tubulin polymerization (such as auristatin and maytansinoids) or induce DNA strand

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scission (such as calicheamicin) have been successfully employed as therapeutic conjugates in cancer therapy (Dosio et al., 2011). Perhaps the two most successful immunoconjugates to date are brentuximab vedotin and trastuzumab emtansine, which hold FDA approvals for the treatment of lymphomas and breast cancers, respectively. Brentuximab vedotin is an immunoconjugate that links an anti-CD30 antibody to the antimicrotubule monomethylauristatin E (MMAE) (Pro et al., 2012). This immunoconjugate is internalized when the antibody binds the CD30 antigen and enters into the lysosome, where MMAE is cleaved off and released; MMAE can then interrupt the microtubule network, inducing cellular apoptosis of the CD30-expressing tumor cell. In patients with anaplastic large cell lymphoma refractory to at least one prior therapy, treatment with brentuximab vedotin demonstrated an 86% objective response rate, with 57% of patients achieving a complete remission (Sutherland et al., 2006). In patients who had relapsed Hodgkin’s disease despite autologous stem cell transplant, treatment with brentuximab vedotin resulted in an overall response rate of 75%, with 34% of patients achieving a complete remission (Younes et al., 2012). Trastuzumab emtansine is an immunoconjugate of the anti-HER2 monoclonal antibody trastuzumab and DM1, a maytansinoid toxic to tumor cells through inhibition of tubulin polymerization (LoRusso et al., 2011). In the 2012 EMILIA study, efficacy of trastuzumab emtansine was compared to the combination of lapatinib and capecitabine in patients with HER2-positive breast cancer whose disease was refractory to trastuzumab therapy (Verma et al., 2012). This study demonstrated a superior progression free survival with the use of trastuzumab emtansine when compared with lapatinib/capecitabine. The activity of trastuzumab emtansine, even in patients who had been previously treated with trastuzumab illustrates the therapeutic potential of immunoconjugates beyond unconjugated antibody therapy. The immunoconjugate gemtuzumab ozogamicin, which links calicheamicin to a CD33-targeting antibody, received accelerated FDA approval in 2000 for the treatment of relapsed acute myeloid leukemias (Bross et al., 2001). This accelerated approval was granted because the agent, in early phase studies, appeared to be well-tolerated and effective, whereas conventional chemotherapy in this setting is often poorly tolerated and ineffective. However, in a regulatory agency–required postapproval study, gemtuzumab ozogamicin did not improve clinical benefit and demonstrated higher rates of toxicity compared to conventional chemotherapy (Petersdorf et al., 2009). Gemtuzumab ozogamicin was subsequently withdrawn in 2010. The immunoconjugate ibritumomab tiuxetan is the only radioimmunoconjugate currently holding FDA approval. This agent has antitumor activity in rituximab-refractory follicular lymphomas. Ibritumomab is a monoclonal antibody that binds the CD20 antigen; tiuxetan is a linker molecule, which allows the antibody to deliver the radioactive isotope 90yttrium to the tumor cell. In non-Hodgkin lymphoma patients who were refractory to therapy with the monoclonal antibody rituximab and were heavily pretreated with systemic therapies, the administration of ibritumomab tiuxetan produced an overall response rate of 74%, with 15% of patients achieving a complete response (Witzig et al., 2002).

Of note, another FDA-approved radionucleotide immunoconjugate, iodine tositumomab, encountered challenges that prompted its manufacturer to remove it from the market in February 2014. Kaminski and colleagues reported in 2001 that iodine tositumomab delivers radioactive 131-iodine to CD20 expressing tumor cells via an anti-CD20 monoclonal antibody and is effective treatment of follicular lymphoma (Kaminski et al., 2001). Reported median survival with this agent is 22.8 months. However, following regulatory approval and marketing, practitioner familiarity with the monoclonal antibody rituximab also may have limited the widespread use of this radionucleotide immunoconjugate, and treatment using the new agent was considered to be complicated to deliver.

Antibodies in Clinical Trials Monoclonal Antibodies Presently, nearly 700 studies are listed in clinicaltrials.gov, recruiting patients with various cancers for treatment with monoclonal antibodies, including 96 studies that have reached Phase III level of investigation. Specific information about clinical trials can be found at the url: http://clinicaltrials.gov/. While many of these trials are exploring the activity of the agents already approved in either other cancer types, lines of therapy, or combinations, a number of new monoclonal antibodies are being developed as well. Rilotumumab is a fully humanized monoclonal antibody that targets hepatocyte growth factor, preventing its interaction with cMET, and thus prevents cMET activation and tumor proliferation (Giordano, 2009). This agent is being investigated in the treatment of gastric, lung, and brain cancers. Bavituximab is a chimeric antibody, which targets the membrane phospholipid phosphatidylserine, resulting in occlusion of tumor blood vessels (DeRose et al., 2011), is being evaluated for the treatment of rectal, lung, liver, and skin cancers. Some of these new monoclonal antibodies are making their way into clinical practice. Ramucirumab, a fully humanized monoclonal antibody directed against the VEGF receptor 2, prevents ligand binding and thus downstreams signaling that promotes tumor angiogenesis. Based on the 2014 results of the REGARD trial (Fuchs et al., 2014) and the RAINBOW trial (Wilke et al., 2014), ramucirumab is approved for the secondline treatment of patients with metastatic gastric cancer. Ramucirumab has approvals for the treatment of certain lung and colon cancers as well (Garon et al., 2014; Tabernero et al., 2015). Siltuximab, a chimeric monoclonal antibodytargeting IL-6, is being investigated for the treatment of both solid tumors (renal cell, prostate, and ovarian cancers) and liquid tumors (non-Hodgkin’s lymphomas, multiple myeloma). In 2014, siltuximab became the first-approved treatment for multicentric Castleman’s disease, through the FDA’s priority review program (Markman and Patel, 2014).

Immunoconjugates A number of pharmacologic immunoconjugates are being developed; these molecules exploit known specific surface markers as targets for therapeutic delivery. Table 2 summarizes the pharmacologic immunoconjugates under active investigation. Such immunoconjugates include other calicheamicin conjugates: the agent inotuzumab ozogamicin, for example, consists of calicheamicin linked to a humanized antibody

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Table 2 Selected pharmacologic immunoconjugates in various stages of development are summarized by disease type, pharmacologic entity, and antibody, with the studies that support their use in these diseases Agent

Diseases treated

Cytotoxic agent

Antibody type

Studies

Inotuzumab ozogamicin

B cell non-Hodgkin’s lymphoma or acute lymphoid leukemias Metastatic breast cancer or unresectable melanoma Ovarian, Merkel cell, and small cell lung cancers NaPi2b expressing ovarian and NSCLC Glioblastoma B cell non-Hodgkin’s lymphoma or Acute lymphoid leukemia

Calicheamicin

Humanized IgG4 targeting CD22

Ricart (2011)

MMAE DM1

Humanized IgG2-targeting transmembrane glycoprotein NMB Humanized IgG1-targeting CD56

Maric et al. (2013), Hamid et al. (2010) Woll (2009)

MMAE MMAF DM4

Humanized IgG-targeting NaPi2b Humanized IgG-targeting EGFR Humanized IgG1-targeting CD19

Burris et al. (2014) Gan et al. (2014) Ribrag et al. (2014)

Glembatumumab vedotin Lorvotuzumab mertansine DNIB0600A ABT-414 SAR3419

directed against CD22 (Ricart, 2011). This agent is being studied for treatment of relapsed or refractory acute lymphoblastic leukemia, in B cell non-Hodgkin’s lymphomas, and as a part of a preparative regimen for stem cell transplant. SAR3419, an immunoconjugate of a CD19 antibody and a maytansinoid (DM4), is under investigation for the treatment of relapsed or refractory B cell non-Hodgkin’s lymphoma and in relapsed or refractory acute lymphocytic leukemia (Ribrag et al., 2014). A number of additional antibody targets in solid tumors are actively being investigated in the development of pharmacologic immunoconjugates as well. One tumor target, glycoprotein nonmetastatic B (GPNMB), is overexpressed in certain solid tumors, including triple-negative breast cancer and melanoma. It is being investigated as the antibody target for the immunoconjugate glembatumumab vedotin in these cancer types (Maric et al., 2013; Hamid et al., 2010). Lorvotuzumab mertansine, which combines the maytansinoid DM1 with an anti-CD56–targeting antibody, is under phase I investigation for the treatment of patients with advanced ovarian, Merkel cell, and small cell lung cancers (Woll, 2009). Gan et al. (2014) have described ABT-414, an immunoconjugate with an Auristatin moiety linked to an antibody-targeting–activated EGFR, which demonstrates preliminary promise in patients with glioblastoma (Gan et al., 2014). Studies of DNIB0600A, which links MMAE to an antibody-targeting NaPi2b, are underway to evaluate for efficacy, following encouraging phase I data presented by Burris and colleagues at the 2014 ASCO meeting for the treatment of ovarian and nonsmall cell lung cancers (Burris et al., 2014). Compared to pharmacologic immunoconjugate therapies, in the wake of the discontinuation of iodine tositumomab

production, further radioimmunoconjugate development has slowed, and it remains to be seen if such agents will find an expanded role in the treatment of human cancers. However, a third class of immunoconjugates, which employs a catalytic toxin as the therapeutic entity, remains interesting, relatively underexplored, and potentially useful. As a systemic treatment, an intact bacterial or other pathologic toxin cannot be readily used as a cancer treatment, due to the significant collateral toxicity to healthy patient tissue. However, if such molecules can be reliably delivered to the target, i.e., the cancer, with high selectivity, then they have the potential to be extremely effective. Pastan and colleagues at the Laboratory of Molecular Biology of the National Cancer Institute’s Center for Cancer Research have described their progress in developing this class of immunoconjugate. For example, Kreitman reported that treatment with moxetumomab pasudotox, an immunoconjugate that delivers pseudomonas exotoxin A to the CD22expressing leukemic cells was well-tolerated without the development of dose-limiting toxicities to treat relapsed/ refractory hairy cell leukemia (Kreitman et al., 2012). Treatment with moxetumomab pasudotox is also being explored in treatment of acute lymphocytic leukemia and non-Hodgkin’s lymphoma. In a separate trial also led by the Laboratory of Molecular Biology, the SS1P anti-mesothelin immunotoxin is being administered to patients along with T cell and B cell– depleting chemotherapies to minimize antitoxin antibody responses. Major tumor regressions were reported in 3 of 10 patients with mesothelioma who were treated with this regimen, supporting the further development of this therapy in solid tumors (Hassan et al., 2013). Immunotoxin immunoconjugates under development are summarized in Table 3.

Table 3 Toxin immunoconjugates in development are summarized by disease type, toxin employed, and antibody, with the studies that support their use in these diseases Agent

Diseases treated

Toxin

Antibody type

References

Moxetumomab pasudotox

Relapsed/refractory hairy cell leukemia Relapsed/refractory acute lymphoid leukemia Non-Hodgkin lymphoma Mesothelioma

Pseudomonas exotoxin A

Fv antibody fragment targeting CD22

Kreitman et al. (2012)

Pseudomonas exotoxin A

Fv antibody fragment targeting mesothelin

Hassan et al. (2013)

SS1P

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New Candidates – Preclinical Evidence In addition to the development of new agents and novel combinations, a number of approaches to optimize radionucleotide immunoconjugate therapy have been explored. One particular approach employed a pretargeting strategy in the treatment of tumor cells with radioimmunotherapies (Press et al., 2001). Pretargeting initially uses an unlabeled antibody to localize the tumor; a radionucleotide–hapten complex is subsequently administered, which reacts with the pretargeted antibody to concentrate the radionucleotide to the tumor. While preclinical evaluation of this method targeting CD20 in this manner demonstrated promise, results were disappointing in a phase II study of the treatment of patients with metastatic colorectal cancer with pretargeted radioimmunotherapy, both in terms of high rates of toxicity and low rate of response (Knox et al., 2000). Preclinical efforts to optimize pretargeting radionucleotide immunoconjugate therapy are ongoing in order to further the development of radioimmunoconjugate therapy as well. The work of Breichbiel and colleagues has focused on the use of targeted-a therapy, in which a particles are delivered by appropriate vectors to tumor cells (Baidoo et al., 2013). Exquisitely cytotoxic and exhibiting a short path length, the a particles are particularly suited to the situations of minimal residual disease or micrometastases.

Identification and Molecular Engineering of Therapeutic Antibodies

Improvements in antibody engineering techniques have allowed for the customization of therapeutic antibodies to overcome traditional mAb limitations. Modification in the antibody structure has resulted in the manipulation of immunogenicity, size, valence, affinity for target antigen, and Fc domain–directed interactions. Specific examples include:

Fab Domain Modification The Fab domain is the antigen-binding domain of antibodies. Antibody engineering has utilized hybridoma technology and phage- or yeast-display libraries to create a 25-kDa, monovalent single chain Fv (scFv) composed of the variable domains (VH and VL) of an antibody fused together with short peptide linker. The scFv became the basic building block of antibodybased fragments such as the enzymatically cleaved fragments, Fab and F(ab’)2, and genetically engineered formats such as scFv, (scFv)2, diabody, and minibody (Figure 1; reviewed by Robinson et al., 2004). Moreover, techniques such as chain-shuffling and site-directed mutagenesis (Swers et al., 2004; Crameri et al., 1996; Marks et al., 1992) are employed to manipulate antigen-binding properties and define the roles of binding affinity and avidity on in vitro and in vivo tumor targeting, immune effector functions such as antibodydependent cell-mediated cytotoxicity (ADCC) and therapeutic efficacy.

Fc Domain Modification

The size of antibodies (typically about 150 kDa) is regarded as a major limitation of their therapeutic application and effectiveness due to its role in hindering tissue penetration and delivery (Chames et al., 2009). Size also attributes to nonspecific uptake of antibodies or immunoconjugates by the reticuloendothelial system, limited efficacy, and the induction of dose-limiting toxicity to the liver and bone marrow (Weiner et al., 2012).

The significance of the Fc domain is due to its role in immune effector cell activation. Fcg on the surface of immune effector cells (NK cells, dendritic cells, neutrophils, and mononuclear phagocytes) recognizes the Fc domain and transduces activating signals through immunoreceptor tyrosine-based activation motifs (ITAMs) or delivers inhibitory signals though immunoreceptor tyrosine-based inhibitor motifs (ITIMs). Most Fc stimulatory signals are transduced by FcgR1 (CD64) and FcgRIIIA (CD16a). Cross-linking of FcgRs on effector cells

Enzymacally derived fragments

F(ab’)2

Fab’

Fc

scFv

(scFv)2

Minibody

Diabody

Triabody

Genecally engineered fragments Figure 1

Structures of enzymatically derived and genetically engineered fragments of Ig molecule.

Tetrabody

Tumor Immunology j Antibody Therapy promotes ADCC and tumor cell destruction (Nimmerjahn and Ravetch, 2012). ADCC has been shown to be an important mechanism for monoclonal antibodies used in cancer therapy. Manipulation of the Fc domain effector functions has been via two general approaches. The first approach entails altering the amino acid structure of the domain to influence binding affinity to Fc receptors (Shields et al., 2001; Lazar et al., 2006; Nimmerjahn and Ravetch, 2012; Stavenhagen et al., 2008). Altered amino acid sequences have led to the creation of Fc domains with higher affinity for FcgRIIIA and enhanced ADCC properties. The second approach is by modifying the oligosaccharide content of the Fc domain. The defucosylation of antibody oligosaccharides has shown enhancement of ADCC in vitro and enhanced in vivo antitumor activity (Kubota et al., 2009).

Ig-Like Scaffolds Our knowledge of the structure and function of the immunoglobulin molecule has allowed us to recognize the limitations of current forms of antibody therapy. In an effort to address such limitations, scientists have utilized current engineering technologies and naturally occurring structures to developed recombinant single chain Ig scaffolds.

Nanobodies Nanobodies are heavy chain antibodies (HCAB) based on distinct Ig structures found naturally in sharks and camelid. The structure of HCAB is unique, composed of a single heavy chain with one variable domain (VHH) (Figure 2(a)).The VHH is equivalent to the Fab fragment of IgG antibodies (Muyldermans et al., 1994). Nanobodies are smaller and have longer VHH domains facilitating their reach to targets inaccessible to conventional antibodies (Muyldermans et al., 1994). They are also easily manipulated yielding

(a)

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bispecific/bifunctional structures and posses advantageous properties such as specificity, stability, thermotolerance, and low immunogenicity. The structural and functional appeal of nanobodies has increased their attractiveness as potential drugs leading to their commercial development and selection by phage display libraries. There are currently six nanobodies undergoing clinical trials, none of which are for anticancer purposes (Muyldermans, 2013).

Domain Antibodies (dAbs) dAbs or engineered antibody domains (eAds) are small engineered monomers (11–15 kDa) based on the variable domain of an antibody heavy chain (VH domain) (Ward et al., 1989) or light chain (VL domain) (Pereira et al., 1998). Their small size facilitates and ease of modification makes them attractive candidates for pharmaceutical development. The goal is to produce stable novel structures with favorable biophysical properties unique to dAb (Holt et al., 2003). Currently, the only dAb undergoing clinical trials is CEP-37247; an antiTNF bivalent dAb fused to a human IgG1 Fc region (Gay et al., 2010), for the treatment of sciatica. In cancer therapy, dAbs are yet to establish therapeutic efficacy and application.

Chemically Programmed Antibodies (cpAbs) CpAbs are novel scaffolds composed of an antibody fragment joined to a synthetic component (peptide or small molecule) (Figure 2(b)). The synthetic component functions as an antigen recognizing and binding region, while the antibody fragment carries out the effector functions, in addition to contributing to the half-life and bivalency. The minimum antibody requirement in cpAbs is the Fc fragment, while the Fab portion is usually replaced by a synthetic component (reviewed by Rader, 2014). The only cpAbs undergoing clinical trials have peptides as their synthetic component and are based on the CovX-Body platform. There has been a halt in the development

(b)

VHH

Pepde or small molecule Fc

VHH (nanobody) Fc

Heavy chain anbody (HCAB)

(c)

BiTes

DaRTs

TandAB

Figure 2 Schematic representation of Ig-like scaffolds (a) Heavy chain antibody (HCAB) and VHH derivative (nanobody) (b) Chemically programmed antibodies (CpAbs), composed of an antibody component and a synthetic component (peptide or small molecule) and (c) Bispecific scaffolds BiTes, DaRTs, and TandABs.

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of cpAbs, yet they still demonstrate potential due to a range of their possible synthetic components.

specific cancers, including melanoma and non-small cell lung cancer (Larkin et al., 2015; Weber et al., 2015; Robert et al., 2015a; Gettinger et al., 2015; Brahmer et al., 2015; Robert et al., 2015b).

Antibodies Targeting the Immune System Targets of antibody therapy are not limited to the tumor cell; they can also be used to modulate a desired immune response or signaling pathway. The immune system is very attractive therapeutically. Manipulating entities such as immunoregulatory coreceptors and other cell surface signaling molecules, could lead to enhanced antitumor response and influence the outcome of lymphocyte-mediated immune responses by modulating antigen-specific T cell receptor and B cell receptor signals (Weiner et al., 2010). Ipilimumab is the first FDA-approved immune-targeting monoclonal antibody against cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), an inhibitory receptor of T cell activation.

Antibodies in Clinical Trials Manipulating the immune system to fight the tumor has proven to be a difficult task. Yet, understanding the mechanisms in which the effector cells operate, and the success of ipilimumab has opened the door for several other targets that are currently in clinical trials. Other targets under study that serve as costimulatory molecules include PD-1/PDL-1, CD40, and CD137 (Pardoll, 2012). There are currently 22 antibodies in clinical trials that target the immune system (Table 4), 19 of which are for the treatment of cancer (Yao et al., 2013). Several of these therapies have garnered approval for treatment of

Table 4

New Candidates The manipulation and stimulation of the immune system can be achieved not only by mAb but also by structurally modified antibodies such as bispecific antibodies (bsAbs) and trispecific antibodies (triomAbs). These modified structures are able to simultaneously engage tumor antigen and an immune activator, redirecting the immune response toward the tumor antigen. A common target of bsAbs and triomAb is CD3, the activating receptor for T cells, whose engagement activates and retargets T cells to the tumor. An example of a developed triomAb is catumaxomab, which binds the tumor antigen EpCAM, CD3, and innate effector cells through its intact Fc portion (Ruf and Lindhofer, 2001). Other tumor antigens targeted by triomAbs include HER2/neu (ertumaxomab), CD20 (Bi20/FBTA05), GD2, and GD3 (Ektomun) (Weiner et al., 2012).

Identification and Genetic Engineering of Therapeutic Antibodies Genetic engineering has also been utilized to create multifunctional protein scaffolds derived from the scFv antibody fragment that are able to modulate the immune system (Figure 2(c)). Bispecific formats have demonstrated notable clinical success (Shahied et al., 2004; Müller et al., 2010;

Antibodies targeting the immune system in clinical trials (Yao et al., 2013)

Name

Antibody type

Target

Activity

Diseases treated

Trial phase

Tremelimumab Galiximab

Human IgG2 Chimeric IgG1 Human IgG4 Humanized IgG1 IgG4 Fusion protein (B7DC þ IgG1) Human IgG4 Engineered human IgG1 Engineered human IgG1 Humanized IgG1 Fusion protein (LAG3 þ IgG1)

CTLA4 CD80 PD1–B7H1, PD1–B7DC PD1–B7H1, PD1–B7DC PD1–B7H1, PD1–B7DC PD1–B7H1, PD1–B7DC PD1–B7H1 PD1–B7H1 PD1–B7H1 B7H3 LAG3–MHCII

Solid tumors Lymphoma Multiple cancers Solid tumors Solid tumors Multiple cancers Solid tumors

II II

Anti-OX40 TRX518 CP-870,893 Lucatumumab

Human IgG4 Human IgG Human IgG1 OX40-specific mouse IgG GITR-specific humanized IgG1 Human IgG1 Human IgG1

CD137 CD137 CD27 OX40 GITR–GITRL CD40 CD40

T cell priming and activation B cell proliferation T cell activation and tolerance T cell activation and tolerance T cell activation and tolerance T cell activation and tolerance T cell activation and tolerance T cell activation and tolerance T cell activation and tolerance T Cell activation and tolerance DC maturation and T cell activation T cell activation T cell activation T cell activation CD4 T cell activation T cell activation APC activation and B cell maturation APC activation and B cell maturation

Dacetuzumab

Humanized IgG1

CD40

AMP224 BMS-936559 MPDL3280A MGA271 IMP321

PF-05082566

Solid tumors Solid tumors Multiple cancers

Solid tumors Lymphoma Multiple cancers Prostate cancer Solid tumors Multiple cancers Lymphoma and leukemia APC activation and B cell maturation Lymphoma and multiple myeloma

I I

I I/II I/II I I II I I I/II II

B7H1, B7 homolog 1 (programmed death-1 ligand-1); B7DC, B7 dendritic cell (programmed death-1 ligand-2); CTLA4, cytotoxic T lymphocyte antigen 4; GITR, glucocorticoidinduced TNFR-related protein; GITRL, GITR ligand; IgG1, immunoglobulin G1; LAG3, lymphocyte activation gene 3; MHCII, major histocompatibility complex class II; OX40L, OX40 ligand; PD1, programmed cell death protein 1.

Tumor Immunology j Antibody Therapy Rossi et al., 2003; Baeuerle et al., 2009; Kellner et al., 2008), the most successful of which is the BiTE (bispecific T cell engager). BiTEs simultaneously engage CD3 and a tumor antigen such as CD19, EpCAM or EGFR. Blinatumomab (MT-103) is the most clinically advanced BiTE targeting CD19 and CD3 (Baeuerle et al., 2009), it is undergoing Phase III clinical trials for treatment of acute lymphoblastic leukemia and has completed Phase I evaluations in non-Hodgkin’s lymphoma. Other bispecific scaffolds being developed are DARTs (dual affinity retargeting) (Rader, 2011) and TandAbs (tetravalent tandem antibodies) (Mølhøj et al., 2007). Their structures and functions resemble BiTes but differ in the linkage of heavy and light chain variable domains. DARTs have yet to reach clinical trials while TandAb have been in clinical development, with the completion of Phase I clinical trial of AFM13 against CD30 and CD16a (FcgRIIIA) (Mølhøj et al., 2007; Baeuerle et al., 2008; Rothe et al., 2011). Clinical testing of several additional anticancer therapies are underway that expand upon and apply the immunoconjugate concept in novel ways. In 1999, Marks et al. developed a method to select antibodies optimal for internalization by cancer cells though the recovery of infectious phage from the cancer cells to be targeted (Becerril et al., 1999). In 2009, this method was used to create highly specific antibodies coupled to liposomes containing doxorubicin, which demonstrated significant antitumor effects in mice (ElBayoumi and Torchilin, 2009). Exploration of this method in human subjects has potential merit. This concept of using antibody-loaded liposomes to deliver a therapeutic entity has also been explored as a novel method of immunoconjugate development. Immunoliposomes also have been employed by Xu and colleagues as delivery vehicles for the p53 tumor suppressor (Xu, 2001). The therapeutic potential of immunoliposomes has been explored in some phase I studies. In addition to this repurposing of antibodies already in clinical use, future advancement of immunoconjugate therapy will draw on new techniques being developed in other areas of cancer medicine that can be adapted to further antibody selection and design. These techniques involve the selection of antigens to serve as cancer-specific antibody targets as well as optimization of the internalization of the antibodies by the tumor cells. Some tumor-specific antigens that could be targeted by immunoconjugates may emerge from intense genomic analysis of tumors, some of which may be identifying driver mutations. The tumor-specific protein products of these mutations would be attractive targets for an immunoconjugate, as toxicity to normal cells based upon on-target toxicity would be minimized. New approaches may further widen the use of immunoconjugates. For example, Adams, Rudnick, and colleagues have reported in 2011, the utility of a novel radiotracer linked to a HER2 receptor antibody for positron emission tomography imaging, and have identified antibody structural features that influence cellular internalization and subsequent tumor catabolism that is associated with variability in antibody affinity for the antigen (Rudnick et al., 2011). Finally, antibody therapy can be incorporated into vaccination strategies. Schlom and colleagues demonstrated in 2012 how vaccine-induced immune responses can be enhanced by trastuzumab therapy in breast cancer patients (Schlom, 2012; Disis et al., 2009; Norton et al., 2014). This

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approach may have additional utility in augmenting immunoconjugate therapy that uses trastuzumab or other monoclonal antibodies as the delivery vehicle of the therapeutic entity to the cancer. Through these and other techniques, the engineering of therapeutic antibodies capable of targeting cancerspecific epitopes with ever-higher degrees of specificity will allow for even greater anticancer effects.

See also: Myeloid and B Cell Development: Regulation of IgL Chain Recombination; Regulation of Igh Recombination and Allelic Exclusion. Structure and Function of Diversifying Receptors: IgG Structure and Function; Structure and Function of Camelid VHH. Tumor Immunology: Cancer Immunosurveillance: Immunoediting; Inflammation and Cancer; Therapeutic and Prophylactic Cancer Vaccines; Tumor Antigens, Viral Origin; Vaccines and Their Role in CD8 T CellMediated Antitumor Immunity.

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