Chapter 1 Antibody Therapies in Cancer

Shengdian Wang and Mingming Jia

Abstract Antibody-based immunotherapy has become a standard treatment for a variety of cancers. Many well-developed antibodies disrupt signaling of various growth factor receptors for the treatment of a number of cancers by targeting surface antigens expressed on tumor cells. In recent years, a new family of antibodies is currently emerging in the clinic, which target immune cells rather than cancer cells. These immune-targeted therapies strive to augment antitumor immune responses by antagonizing immunosuppressive pathways or providing exogenous immune-activating stimuli, which have achieved dramatic results in several cancers. The future of cancer therapies is likely to combine these approaches with other treatments, including conventional therapies, to generate more effective treatments.

Keywords Immunotherapy • Therapeutic antibody • Cancer

Over the past 20 years, antibodies have been used as passive immunotherapy strategies as part of the standard treatment of many cancers. Many of these antibodies are specific for surface antigens expressed by tumor cells. A major class of them targeting growth factor receptors, such as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), are commonly used for the treatment of non-leukemic cancers. The antibodies targeting the lineage markers of hematopoietic cells, such as CD20, have shown the thera- peutic efficacy in hematological malignancies. By directly binding to these membrane-bound receptors, these antibodies result in tumor cell death through dampening the downstream signaling cascades that promote cell cycle and function and Fc-mediated innate immunological effector mechanisms, such as antibody- dependent cell-mediated cytotoxicity (ADCC). In addition, therapies of these tumor-targeted antibodies can induce endogenous adaptive antitumor immune responses which were recently shown to play important roles in the therapeutic efficacy.

S. Wang (*) • M. Jia CAS Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Datun Road #15, Chaoyang District, 100101 Beijing, China e-mail: [email protected]

© Springer Science+Business Media Dordrecht 2016 1 S. Zhang (ed.), Progress in Cancer Immunotherapy, Advances in Experimental Medicine and Biology 909, DOI 10.1007/978-94-017-7555-7_1 2 S. Wang and M. Jia

In recent years, it has been demonstrated that antibodies could be used to manipulate the host’s immune responses by targeting the immune cells to generate active antitumor immunity in cancer patients. Such immunomodulatory antibodies may be either agonistic, targeting costimulatory molecules, or antagonistic, “blocking” inhibitory molecules expressed on the surface of immune cells. The aim of these approaches is to augment endogenous antitumor immune responses, either by providing direct immune stimulation or by releasing immunosuppressive mechanisms. They have resulted in a paradigm shift in cancer therapy, where instead of using drugs to target the tumor cells, molecules are designed to target the immune system in order to break the tumor tolerance and stimulate the antitumor immune response. The diversity of these targeted approaches reflects the versatility of antibodies as platforms for therapeutic development.

1 Historical Review of Antibody Therapeutics in Cancer

1.1 Characteristics of Antibody

Antibodies are composed of two identical light chains and two identical heavy chains and comprised of two distinct functional units: the fragment of antigen binding (Fab) and the constant fragment (Fc). Heavy and light chains each have variable and constant regions. The variable regions of a heavy chain and a light chain combine to form an antigen-binding site, so that an antibody molecule has two identical antigen-binding sites (Fig. 1.1). The Fc implements immune effector functions by binding to Fc receptors (FcRs) expressed on immune cells or initiating complement-dependent cytotoxicity. Based on the sequence of the heavy-chain constant regions, antibodies are grouped into five classes: IgM, IgD, IgG, IgE, and IgA. IgG can be further subdivided into four subclasses (IgG1, IgG2, IgG3, and IgG4). Most of the approved antibodies in oncology are of the human IgG1 subclass, which is the most effective at engaging Fcγ receptors (FcγRs) on natural killer (NK) cells, macrophages, and neutrophils. Antibody engagement of these receptors leads to the killing of antibody-bound target cells by ADCC or antibody-dependent phago- cytosis. In addition, IgG1 and IgG3 are potent activators of the classical comple- ment pathway. The binding of two or more IgG molecules to the cell surface leads to high-affinity binding of complement component 1q (C1q) to the Fc domain, followed by activation of C1r enzymatic activity and subsequent activation of downstream complement proteins, resulting in cell lysis. ADCC can be augmented through modification of the antibody Fc region to produce a more favorable binding profile for the FcRs expressed on monocytes and NK cells. These modifications include mutations in the amino acids and alterations in the glycosylation pattern of the Fc region. A triple alanine substitution mutant trastuzumab (S298A/E333A/K334A), an anti-HER2/neu antibody, has significantly 1 Antibody Therapies in Cancer 3

Fig. 1.1 The structure of antibody (IgG). Antibody is composed of two heavy (H) and two light (L) chains. These chains comprise constant (C) regions, which constitute the Fc domain, and variable (V) regions, which constitute the Fab domain and allow antigen specificity

improved binding to FcγRIIIA, the principal activating FcR on monocytes and NK cells. Consistent with the improved binding, this substituted trastuzumab has a superior ability to activate ADCC in vitro. Most of the currently used therapeutic antibodies are highly fucosylated owing to the nature of the cell lines used for manufacturing. However, antibodies with defucosylated oligosaccharides can pro- mote FcγRIIIA binding and show a significant enhancement in ADCC in vitro and enhanced in vivo antitumor activity. Antibody-mediated killing can also be enhanced by decreasing binding to the inhibitory FcγRIIB. Conversely, ADCC can be eliminated by modifying specific residues in the Fc domain that bind to FcγR or by producing recombinant antibodies that lack the N-glycosylation of Fc regions. The IgG4 subclass has also been used for reducing ADCC. The neonatal FcR (FcRn) is structurally distinct from FcγR. By binding to Fc, FcRn expressed on the vascular endothelium can protect antibody from transcytotic lysosomal catabolism after antibody internalization by endothelial cells and return it to the circulation (Roopenian and Akilesh 2007). FcRn is largely responsible for the serum half-life of antibodies. Thus, antibody half-life can be extended or reduced by introducing mutations into Fc region that enhance or diminish FcRn binding. These may prove to be important considerations in controlling the phar- macokinetic exposure levels of a given antibody, with a potential toxicity possibly mitigated by faster clearance (Yeung et al. 2009).

1.2 Development of Antibody Therapies for Cancer

Since the first description of monoclonal antibodies (mAbs) in 1975 (Kohler and Milstein 1975), mAbs were recognized as unique biological tools and quickly became invaluable in pathological diagnosis. Meanwhile there was equal excite- ment about their therapeutic potential based on the ability to manufacture mAbs of 4 S. Wang and M. Jia defined specificity and class in essentially unlimited amounts. This would allow for highly specific targeting of cancer cells on the basis of their molecular phenotype. However, early clinical results exploring mAb-based therapeutics were disap- pointing (Vaickus and Foon 1991). The first mAb evaluated in clinic as cancer treatments was a murine mAb. Although there were intriguing hints that antibody therapy could be successful, the treatments with murine mAbs were often associ- ated with the development of an immune response against the therapeutic antibody itself and the rapid clearance of the antibody due to their immunogenicity for human, which limited their clinical applicability. To overcome these side effects, chimeric mouse-human antibodies were developed by grafting the entire antigen- specific domain of a mouse antibody onto the constant domains of a human antibody using genetic engineering techniques (Morrison et al. 1984) (Fig. 1.2). In 1997, rituximab (Rituxan), a mouse-human chimeric mAb against the B-cell lineage marker CD20, was approved by FDA for treatment of B-cell non-Hodgkin lymphoma. This is the first antibody approved for cancer therapy. Since then, no less than 15 distinct antibodies have been approved for the treatment of hemato- logic and solid tumors. In 2004, cetuximab (Erbitux), another chimeric mAb against Her-1, a member of epidermal growth factor receptor (EGFR) family, was approved for treatment of colorectal carcinoma (Galizia et al. 2007). With the advent of in vitro phage display technology and the generation of transgenic rodents expressing human immunoglobulin genes, the humanized anti- bodies and fully human antibodies were generated (Fig. 1.2). In 1998, trastuzumab (Herceptin), a humanized antibody binding the extracellular domain of the HER2, was approved for the treatment of metastatic HER2-overexpressing breast cancer (Hudis 2007). In 2001, alemtuzumab (Campath), a humanized mAb against CD52, a cellular surface glycoprotein expressed on both normal and malignant B and T lymphocytes, was approved by FDA for treatment of drug-resistant chronic lym- phocytic leukemia (Alinari et al. 2007). In 2004, the first anti-angiogenic agent, bevacizumab (Avastin), was approved by FDA (Fig. 1.3). Bevacizumab is a humanized version of a murine mAb against VEGF, which binds and neutralizes all human VEGF isoforms and bioactive proteolytic fragments. Bevacizumab has been used in combination with conventional chemotherapy and/or targeted

Fig. 1.2 Timeline of antibody development for cancer therapy. Box outline: blue, chimeric antibody; red, humanized antibody; yellow, human antibody 1 Antibody Therapies in Cancer 5 anticancer agents for colorectal cancer, acute myeloid leukemia, multiple myeloma, head and neck squamous cell carcinoma, etc. (Hurwitz et al. 2004). In 2013, the use of antibodies to harness the power of the immune system to fight cancers was heralded in science as the “breakthrough of the year.” This was mainly due to the great early successes of antibodies to two co-inhibitory receptors, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1), expressed on activated T cells (Couzin-Frankel 2013). Ipilimumab, a fully human IgG1 monoclonal antibody targeting CTLA-4, is the first immune checkpoint inhibitor approved by FDA for treatment of cancer in 2011. Ipilimumab blocks CTLA-4 signaling pathway in activated T cells and can induce sustained antitumor responses (Hodi et al. 2010). The next generation of immune checkpoint inhibitors blocks the interaction of co-inhibitory receptor PD-1 on T cells and its ligand PD-L1 on tumor cells and antigen-presenting cells (APCs). Multiple break- through designations for PD-1- and PD-L1-blocking antibodies have been granted by the FDA in melanoma, non-small cell lung cancer (NSCLC), Hodgkin

Fig. 1.3 Mechanisms of tumor-targeted antibody therapy in cancers. (a) Antibodies directed against TAA (such as EGFR and HER2) inhibit oncogene signaling. (b) The complex of antibody and tumor antigen initiates soluble complement-mediated cytotoxicity. (c) Antibody-coated apo- ptotic tumor cells can bind Fc receptors on phagocytes and initiate Fc-dependent phagocytosis. (d) Recognition of antibody-coated tumors by Fcγ receptors (FcγRs) on effector immune cells such as natural killer (NK) cells, macrophages, and neutrophils leads to ADCC and tumor cell apoptosis, which is mediated by the delivery of perforin and granzymes to the tumor cell. (e) Antibody-coated tumor antigens released by dying cells are taken up by DCs, processed, and cross-presented to T cells 6 S. Wang and M. Jia lymphoma, bladder cancer, renal cell carcinoma, etc. Such designations have led to the accelerated FDA approval of fully human anti-PD-1 mAb prembrolizumab for patients with melanoma in 2014 (Hamid et al. 2013) and nivolumab for patients with melanoma or squamous cell NSCLC in 2014 and 2015, respectively. This class of immunomodulatory antibodies blocking PD-1 co-inhibitory pathway are argu- ably the most exciting development in current cancer drug development.

1.3 Classes of Antibody Therapeutics in Cancer

Anticancer immunotherapies are generally classified as “passive” or “active” based on their ability to activate the host immune system against malignant cells. From this standpoint, tumor-targeting antibody therapeutics are considered passive immunotherapy, as they are endowed with intrinsic antineoplastic activity. Con- versely, immunostimulatory antibodies and checkpoint inhibitors constitute clear examples of active immunotherapy as they exert anticancer effects by modulating antitumor immune responses only upon the engagement of the host immune system. Tumor-targeting antibodies exist in at least four functionally distinct variants. First, the antibodies inhibit signaling pathways required for the survival or progres- sion of neoplastic cells, such as the EGFR-specific antibody (cetuximab) for the treatment of head and neck cancer and colorectal carcinoma (CRC) (Weiner et al. 2008). Second, the TAA-specific antibodies opsonize cancer cells and hence activate ADCC, CDC, and antibody-dependent cellular phagocytosis, such as the CD20-specific antibody rituximab for the treatment of chronic lymphocytic leuke- mia (CLL) and non-Hodgkin lymphoma (Scott 1998; Jones 2013). Third, immunoconjugates, i.e., TAA-specific antibodies, coupled with toxins or radionu- clides, such as gemtuzumab ozogamicin, an anti-CD33 calicheamicin conjugate for the treatment of acute myeloid leukemia (Hughes 2010). Fourth, the “bispecific T-cell engagers” (BiTEs) consist of two single-chain variable fragments from distinct mAbs, one targeting a TAA and one specific for a T-cell surface antigen, such as blinatumomab, a CD19 and CD3 BiTE recently approved for the therapy of Philadelphia chromosome-negative precursor B-cell acute lymphoblastic leukemia (Walter 2014). The immunomodulating antibodies operate by interacting with the immune system to elicit a novel or reinstate an existing anticancer immune response. So far, this has been achieved through four kinds of antibodies: (1) Antagonistic antibodies block immunosuppressive receptors expressed by activated T lympho- cytes, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) or NK cells, like various members of the killer cell immunoglobulin-like receptor (KIR) family (Long 2008); (2) the antibodies against the ligands of these immunosuppressive receptors block the interactions of these receptors and ligands (Zou and Chen 2008); (3) agonistic antibodies activate the costimulatory receptors expressed on the surface of immune effector cells, such as tumor necrosis factor receptor superfamily, member 4 (TNFRSF4, best known as 1 Antibody Therapies in Cancer 7

OX40), TNFRSF9 (best known as CD137 or 4-1BB), and TNFRSF18 (best known as GITR) (Croft 2009); and (4) neutralizing antibodies neutralize the activities of immunosuppressive factors released in the tumor microenvironment, such as transforming growth factor β1 (TGFβ1) (Pickup et al. 2013).

2 Tumor-Targeted Antibody Therapies

Tumor-targeted antibody therapy has shown efficacy in the past 30 years and is now one of the most successful and leading strategies for targeted treatment of patients harboring hematological malignancies and solid tumors. Tumor-targeting anti- bodies are the best-characterized form of anticancer immunotherapy. These thera- peutics include unconjugated antibodies or antibody fragments targeting TAA, as well as antibody-drug conjugates, radioimmunoconjugates, and bispecific/ trispecific molecules targeting TAA. Currently several FDA-approved monoclonal antibodies are used in the clinic, either alone or in combination with chemotherapy or radiation.

2.1 An Outline of Tumor-Targeted Antibody Therapeutics

Many of the tumor-expressed targets for therapeutic antibodies are growth factor receptors and differentiation antigens that are involved in growth and differentia- tion signaling, such as EGFR, HER2, CD20, CD30, etc. By blocking ligand binding and/or signaling pathways through these receptors, monoclonal antibodies may serve to normalize growth rates, induce apoptosis, and/or help sensitize tumors to chemotherapeutic agents. These include antibodies that target receptors expressed on the tumor cells. In addition, antibodies that target tumor microenvironment and inhibit processes such as angiogenesis have shown therapeutic promise.

2.1.1 Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor overexpressed in many different malignancies, including those originating in the colon, head and neck, ovary, lung, and brain. Ligand binding causes EGFR dimer- ization, leading to activation of the tyrosine kinase domain which promotes cell proliferation, migration, and invasion via activation of the MAPK and AKT path- ways (Li et al. 2005). Some EGFR-expressing tumors have rearrangements of the EGFR gene that lead to the expression of constitutively activated mutant receptors. The most common EGFR mutation in the extracellular domain is EGFRvIII, which has an in-frame deletion of exons II–IV. EGFRvIII is found in glioblastoma, head and neck cancers, and NSCLC (Li et al. 2007). This mutated receptor has 8 S. Wang and M. Jia constitutive tyrosine kinase activity and has important pro-oncogenic effects including proliferation and chemotherapeutic resistance (Fan et al. 2013). Cetuximab (Erbitux, ImClone Systems/Bristol-Myers Squibb) and panitumumab (Vectibix, Amgen, Inc.) are both EGFR-specific antibodies. The former is a chimeric IgG1 monoclonal antibody and the latter is a fully humanized IgG2 isotype. Both inhibit EGFR-mediated signal transduction by preventing ligand binding and receptor dimerization, which induce cell cycle arrest and apoptosis in tumor cells (Li et al. 2005; Kim 2009). Cetuximab and panitumumab have been used as second- or third-line therapy for the treatment of metastatic colorectal cancer. Cetuximab is often used in combination with other chemother- apeutic regimens. The combination of cetuximab with folinic acid, 5-fluorouracil, and irinotecan (FOLFIRI chemotherapy) has been shown to prolong progression- free survival of patients with metastatic CRC harboring wild-type KRAS alleles (Van Cutsem et al. 2009). Necitumumab and nimotuzumab are another two anti- EGFR antibodies, which are competitive inhibitors of EGFR’s ligand. Necitumumab combined with pemetrexed and cisplatin recently failed to show a benefit in overall survival of patients with NSCLC compared to pemetrexed and cisplatin alone (Paz-Ares et al. 2015). Nimotuzumab is approved for the treatment of various epithelial malignancies in Europe. For example, it is approved for pancreatic cancer treatment in Germany. It is also approved for use in some countries in Asia, South America, and Africa for the treatment of head and neck cancer, glioma, and nasopharyngeal cancer. Efforts are underway to target a truncated form of EGFR, EGFRvIII. A phase I study using the monoclonal antibody 806 (Zymed) targeting EGFRvIII showed good antibody penetration of tumor tissue and no significant toxicities in patients with metastatic disease (Scott et al. 2007).

2.1.2 Human Epidermal Growth Factor Receptor

Human epidermal growth factor receptor 2 (HER2) is a member of the ErbB/HER growth factor superfamily, which is composed of HER1 EGFR (HER1, ErbB1), HER2 (ErbB2), HER3, and HER4. HER2 has no known ligand and constitutively adopts an open configuration priming it for heterodimerization and increased mitogenic signaling. It is gene amplified and overexpressed in approximately 30 % of breast cancers and is overexpressed, although rarely gene amplified, by some gastrointestinal, lung, prostate, and ovarian adenocarcinomas (Chen et al. 2003). The expression of HER2 in breast cancer is associated with aggressive disease, a high recurrence rate, and reduced patient survival. Overexpression of HER2 leads to increased signal transduction and activation of the MAPK and P13K/AKT pathways (Yarden and Sliwkowski 2001). Trastuzumab (Herceptin, Genentech/Roche) was the first FDA-approved anti-HER2 antibody for HER2+ breast cancer, and pertuzumab (Omnitarg, Genentech/Roche) is a newer one. Both are humanized IgG1 anti-HER2 antibodies. Trastuzumab binds the juxtamembrane domain IV region of HER2 and inhibits homo- and heterodimerization and inter- nalization of HER2, whereas pertuzumab binds HER2 at the extracellular 1 Antibody Therapies in Cancer 9 dimerization subdomain II which is critical for heterodimerization of HER2 with other HER-family receptors, most notably HER3 (Hudis 2007; Franklin et al. 2004). A new anti-HER3 antibody MM-121 (Merrimack Pharmaceuticals), which is currently being developed, has been shown to inhibit growth of human tumor xenograft in mice (Schoeberl et al. 2009).

2.1.3 VEGF

Vascular endothelial growth factor (VEGF) is a glycoprotein produced by normal and malignant cells. VEGF and its isoforms are mitogens which bind and activate three different tyrosine kinase receptors, VEGFR1, VEGFR2, and VEGFR3, and play a very important role in the regulation of angiogenesis for both normal and malignant tissues. VEGFR2 is mainly expressed on the surface of vascular endo- thelial cells and highly expressed in many tumor types, including cancers of the gastrointestinal tract. VEGFA binding to VEGFR2 leads to autophosphorylation of tyrosine residues at the carboxy-terminal of the receptor, initiating cell signaling and angiogenesis (Sia et al. 2014). VEGF binds to its receptor on the vascular endothelium to stimulate the growth of new blood vessels to allow for tumor growth, and it also maintains the immature blood vessels. Bevacizumab (Avastin, Genentech) is the first VEGFA-specific antibody that effectively blocks the activation of key pathways required in tumor angiogenesis by blocking the binding of VEGF to its receptor (Sullivan and Brekken 2010). It exerts its antitumor effect by functionally altering or slowing the formation of the tumor vasculature. Bevacizumab is approved for the treatment of breast, colorectal, and non-small cell lung cancer in combination with cytotoxic chemotherapy (Ellis and Hicklin 2008). The treatment of bevacizumab has led to production of bevacizumab-resistant tumors owing to upregulation of other pro-angiogenic medi- ators, such as platelet-derived growth factor (PDGF). PDGF receptor (PDGFR) signaling plays an important role in maintaining the endothelial support system, which stabilizes and promotes the growth of new blood vessels (Hirschi et al. 1998). Blockade of PDGFR signaling by a PDGFRβ-specific human antibody has been shown to synergize with anti-VEGFR2 therapy in preclinical models and suggests the utility of anti-PDGFRβ therapy in the setting of bevacizumab resistance (Shen et al. 2009). Ramucirumab (IMC-1121B, ImClone Systems) is a humanized anti- VEGFR2 antibody that blocks the VEGFR2-related signaling and activating path- ways (Spratlin 2011). Ramucirumab was approved for use in advanced cases of gastric and gastroesophageal adenocarcinomas that have been refractory to first- line treatments. The therapeutic antibodies targeting VEGFR1 (IMC-18F1) are currently underway and have shown preclinical promise (Wu et al. 2006). 10 S. Wang and M. Jia

2.1.4 Hematopoietic Differentiation Antigens

Hematopoietic differentiation antigens are glycoproteins that are usually associated with cluster of differentiation (CD) grouping selectively expressed on hematopoi- etic cells. Some of them, such as CD20, CD52, CD19, etc., can be targeted by therapeutic antibodies for treatment of hematopoietic malignancies.

2.1.4.1 CD20

CD20 is a B-cell lineage marker expressed on the surface of normal B cells, but not mature plasma cells. It is also expressed on more than 90 % of B-cell neoplasms. Rituximab (Rituxan), a mouse-human chimeric monoclonal antibody against CD20, was initially developed in the early 1990s by FDA approved in 1997 for treatment of non-Hodgkin B-cell lymphoma and approved. Rituximab is the first antibody approved for malignancy therapy and perhaps the most studied (Grillo- Lopez et al. 2002). Ofatumumab (Arzerra, Genmab/GlaxoSmithKline), the first humanized anti-CD20 antibody, received accelerated approval in 2009 for the treatment of relapsed or refractory CLL which has failed to fludarabine and alemtuzumab (Gupta and Jewell 2012). Rituximab binds to the large loop of CD20 antigen alone, whereas ofatumumab binds to a novel epitope that includes both small and large loops. Binding kinetics of ofatumumab is superior, resulting in a lower off-rate when bounding to CD20 (Teeling et al. 2004). Accordingly, in vitro studies showed that ofatumumab activates complement more efficiently than rituximab (Pawluczkowycz et al. 2009). Ofatumumab has been shown to be more potent than rituximab against both rituximab-sensitive and rituximab-resistant cells (Barth et al. 2012). Its activity against rituximab-resistant cells and the potent cytotoxic effect are believed to be due to the proximal epitope of the small loop of CD20 molecule and the high capacity for C1q activation. The newer-generation humanized anti-CD20 antibodies have been developed to increase their binding affinity for the FcγRIIIA expressed on NK cells by engineering Fc region. These antibodies include obinutuzumab (GA-101), ocrelizumab (2H7, Genentech/Roche/ Biogen Idec), and AME-133 (Applied Molecular Evolution/Eli Lilly), which are undergoing active clinical development (Cang et al. 2012). Obinutuzumab is a glycol-engineered anti-CD20 Ab in which Fc region was engineered to contain less fucose (Peipp et al. 2008). Obinutuzumab was approved by FDA for the treatment of CLL, and its activity in various B-cell malignancies is under clinical investigation.

2.1.4.2 CD52

CD52 is a cellular surface glycoprotein expressed on both normal and malignant B and T lymphocytes, but not on hematopoietic stem cells. It is also highly expressed 1 Antibody Therapies in Cancer 11 on B cell. Alemtuzumab (Campath), a humanized IgG1 antibody against CD52, was initially developed for the prevention of graft-versus-host disease (GVHD) in allogeneic bone marrow transplant. By binding to CD52, it induces ADCC of CLL cells (Hallek 2013). In 1997, a phase II trial of alemtuzumab was undertaken to evaluate the safety and efficacy in CLL patients who relapsed after standard chemotherapy. Alemtuzumab was approved in 2001 by FDA for the treatment of drug-resistant chronic lymphocytic leukemia (Ferrajoli et al. 2001). But it was withdrawn from the market in 2012. However, it is still available to patients with refractory CLL who have failed therapy with alkylating agents and second-line therapy with fludarabine.

2.1.4.3 CD19

CD19, a transmembrane protein, is a specific B-cell marker expressed on B cells along all differentiation stages of the lineage. In parallel, all cells derived from mature B-cell malignancies express CD19, except for plasma cell disorders, although the levels of CD19 expression are lower in CLL, mantle cell lymphoma, B-prolymphocytic leukemia, follicular lymphoma, and diffuse large B-cell lym- phoma samples, compared with normal B cells. CD19 staining is considered mandatory in the immunophenotyping schemes of the acute lymphoblastic leuke- mias (ALLs). The fact that CD19 is expressed by a wide range of B-lymphoid malignancies, but not by hematopoietic stem cells and pro-B cells (van Zelm et al. 2005), makes it an attractive target for antibody-mediated therapy. Humanized anti-CD19 antibodies have been designed to attract components of the immune system, predominantly T cells, to eliminate CD19+ target cells, such as modified anti-CD19 antibodies (Awan et al. 2010) and bispecific anti-CD19/anti-CD3 anti- bodies (Topp et al. 2011). One of the most attractive approaches to target malignant B cells is the introduction of chimeric antigen receptors (CARs), composed of single-chain anti-CD19 antibody and intracellular signaling components for T-cell activation, into patient-derived T cells (Porter et al. 2011). The novel anti-B-cell therapeutics have shown promising clinical effects against various B-cell malig- nancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL).

2.1.4.4 CD30

CD30, a membrane glycoprotein, is a member of TNF receptor family which is expressed on activated, but not resting, T and B cells. CD30 expression is very low in normal tissues. However, CD30 shows highly selective expression on tumor cells. In particular, CD30 is broadly expressed in a variety of lymphoid malignan- cies. CD30 expression is also observed on nonlymphoid embryonal carcinomas and occasionally in nasopharyngeal cancers. Brentuximab vedotin (Adcetris, Seattle Genetics) is a chimeric anti-CD30 antibody conjugated to the highly potent 12 S. Wang and M. Jia auristatin derivative MMAE through the cleavable linker (Sievers and Senter 2013). Brentuximab vedotin treatment causes complete regression of established tumors in xenograft models of Hodgkin lymphoma and anaplastic large-cell lymphoma. Brentuximab vedotin was approved in 2011 for the treatment of patients with Hodgkin lymphoma and systemic anaplastic large-cell lymphoma (Katz et al. 2011).

2.2 Clinical Efficacy of Tumor-Targeted Antibody Therapeutics

Over 13 tumor-targeted antibodies have been approved by the FDA for the treat- ment of a variety of solid tumors and hematological malignancies (Table 1.1). Meanwhile a large number of therapeutic antibodies are currently being tested in early and late-stage clinical trials. The most successful therapeutic antibodies in patients with solid tumors are the classes of antibodies targeting the members of EGFR family (such as EGFR and HER2) and VEGF. More importantly, there are some predictive biomarkers that are pivotal in optimal selection of patients for these therapeutics. For example, colorectal cancers bearing wild-type KRAS (Kirsten rat sarcoma viral oncogene) tumor treated with anti-EGFR antibodies have improved responses and survival (Van Cutsem et al. 2009; Amado et al. 2008). The use of trastuzumab has also been restricted to patients with either 3+ immunohistochem- ical staining or fluorescence in situ hybridization positive for ErbB2 (HER2) expression. In the hematologic realm, the antibody against CD20 has enjoyed considerable success in patients with non-Hodgkin B-cell lymphoma and chronic lymphocytic leukemia.

2.2.1 Hematological Malignancies

There are currently two chimeric antibodies (rituximab and brentuximab vedotin) and three fully humanized antibodies (alemtuzumab, eculizumab, and ofatumumab) that are FDA approved for treatment of hematologic diseases. The first approved antibody was rituximab, which was initially approved for the treatment of non-Hodgkin B-cell lymphoma. Since then, the use of rituximab has grown widely to encompass not only a variety of B-cell malignancies but also immune-mediated disorders (i.e., rheumatoid arthritis, systemic lupus erythematosus, immune- mediated thrombocytopenia, autoimmune hemolytic anemia, cryoglobulinemia, etc.). Rituximab has been studied in a number of clinical trials, which have successfully demonstrated improvement in progression-free and overall survival in non-Hodgkin lymphoma (including follicular lymphoma and diffuse large B-cell lymphoma) as well as improvement in progression-free survival in chronic lym- phocytic leukemia. Rituximab in combination with cyclophosphamide, 1 Antibody Therapies in Cancer 13

Table 1.1 The approved antibodies for clinical treatment of cancers Antibody Initial format Target FDA-approved indication approval Trastuzumab Humanized HER2 HER2-positive breast cancer, 1998 (Herceptin, IgG1 as a single agent or in combi- Genentech/Roche) nation with chemotherapy for adjuvant or palliative treatment HER2-positive gastric or gastroesophageal junction carcinoma as first-line treat- ment in combination with cisplatin and capecitabine or 5-fluorouracil Pertuzumab Humanized HER2 For use in combination with 2012 (Perjeta, IgG1 trastuzumab and docetaxel for Genentech, Inc.) the neoadjuvant treatment of patients with HER2-positive, locally advanced, inflamma- tory, or early stage breast can- cer (either greater than 2 cm in diameter or node positive) as part of a complete treatment regimen for early breast cancer Ado-trastuzumab Humanized HER2 For use as a single agent for 2013 emtansine IgG1 (DM1 the treatment of patients with (KADCYLA, conjugated) HER2-positive, metastatic Genentech, Inc.) breast cancer who previously received trastuzumab and a taxane, separately or in com- bination. Patients should have either received prior therapy for metastatic disease or developed disease recurrence during or within 6 months of completing adjuvant therapy Bevacizumab Humanized VEGFA For first-line and second-line 2004 (Avastin, IgG1 treatment of metastatic colon Genentech/Roche) cancer, in conjunction with (continued) 14 S. Wang and M. Jia

Table 1.1 (continued) Antibody Initial format Target FDA-approved indication approval 5-fluorouracil-based chemo- therapy; for first-line treatment of advanced NSCLC, in com- bination with carboplatin and paclitaxel, in patients who have not yet received chemo- therapy; as a single agent in adult patients with glioblas- toma whose tumor has progressed after initial treat- ment; and in conjunction with IFN-α to treat metastatic kid- ney cancer In combination with pacli- taxel, pegylated liposomal doxorubicin, or topotecan for the treatment of patients with platinum-resistant, recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer For the treatment of persis- tent, recurrent, or metastatic cervical cancer, in combina- tion with paclitaxel and cis- platin or paclitaxel and topotecan For use in combination with fluoropyrimidine-irinotecan- or fluoropyrimidine- oxaliplatin-based chemother- apy for the treatment of patients with metastatic colo- rectal cancer (mCRC) whose disease has progressed on a first-line bevacizumab- containing regimen Ramucirumab Humanized VEGFR2 In combination with FOLFIRI 2014 (CYRAMZA, Eli IgG1 for the treatment of patients Lilly and Company) with metastatic colorectal cancer (mCRC) whose disease has progressed on a first-line bevacizumab-, oxaliplatin-, and fluoropyrimidine- containing regimen For use in combination with docetaxel for the treatment of patients with metastatic (continued) 1 Antibody Therapies in Cancer 15

Table 1.1 (continued) Antibody Initial format Target FDA-approved indication approval non-small cell lung cancer (NSCLC) with disease pro- gression on or after platinum- based chemotherapy For use in combination with paclitaxel for the treatment of patients with advanced gastric or gastroesophageal junction For use in combination with paclitaxel for the treatment of patients with advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma For use as a single agent for the treatment of patients with advanced or metastatic, gas- tric, or gastroesophageal junction (GEJ) adenocarci- noma with disease progres- sion on or after prior treatment with fluoropyrimidine- or platinum-containing chemotherapy Cetuximab Chimeric IgG1 EGFR In combination with radiation 2004 (Erbitux, ImClone therapy for the initial treat- Systems/Bristol- ment of locally or regionally Myers Squibb) advanced SCCHN, as a single agent for patients with SCCHN for whom prior platinum-based therapy has failed, and palliative treatment of pretreated metastatic EGFR-positive colorectal cancer For use in combination with FOLFIRI (irinotecan, 5-fluorouracil, leucovorin) for first-line treatment of patients with KRAS mutation- negative (wild-type), EGFR- expressing metastatic colo- rectal cancer (mCRC) as determined by FDA-approved tests for this use In combination with platinum-based therapy plus 5-florouracil (5-FU) for the (continued) 16 S. Wang and M. Jia

Table 1.1 (continued) Antibody Initial format Target FDA-approved indication approval first-line treatment of patients with recurrent locoregional disease and/or metastatic squamous cell carcinoma of the head and neck (SCCHN) Panitumumab Human IgG2 EGFR As a single agent for the treat- 2006 (Vectibix, Amgen, ment of pretreated EGFR- Inc.) expressing, metastatic colo- rectal carcinoma Dinutuximab Chimeric IgG1 GD2 In combination with 2015 (Unituxin, United granulocyte-macrophage col- Therapeutics ony-stimulating factor Corporation) (GM-CSF), interleukin-2 (IL-2), and 13-cis retinoic acid (RA), for the treatment of pediatric patients with high- risk neuroblastoma who achieve at least a partial response to prior first-line multi-agent, multimodality therapy Denosumab (Xgeva Human IgG2 RANKL For the treatment of adults and 2010 injection, Amgen, skeletally mature adolescents Inc.) with giant cell tumor of the bone that is unresectable or where surgical resection is likely to result in severe morbidity As a treatment to increase bone mass in patients at high risk for fracture receiving androgen deprivation therapy (ADT) for nonmetastatic prostate cancer or adjuvant aromatase inhibitor (AI) therapy for breast cancer. In men with nonmetastatic prostate cancer, denosumab also reduced the incidence of vertebral fracture Rituximab Chimeric IgG1 CD20 For the treatment of CD20- 1997 (Rituxan/ positive B-cell NHL and CLL MabThera, and for maintenance therapy Genentech/Roche/ for untreated follicular CD20- Biogen Idec) positive NHL A 90-min infusion starting at cycle 2 for patients with non-Hodgkin lymphoma (continued) 1 Antibody Therapies in Cancer 17

Table 1.1 (continued) Antibody Initial format Target FDA-approved indication approval (NHL) who did not experi- ence a grade 3 or 4 infusion- related adverse reaction dur- ing cycle 1. Patients with clinically significant cardio- vascular disease and high cir- culating lymphocyte counts (>5000/mcL) are not recommended to receive the faster infusion Alemtuzumab Humanized CD52 As a single agent for the treat- 2001 (Campath, IgG1 ment of B-cell chronic lym- Genzyme/Bayer) phocytic leukemia Ofatumumab Human CD20 Treatment of patients with 2009 (Arzerra, Genmab/ (XenoMouse) CLL refractory to fludarabine GlaxoSmithKline) IgG1 and alemtuzumab In combination with chlorambucil, for the treat- ment of previously untreated patients with chronic lym- phocytic leukemia (CLL), for whom fludarabine-based therapy is considered inappropriate Obinutuzumab Humanized CD20 For use in combination with 2013 (Gazyva, IgG1 chlorambucil for the treatment Genentech) of patients with previously untreated chronic lymphocytic leukemia (CLL) Blinatumomab Bispecific CD19 For the treatment of Philadel- (BLINCYTO, T-cell engagers phia chromosome-negative Amgen, Inc.) relapsed or refractory B-cell precursor acute lymphoblastic leukemia (R/R ALL) Gemtuzumab Humanized CD33 For the treatment of patients 2000 ozogamicin IgG4 with CD33-positive acute (Mylotarg, Pfizer) (Calicheamicin myeloid leukemia in the first conjugated) relapse who are 60 years of age or older and who are not considered candidates for other cytotoxic chemotherapy; withdrawn from use in June 2010 Brentuximab Chimeric IgG1 CD30 For the treatment of relapsed 2011 vedotin (Adcetris, (MMAE or refractory Hodgkin lym- Seattle Genetics) conjugated) phoma and systemic anaplastic lymphoma (continued) 18 S. Wang and M. Jia

Table 1.1 (continued) Antibody Initial format Target FDA-approved indication approval Ibritumomab Murine IgG1 CD20 Treatment of relapsed or 2002 tiuxetan (Zevalin, (90Y labeled) refractory, low-grade, or fol- Biogen Idec) licular B-cell NHL Previously untreated follicu- lar NHL in patients who achieve a partial or complete response to first-line chemotherapy Tositumomab Murine IgG2 CD20 Treatment of patients with (Bexxar, (131I labeled) CD20 antigen-expressing GlaxoSmithKline) relapsed or refractory, low-grade, follicular, or transformed NHL Ipilimumab Human IgG1 CTLA-4 For the treatment of 2011 (Yervoy, Bristol- unresectable or metastatic Myers Squibb) melanoma Pembrolizumab Humanized PD-1 For the treatment of patients 2014 (KEYTRUDA, IgG4 with unresectable or meta- Merck Sharp & static melanoma and disease Dohme Corp.) progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor Nivolumab Human IgG4 PD-1 For the treatment of patients 2014 (OPDIVO, Bristol- with metastatic squamous Myers Squibb) non-small cell lung cancer (NSCLC) with progression on or after platinum-based chemotherapy For the treatment of patients with unresectable or meta- static melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor

doxorubicin, vincristine, and prednisone (CHOP) remains the standard frontline regimen for diffuse large B-cell lymphoma (Coiffier et al. 2002). However, suboptimal response and resistance to rituximab have remained a challenge in the therapy of B-cell non-Hodgkin lymphoma. Rituximab has also been studied in new combination regimens, particularly for relapsed and refractory lymphomas (Recher et al. 2011). Brentuximab vedotin, a CD30-directed antibody-drug conjugate, has been eval- uated in Hodgkin lymphoma and anaplastic large-cell lymphoma in clinical studies. 1 Antibody Therapies in Cancer 19

Results of phase I studies provided very encouraging evidence of antitumor activity and indicated that the antibody-drug conjugate was well tolerated. Then, two open- label single-arm phase II studies were initiated with relapsed/refractory systemic Hodgkin lymphoma or anaplastic large-cell lymphoma. The Hodgkin lymphoma trail reported an ORR of 75 % and a durable CR of 34 % (Younes et al. 2012), and the anaplastic large-cell lymphoma trial demonstrated an ORR of 86 % and a durable CR of 57 % (Pro et al. 2012). Based on these encouraging data, brentuximab vedotin was granted an accelerated approval in 2011 by FDA for the treatment of two indications: the patients with Hodgkin lymphoma after failure of autologous stem cell transplant or after failure of at least two prior multi-agent chemotherapy regimens in those patients who are not eligible for stem cell trans- plant and the patients with systemic anaplastic large-cell lymphoma after failure of at least one prior multi-agent chemotherapy regimen. Ofatumumab, a humanized anti-CD20 antibody, received accelerated approval in 2009 for the treatment of patients with CLL who have failed fludarabine and alemtuzumab (Cheson 2010). A total of nine clinical trials have been completed evaluating ofatumumab in CLL. The overall response rate ranged from 40 to 51 % in these trials even in the rituximab-refractory patients (Wierda et al. 2011). The most common adverse events were infusion-related reactions, which occurred in 63 % of patients (the vast majority were grade 1 or 2). Other common or important adverse events were rash, fatigue, cough, fever, and infections. Severe adverse events included neutropenia, anemia, and thrombocytopenia in a subset of patients. Ofatumumab has been exploring in combination with other agents in various B-cell neoplasms. A phase II trial was conducted to evaluate the combination of ofatumumab with CHOP therapy for frontline treatment of follicular lymphoma (Czuczman et al. 2012). Tositumomab, a humanized anti-CD20 antibody coupled with iodine I131 (I131) (brand name Bexxar), was approved in 2003 for patients with CD20-positive non-Hodgkin lymphoma that have progressed during or after rituximab therapy. Efficacy of tositumomab conjugated to I131 was established in a clinical trial of 40 patients with low-grade, transformed low-grade, or follicular large-cell lym- phoma who had all progressed following at least four cycles of rituximab therapy. The overall response rate was 68 % with a complete response rate of 33 % (Davies et al. 2004). Subsequently four other single-arm studies showed similar results. It has also been effectively used as consolidation postchemotherapy and is being evaluated for use in conditioning regimens prior to autologous stem cell transplant.

2.3 Gastrointestinal Tumors

There are four FDA-approved monoclonal antibodies used in the treatment of gastrointestinal malignancies. Bevacizumab is a humanized neutralizing anti- VEGF antibody. A phase III trial published in 2004 showed a significant improve- ment in progression-free survival and overall survival in patients with metastatic 20 S. Wang and M. Jia colorectal cancer who were treated with a combination of bevacizumab, irinotecan, bolus fluorouracil, and leucovorin as first-line therapy (Hurwitz et al. 2004). Bevacizumab has also shown efficacy in combination with oxaliplatin in both first- and second-line settings for metastatic colorectal cancer (Saltz et al. 2008). Currently, bevacizumab-containing regimens are considered to be standard of care in the treatment of advanced colorectal cancer. The main side effects associated with bevacizumab include hypertension, proteinuria, bowel perforation (1.5–2 %), arterial thrombotic events (4–5 %), and delayed wound healing. Panitumumab, a humanized anti-EGFR antibody, was approved in 2006 after an open-label phase III trial of panitumumab showed improved progression-free survival over the best supportive care alone in patients with chemotherapy- refractory metastatic colorectal cancer. The patients who benefitted the most were noted to be KRAS wild type (Van Cutsem et al. 2007). Determination of the presence or absence of wild-type Kirsten rat sarcoma viral oncogene (KRAS) via fluorescent in situ hybridization (FISH) is a prerequisite prior to starting panitumumab therapy. Panitumumab can be given by itself in the second-line setting or in conjunction with chemotherapy in the first- and second-line settings. It is not indicated for use in the adjuvant setting. Side effects associated with panitumumab include infusion reactions, hypomagnesia, diarrhea, hypersensitivity reactions, dermatological toxicities, and ocular toxicities. Cetuximab, a chimeric anti-EGFR antibody, is currently approved as monotherapy in the third-line setting or in conjunction with systemic chemotherapy in the first- and second-line settings for treatment of metastatic colorectal cancer (Van Cutsem et al. 2009). Combining cetuximab therapy with folinic acid, 5-fluorouracil, and irinotecan (FOLFIRI chemotherapy) has been shown to prolong progression-free survival in patients with metastatic colon cancer, whose tumors harbor wild-type KRAS alleles (Van Cutsem et al. 2011). Biomarker testing to rule out KRAS mutation is also required prior to the use of cetuximab. Cetuximab is not currently approved for use in the adjuvant setting. Besides, side effects of cetuximab are similar to panitumumab. Trastuzumab, an anti-HER2 antibody, has been approved for treatment of HER2+ breast cancer. However, trastuzumab in combination with chemotherapy has also demonstrated efficacy in metastatic or locally advanced unresectable gastric cancer and gastroesophageal cancer. A phase III clinical trial showed statistically significant efficacy with the use of trastuzumab, including improved overall survival and progression-free survival (Bang et al. 2010). The use of trastuzumab requires dem- onstration of HER2 expression by immunohistochemistry of at least 3+ or FISH positivity (HER2: CEP 17 ratio of 2). In the metastatic setting, it is used in conjunction with chemotherapy in first- and second-line settings. Side effects of trastuzumab include left ventricular dysfunction, which is reversible with cessation of the therapy. Therapy has been successfully reinitiated once the ventricular function normalizes without further dysfunction. 1 Antibody Therapies in Cancer 21

2.3.1 Breast Cancer

There are approximately 25 % breast cancers that overexpress HER2/neu. Trastuzumab, a humanized anti-HER2 antibody, has been approved for use as a single agent as well as in combination with chemotherapy in patients with breast cancer. Trastuzumab monotherapy showed a 35 % objective response rate in patients with metastatic breast cancer who have not previously received chemo- therapy (Vogel et al. 2002) and resulted in a response rate of 21 % in patients with HER2+ tumors previously treated with cytotoxic chemotherapy (Cobleigh et al. 1999). The combination of trastuzumab with chemotherapy had an overall survival advantage as compared with chemotherapy alone (25.1 vs. 20.3 months) in the patients with metastatic disease (Slamon et al. 2001). Trastuzumab is associated with significant cardiac toxicity when combined with doxorubicin (Seidman et al. 2002). Patients with metastatic breast cancer with substantial overexpression of HER2/neu are candidates for treatment with the combination of trastuzumab and paclitaxel. In one randomized study of patients with metastatic breast cancer treated with trastuzumab, paclitaxel, and carboplatin, patients tolerated the combination well and had a longer time to progression, compared to trastuzumab and paclitaxel alone (Robert et al. 2006). Pertuzumab is another humanized anti-HER2 antibody. Because trastuzumab and pertuzumab bind to different regions of HER2 and block HER2 heterodi- merization with different HER2 family receptors, dual-antibody therapy should allow for simultaneous antagonism of both activated forms of HER2. A phase II clinical trial testing the combination antibody therapy in patients whose tumors progressed on trastuzumab and cytotoxic chemotherapy demonstrated an objective response of 25 % and a clinical benefit rate of 50 % (Baselga et al. 2010). A neoadjuvant trial further confirmed that the antibody combination was also effec- tive in patients who were naı¨ve to therapy (Gianni et al. 2012). A phase III study that compared the antibody combination plus docetaxel versus trastuzumab plus docetaxel in the first-line HER2+ metastatic setting showed that the median progression-free survival was 12.4 months in the control group versus 18.5 months in the pertuzumab group and overall survival is greater than what was observed for the initial approval of trastuzumab. The toxicity profile was similar in both treat- ment groups with no increase in cardiac toxic effects seen in the pertuzumab combination arm (Baselga et al. 2012). In 2012, the combination of pertuzumab with trastuzumab and docetaxel is approved as first-line therapy for HER2-positive metastatic breast cancer and for neoadjuvant treatment of HER2-positive breast cancer.

2.3.2 Lung Cancer

Bevacizumab, a VEGFA-specific antibody, was approved by FDA for use with carboplatin and paclitaxel as first-line treatment of unresectable, locally advanced, 22 S. Wang and M. Jia recurrent, or metastatic non-small cell lung cancer (NSCLC). The approval came after a single, large, randomized, open-label study evaluating the efficacy of bevacizumab in combination with paclitaxel and carboplatin in patients with locally advanced, metastatic, or recurrent nonsquamous NSCLC. The median duration of overall survival in the chemotherapy plus bevacizumab group was 12.3 months compared with 10.3 months in the chemotherapy alone group ( p ¼ 0.003) (Sandler et al. 2006). However, the trial evaluating the combination of bevacizumab with cisplatin and gemcitabine failed to show a benefit to the addition of bevacizumab, and thus it is not FDA approved for use in this combination. The toxicity profile of bevacizumab in lung cancer is similar to that seen in patients with colorectal or breast cancer, including hypertension, proteinuria, cerebrovascular ischemia, and infection.

2.3.3 Head and Neck Cancers

Cetuximab also received full FDA approval for the treatment of patients with locally advanced (with radiation) or metastatic squamous cell carcinoma of the head and neck (HNSCC) (as a single agent). It is also approved for use in a triple drug combination with a platinum and 5-FU, again for patients with recurrent or metastatic disease. The approval came after three pivotal trials. The initial trial compared cetuximab plus radiation with radiation alone. The overall survival in the antibody arm was 50 months compared to 30 months in the radiation alone arm. Locoregional control also showed a statistically significant improvement with the addition of cetuximab (Bonner et al. 2006). A second trial evaluated the triple combination of cetuximab, platinum, and 5-FU versus platinum and 5-FU. Overall survival was improved by 3 months in the experimental arm with a statistically significant improvement in objective response rate (Vermorken et al. 2008). The third study was a single-arm study evaluating the use of cetuximab as a single agent in patients with recurrent or metastatic HNSCC who failed to respond to platinum- based therapy (Vermorken et al. 2007). The objective response rate was unimpressive at 13 % but still meaningful for this difficult population.

2.3.4 Genitourinary

Bevacizumab has also been approved by FDA for treatment of clear-cell renal cell carcinomas. Bevacizumab delayed progression of clear-cell renal cell carcinoma when compared with placebo in patients with disease refractory to biological therapy (Coppin et al. 2011). Similarly, the combination of bevacizumab with interferon alpha as first-line therapy in patients with metastatic renal cell carcinoma resulted in longer progression-free survival but not overall survival compared with interferon alpha alone in two similarly designed randomized trials (Rini et al. 2008; Escudier et al. 2007). 1 Antibody Therapies in Cancer 23

2.4 Mechanisms Underlying the Antitumor Activity of Tumor-Targeted Antibodies

Many mechanisms have been proposed to explain the clinical antitumor activity of tumor-targeted antibodies. Although the ability of antibodies to disrupt signaling pathways involved in the maintenance of the malignant phenotype has received widespread attention, the antitumor effects of therapeutic antibodies were also shown to be dependent on several immune-mediated mechanisms, including ADCC, complement activation, and antibody-mediated phagocytosis. Importantly, it has been demonstrated that tumor-targeted antibody therapies can initiate tumor- specific immune responses that play critical roles in clinical efficacy. Each of these mechanisms may play a role in the antitumor activity of the antibodies. These roles are altered by different characteristics of the antibody themselves and the physio- logic environment in ways that remain to be fully elucidated.

2.4.1 Blockade of Ligand Binding and Signaling

Many targets for therapeutic antibodies are growth factor receptors that are overexpressed during tumorigenesis. By blocking the binding of ligands to recep- tors and/or signaling through these receptors, monoclonal antibodies may normal- ize tumor cell growth, induce tumor cell apoptosis, and/or help sensitize tumors to chemotherapeutic agents. EGFR and HER2 are tyrosine kinase receptors that belong to the ErbB/HER receptor family. Ligand binding or receptor dimerization initiate signaling through several pathways, including the phosphatidylinositol 3-kinase (PI3K)/AKT and Ras/mitogen-activated protein (MAP) kinase pathway, which promote cell survival and proliferation (Harari et al. 2007). The therapeutic anti-EGFR antibodies, cetuximab and panitumumab, induces cell cycle arrest and apoptosis in tumor cells by preventing ligand binding and receptor dimerization, a crucial step for initiating EGFR-mediated signal transduction (Shuptrine et al. 2012). Colon cancer commonly carries an activating mutation in exon 2 of the KARS gene encoding a GTPase, which functions downstream of EGFR signaling pathway (Harari et al. 2007). These antibodies are ineffective when used to treat patients with cancers that possess activating KRAS mutations. In contrast to EGFR, HER2 has no known ligand, and the antibodies targeting this receptor, trastuzumab and pertuzumab, function mainly to inhibit receptor homo- and heterodimerization and internalization (Chen et al. 2003). CD20, the targeted antigen of rituximab, has been suggested to trigger anti-apoptotic pathways in B cells through Bcl-2 (Bonavida 2007). Antibodies that target the tumor microenvironment and inhibit crucial events such as angiogenesis have shown therapeutic promise. For example, many tumors highly express VEGFs, which bind to their receptors on the vascular endothelium to stimulate angiogenesis. The anti-VEGF antibody, bevacizumab, and anti-VEGFR2 24 S. Wang and M. Jia antibody, ramucirumab, block the binding of VEGF to VEGFR highly expressed on vascular endothelial cells in tumors to inhibit tumor angiogenesis. Bevacizumab has shown some efficacy in a range of solid tumors, including nonsquamous non-small cell lung cancer (NSCLC), metastatic colon cancer, metastatic HER2/ neu-negative breast cancer, renal cancer, and pancreatic cancer.

2.4.2 Antibody-Dependent Cellular Cytotoxicity and Phagocytosis

Many evidences suggest that blockade of signal transduction may not be the only mechanism for antibody therapy in cancer patients. The potential roles of immu- nological mechanisms in the therapeutic efficacy of anti-HER2 and EGFR anti- bodies are supported by several line evidences. First, the clinical responses of the antibodies are correlated with certain polymorphisms of FcγR on NK cells, mono- cytes, and granulocytes known to have lytic activity in patients. Second, levels of expression, activation, or genomic amplification of EGFR are not consistently correlated with the clinical response to the therapy of antibodies targeting these receptors. Last, tumor cell apoptosis is not observed in vitro culture system without lymphocytes (Ferris et al. 2010). Several studies have established the importance of Fc-FcR interactions for the antitumor effects of certain antibodies in murine tumor models and cancer patients. The antitumor activities of trastuzumab and rituximab were lower in FcγR-deficient mice than wild-type mice. The FcR polymorphisms that enhance antibody binding are highly correlated with the clinical response to rituximab, cetuximab, and trastuzumab. One year following rituximab treatment, non-Hodgkin lymphoma patients homozygous for FCGR3A-158V, encoding the high-affinity FcγRIIIA, showed a 90 % objective response rate, compared with a 67 % objective response rate for patients carrying the low-affinity FCGR3A-158F polymorphism (Cartron et al. 2002). In a smaller study of cetuximab treatment in colorectal cancer, the patients with either of the high-affinity polymorphisms, FCGR3A-158V or FCGR3A-131R, had a median progression-free survival of 3.7 months compared with 1.1 months for patients who carried neither high-affinity polymorphism (Zhang et al. 2007). Similarly, breast cancer patients homozygous for FCGR3A- 158V had significantly higher objective response rates following trastuzumab treatment compared with patients carrying FCGR3A-158F (82 % versus 40 %), and this higher response rate was associated with significantly longer progression-free survival (Tamura et al. 2011). Once antibodies are bound to their cognate antigen expressed at the surface of tumor cells, their Fc domain can bind to FcRs on natural killer (NK) cells, mono- cytes, macrophages, and granulocytes to trigger antibody-mediated cellular cyto- toxicity (ADCC), leading to the destruction of the targeted cell. The result of the Fc-FcR interaction depends on both the IgG subclass and the type of FcRs. In humans, the subclasses of IgG that function in ADCC are IgG1 and IgG3. Their binding to FcγRIIIA on NK cells causes recruitment of adapter proteins and activation of the NK cell, followed by a cascade that leads to destruction of the 1 Antibody Therapies in Cancer 25 target cell via release of lytic factors. Antibodies also can mediate antibody- dependent cellular phagocytosis when their variable regions bind tumor cells and Fc regions bind to FcγRI expressed on macrophages, neutrophils, and eosinophils. Some studies showed that antibody-dependent phagocytosis plays a significant role in destruction of antibody-coated tumor cells via FcγRI and other FcγRs expressed on macrophages. In addition, antibody-coated target cells can induce the production and release of cytokines by immune effector cells that express FcRs. These cyto- kines can activate other immune effector cells in the tumor microenvironment. Thus, immune cell activation via FcRs can contribute to direct ADCC, as well as to the production of cytokines that contribute to the control of tumor growth in other ways.

2.4.3 Complement-Dependent Cytotoxicity

In addition to mediate ADCC, the most clinically approved antibodies also can activate the complement system causing tumor cell destruction. The ability of a given antibody to fix complement and to induce cytotoxicity is partly dependent on antigen concentration, the orientation of the antigen in the membrane, and whether the antigen is present on the surface as a monomer or a polymer. When two or more IgG are bound to tumor cell, the binding of multiple IgG Fc to C1q subunit of complement factor 1 (C1) initiates the cascade of complement system, leading to direct cytotoxicity through the formation of complement pores in the membrane of antibody-coated cells. In addition, the highly chemotactic complement molecules C3a and C5a produced during the complement reaction lead to the recruitment and activation of immune effector cells, such as macrophages, neutrophils, basophils, mast cells, and eosinophils. The relationship between complement activation and therapeutic activity is suggested from the studies with several clinically approved therapeutic antibodies. Rituximab has been found to be dependent, in part, on complement-dependent cytotoxicity for its in vivo efficacy. Depletion of complement decreased the ther- apeutic activity of rituximab in a xenograft model of human B-cell lymphoma (Cragg and Glennie 2004). The genetic polymorphisms in the C1QA gene (encoding complement C1q subcomponent subunit A) correlate with clinical response to rituximab therapy in patients with follicular lymphoma (Racila et al. 2008). Optimization of antibody-based complement activities can enhance antitumor activity of antibodies. Ofatumumab binds CD20 at a different epitope from rituximab with improved binding kinetics and induces potent tumor cell lysis through improved activation of the classical complement pathway (Coiffier et al. 2008). Both complement-dependent cytotoxicity and ADCC can contribute to mono- clonal antibody-induced tumor cell lysis. However it is generally accepted that complement-dependent cytotoxicity has a limited role in the therapeutic efficacy of antibodies that recognize target antigens on solid tumors. 26 S. Wang and M. Jia

2.4.4 Induction of T-Cell Immunity Through Cross-Presentation

Early studies on the antitumor effects of therapeutic antibodies focused on the potential roles of passive immunotherapy provided through blockade of signaling pathway, ADCC, and complement-dependent cytotoxicity. However, there is increasing evidence to suggest a role for the adaptive immune system in mediating the long-term benefit of antibody therapies. An increasing number of results in animal model systems and in clinical settings indicate that tumor-targeted anti- bodies trigger or enhance tumor-specific cellular immune responses, involving cytotoxic T lymphocytes (CTLs) and Th cells. Beyond directly inducing tumor cell death by blocking survival pathways and Fc-mediated innate immune effects, antibody therapy can indirectly stimulate persistent responses against tumor-associated antigens through induction of adap- tive immunity. Therapeutic antibodies are effective in enhancing antigen cross- presentation by DCs to T cells in vitro and in vivo, resulting in argumentation of tumor antigen-triggered CTL generation. The uptake, internalization, and presen- tation of apoptotic cell-derived or soluble tumor antigens to CD8+ T cells by DC are enhanced by various receptors on DC that have endocytic activity and by activating FcγR such as FcγRI, FcγRIIa, and FcγRIII (Burgdorf et al. 2006; Dhodapkar et al. 2002). ADCC might trigger cross-presentation by DCs and promote adaptive immune responses, as DCs can engulf the resultant apoptotic tumor cells and subsequently present tumor antigens on MHC class I and II molecules. The potential mechanisms of enhanced cross-presentation induced by antibody therapy include facilitation of tumor antigen-antibody complex uptake by DC, enhancement of FcR ligation and stimulation of DCs (Dhodapkar et al. 2002; Amigorena and Bonnerot 1999), induction of costimulatory and adhesion molecules on the DC, and upregulation of antigen-processing machinery components known to be crucial for optimal antigen processing and presentation (Whiteside et al. 2004). Tumor- targeted antibodies, such as cetuximab, rituximab, and trastuzumab can effectively trigger tumor-specific CTL responses. In preclinical studies, antibodies against tumor antigens have been shown to activate targeted antigen-specific CD8+ T-cell responses in preclinical studies. An antibody recognizing the rat HER2/neu antigen that expressed murine mammary tumor cells can induce tumor antigen uptake and cross-priming that correlated with improved in vivo tumor rejection (Kim et al. 2008). The studies from our and other groups showed in mouse tumor models that anti-HER2 antibody therapy required host HMGB-1, MyD88 signaling, CD8+ cells, and adaptive (RAG-dependent) immunity to mediate its optimal effect except for FcγR (Park et al. 2010; Stagg et al. 2011). More importantly, our study suggested that an inappropriate combination of chemotherapeutic drugs with anti- body therapy, although capable of enhancing the reduction of tumor burden, could abrogate antibody-initiated antitumor immunity leading to decreased resistance to rechallenge or earlier relapse (Park et al. 2010). The capacity of therapeutic antibodies to induce tumor-directed CTL responses is intriguing. Antibody-initiated cross-presentation of tumor antigens can be 1 Antibody Therapies in Cancer 27 exploited to induce adaptive immunity that may extend beyond the targeted anti- gen. CTLs can target intracellular antigens that are inaccessible to therapeutic antibodies. This strategy has been described as the “vaccinal effect” in rituximab therapy of lymphoma and has been shown to be relevant in antibody therapy of solid tumors (Hilchey et al. 2009). The antibody-dependent promotion of adaptive immunity remains an active and very promising area of research, since the induc- tion of adaptive immunity can be accompanied by efforts to expand, shape, and prolong the host immune response. There are extensive interactions, including both synergistic and antagonistic, between various mechanisms of action that can affect the antitumor effects of an individual antibody. An antibody may be developed with one mechanism in mind, but other mechanisms may also be important for its antitumor activity. Disrupting signaling pathway that results from antibody binding to a receptor on a cancer cell can alter the sensitivity of the targeted tumor cells to ADCC. Antibody-induced cancer cell lysis can enhance uptake of tumor antigens and subsequent cross- presentation by antigen-presenting cells, leading to an enhanced tumor-specific T-cell response.

2.5 Immunoconjugates: Targeting Cytotoxic Agents to Tumor Cells

Early efforts to enhance the antitumor effects of mAbs focused on boosting their direct cytotoxic effects on targeted cells. Conjugation of radionuclides (radioimmu- notherapies or RITs), drugs (antibody-drug conjugates or ADCs), toxins (immunotoxins), and enzymes (antibody-directed enzyme prodrug therapy or ADEPT) yielded a multitude of antibodies or antibody-like molecules with varying clinical efficacy. Three conjugated antibodies have translated into FDA-approved therapies, all for hematological malignancies. Yttrium-90 (90Y)-ibritumomab tiuxetan and 131I–tositumomab are RIT agents targeting CD20 and are indicated for treatment of relapsed and/or rituximab-refractory follicular or low-grade lym- phomas. The third approved immunoconjugate, brentuximab vedotin, is an ADC targeting CD30 and carrying the antimitotic drug, monomethylauristatin E. Brentuximab vedotin was recently approved for treatment of anaplastic large- cell lymphoma and Hodgkin lymphoma.

2.5.1 Antibody-Drug Conjugates

Conjugation of antibodies to a variety of very potent protein toxins, such as ricin, pseudomonas exotoxin, and diphtheria toxin, was developed and tested in the 1970s and 1980s. The immunogenicity and nonspecific toxicity of protein toxins posed major problems and limited further development (Dosio et al. 2014). However, the 28 S. Wang and M. Jia lessons learned from the studies of immunotoxins were applied to the development of antibody-drug conjugates (ADCs). ADCs use small molecules instead of protein toxins and thereby reduce immunogenicity. Drugs used as components of ADCs include potent drugs such as calicheamicin, which binds to the minor groove in DNA and causes strand scission; monomethylauristatin E (MMAE), which blocks polymerization of tubulin; maytansine (DM1), which inhibits the assembly of microtubules; and, most recently, pyrrolobenzodiazepines, which cross-link DNA. The linker that connects antibody to drug is vital. It needs to attach the drug to antibody in a manner that does not alter the specificity of antibody, to render the drug nontoxic while bound to antibody, to remain stable in the circulation, and to release the drug in the appropriate intracellular compartment when the ADC is internalized. The first approved ADC was gemtuzumab ozogamicin that was an anti-CD33 calicheamicin conjugate. It was approved by FDA in 2000 for the treatment of patients with CD33-positive acute myeloid leukemia, but was voluntarily with- drawn in 2010 when post-marketing studies indicated that the ADC did not improve survival and had greater toxicity than chemotherapy alone. Brentuximab vedotin (Adcetris@, Seattle Genetics) is an anti-CD30 antibody conjugated to MMAE. The linker contains a peptidic moiety which can be cleaved within the cathepsin B-containing endosomes and lysosomes, where MMAE disrupts microtubule func- tion and leads to cell apoptosis. Under the FDA’s accelerated approval process based on efficacy and good safety profile in clinical, brentuximab vedotin was approved in 2011 for use in Hodgkin lymphoma patients after failure of autologous stem cell transplant or in those ineligible for transplant who have failed at least two chemotherapy regimens and in patients with anaplastic large-cell lymphomas after failure of at least one prior multi-agent chemotherapy regimen (Katz et al. 2011; Younes et al. 2010). Its efficacy in other malignancies is yet to be validated; however preclinical studies suggest that it may have activity against mesothelioma. Trastuzumab emtansine (T-DM1, Roche/Chugai) is the first approved ADC for solid tumors. T-DM1 combines trastuzumab with a potent anti-microtubule cyto- toxic agent, maytansinoid DM1, via a highly stable non-cleavable linker. In 2013, T-DM1 was approved by FDA as a new therapy for patients with HER2-positive metastatic breast cancer. It is important to note that T-DM1 was active against metastatic breast cancer that was previously resistant to trastuzumab-based therapy in the clinical trials (LoRusso et al. 2011; Verma et al. 2012). Besides breast cancer, a randomized phase II/III study comparing the efficacy and safety of T-DM1 to standard taxane regimen is underway in HER2-positive gastric cancer. The clinical successes of brentuximab vedotin and T-DM1 have stimulated a great deal of activity in developing ADCs to different solid and hematologic tumor targets. There is now a relatively long clinical pipeline of nearly 30 additional ADCs against over 24 targets (Mullard 2013). In addition, new toxic payloads and linkers are under development in an effort to further increase therapeutic index and provide for combination therapy. 1 Antibody Therapies in Cancer 29

2.5.2 Radioimmunoconjugates

The successful treatment of thyroid cancer with 131I leads to explore approaches to deliver radioisotopes directly to cancers by various carrier molecules, including antibodies. Two radioimmunoconjugates, ibritumomab tiuxetan and tositumomab, have been approved by the FDA since 2002 and have clinical activity against refractory lymphoma. They are both based on CD20-specific mAbs but use differ- ent isotopes (90Y for ibritumomab tiuxetan and 131I for tositumomab) (Witzig et al. 2002; Kaminski et al. 1993). Radioisotopes continually decay and cause nonspecific radiation damage to normal tissues, especially to the bone marrow which is particularly radiosensitive. The high doses of radiation reach the kidney and the liver for clearance, and only a small fraction of radiation does end up the tumor. These challenges have limited the clinical utility of these radioimmu- notherapies. In addition, the complex of preparing and delivering these therapies also limited their widespread uses.

3 Immunomodulating Antibody Therapies

In addition to directly targeting tumor cells, numerous antibody-based therapeutic strategies have been developed to target cells of the immune system with the goal of enhancing antitumor immune responses. Targeting the immune system offers the attraction of potentially generating active and long-lasting antitumor immunity. Furthermore, such antibodies are not tumor or patient specific and potentially achieve broad, polyclonal antitumor immunity, directed against multiple tumor antigens and reducing the chances of immune escape.

3.1 Therapy Targeting Immune Checkpoints in Cancer

The classical cancer immunotherapies have focused on boosting the immune system to produce new tumor-specific T cells. The general thinking was that the cancer patients were lack of recognition and induction of an antitumor immune response. However, in the mid-1990s, it became clear that the immune system indeed recognizes tumor antigens but remained quiescent in spite of the persistent presence of tumor antigens. Meanwhile, it was discovered that negative regulatory T-cell surface molecules were upregulated in activated T cells to dampen their activity, resulting in less effective killing of tumor cells. The first discovered molecule is CTLA-4, followed by PD-1, Tim-3, BTLA, etc. In various animal models, administration of these immune checkpoint inhibitors unleashed immunity to tumors, viruses, and other pathogens. These seminal studies ultimately led to the development and FDA approval of the first antibody-based immunotherapy that 30 S. Wang and M. Jia targets negative regulator of T cells, such as CTLA-4 and PD-1, in patients with metastatic melanoma and non-small cell lung cancer (NSCLC).

3.1.1 CTLA-4

3.1.1.1 Biology of CTLA-4 Pathway

CTLA-4 is a key negative regulatory receptor mainly expressed on activated T cells where it binds to B7-1 and B7-2, the members of the B7 immunoglobulin super- family, expressed by dendritic cells and other antigen-presenting cells. CTLA-4 ligation effectively inhibits further activation and expansion of activated T cells, thereby controlling the progress of an immune response and attenuating the chances for chronic autoimmune inflammation (Wang and Chen 2004). In addition, CTLA- 4 is constitutively expressed by Tregs, which is important for the immunosuppres- sive function of Treg cells (Peggs et al. 2009). The fundamental importance of CTLA-4 in controlling T-cell function is well illustrated by the phenotype of CTLA-4-/- mice, which develop a lethal multi-organ inflammatory disease. Studies in animal model showed that treatment with a CTLA-4-specific antibody can prevent and reverse antigen-specific CD8+ T-cell tolerance in a CD4+ T-cell- dependent manner (Scalapino and Daikh 2008). A serial of works revealed that enhancement of T-cell effector functions, combined with the inhibition of regula- tory T (Treg) cells, might be responsible for the antitumor effects of CTLA-4 blockade (Allison et al. 1995). These preclinical data show that CTLA-4-specific treatment can enhance adaptive immunity and promote tumor regression.

3.1.1.2 Clinical Impact of CTLA-4 Blockade

Preclinical studies using mouse models provided the evidence that the CTLA-4 blockade could result in significant antitumor activity by enhancing naturally or vaccine-induced T cells, which led two companies (BMS/Medarex and Pfizer) to put two fully human anti-CTLA-4 antibodies, ipilimumab (MDX-010) and tremelimumab (CP-675), into the clinical trials. While ipilimumab is an IgG1 antibody and tremelimumab is an IgG2 antibody, both bind to CTLA-4 with affinities less than 1 nmol/L. Phase I/II trials showed clinical responses in patients with melanoma, renal cell carcinoma, prostate cancer, urothelial carcinoma, and ovarian cancer (Weber et al. 2008). Two phase III clinical trials with ipilimumab were conducted in patients with advanced melanoma. One trial evaluated ipilimumab at 3 mg/kg every 3 weeks for up to four treatments with or without gp100 peptide vaccine versus gp100 peptide vaccine alone for patients with previ- ously treated unresectable stage III or stage IV melanoma. Median overall survival (OS) in the ipilimumab plus gp100 and ipilimumab cohorts was 10.0 and 10.1 months, respectively, compared to 6.4 months for the gp100 control arm [hazard ratio (HR) 0.68, p < 0.001]. More impressive than the mean survival benefit 1 Antibody Therapies in Cancer 31 was the effect of ipilimumab on long-term survival: 18 % of the ipilimumab-treated patients survived beyond 2 years, compared with 5 % of patients receiving the vaccine alone (Hodi et al. 2010). The subsequent first-line trial compared ipilimumab plus dacarbazine versus dacarbazine alone in previously untreated patients with metastatic melanoma. This trial used a higher dose of ipilimumab at 10 mg/kg every 3 weeks for four doses followed by maintenance therapy every 12 weeks. The overall survival was significantly longer in the ipilimumab group (11.2 months) compared with dacarbazine alone (9.1 months). Adding ipilimumab to dacarbazine also increased liver toxicity, presumably due to enhancement of known single-agent hepatotoxicity for both drugs (Robert et al. 2011). These studies led to the approval of ipilimumab in the United States and Europe as therapy for patients with metastatic melanoma or unresectable disease in 2011. This was the first approval of a medication that demonstrated a survival benefit in randomized phase III studies for patients with advanced unresectable or metastatic melanoma. The success in the metastatic melanoma setting led to investigating ipilimumab- resected stage III patients at high risk of recurrence. A phase III study investigating adjuvant ipilimumab versus placebo in patients with stage III melanoma prelimi- narily reported a relapse-free survival of 26.1 months in the ipilimumab arm compared with 17.1 months in placebo group, although the duration of follow-up was only 2.7 years. However, tremelimumab failed to show a statistically signifi- cant survival advantage compared to chemotherapeutic, dacarbazine, in a first-line phase III trial involving 655 patients with advanced metastatic melanoma although it showed promising results in early phase I and II study (Ribas et al. 2013). Currently, tremelimumab continues to be evaluated as a treatment in combination with other anticancer agents for melanoma and other tumor types. Ipilimumab showed only modest antitumor effects in nonmelanoma cancers. A phase II study of metastatic renal cell carcinoma revealed partial response rate of 10 % in ipilimumab monotherapy group (Yang et al. 2007). In two randomized double-blind phase II trials of naı¨ve patients with non-small cell lung cancer or extensive disease small cell lung cancer that received standard chemotherapy alone or combined with ipilimumab, ipilimumab did not have a significant impact on overall survival in both diseases. But a subset analysis showed improved activity in patients with squamous non-small cell lung cancer (Reck et al. 2013; Lynch et al. 2012). The trials of ipilimumab in metastatic castration-resistant prostate cancer also yielded weak but positive signals of activity. Similar responses have been observed when combining ipilimumab with GM-CSF-based vaccines (GVAX) in patients with previously treated pancreatic ductal adenocarcinoma (Le et al. 2013).

3.1.1.3 The Unique Kinetics of Responses to Ipilimumab

Although radiographic responses to ipilimumab are relatively infrequent, the dura- bility of these responses can be measured in years rather than months. There have been several longer-term follow-up studies of patients with advanced melanoma 32 S. Wang and M. Jia who have received ipilimumab. They confirmed relatively low objective response rates (13 %), but higher percentages of stable disease. A pooled meta-analysis of 1861 melanoma patients receiving ipilimumab revealed durable responses in about 20 % of patients living for more than 4 years and a subset of patients for 10 years or more (Ascierto et al. 2014; Lebbe et al. 2014; Schadendorf et al. 2015). In a retrospective evaluation of 177 patients treated on some of the earliest clinical trials, 15 patients achieved a complete response, and 14 of these are ongoing, with the longest lasting 99 or more months (median, 83 months). Interestingly, nine patients who achieved partial responses are alive many years after ipilimumab treatment, three without any further treatment (Prieto et al. 2012). A recent phase III trial of ipilimumab (at 10 mg/kg) versus placebo for treatment of 951 patients with resected melanoma demonstrated a significant improvement in progression- free survival at 3 years of 46.5 versus 34.8 %, respectively. Interestingly, the responses in a small number of patients (~10–15 %) who showed complete tumor regression are extremely durable, lasting several years, indicating that protective immunity nay have been established. In addition to the remarkable durability of responses to ipilimumab, unusual patterns of radiographic responses were seen. Unlike chemotherapy and tyrosine kinase inhibitors where tumor regression is usually evident within weeks of initial administration, the responses to ipilimumab are slower and, in many patients, delayed (up to 6 months after treatment initiation) (Saenger and Wolchok 2008). Furthermore, the major and durable tumor regressions could occur after an apparent increase in size on computed tomography (CT) or magnetic resonance imaging (MRI). Tumor enlargement may result from drug-induced inflammation at tumor sites or could reflect actual tumor growth followed by delayed regression. Such phenomena pose challenges for the conventional response evaluation criteria in solid tumors. After the analysis of the distinct response patterns associated with ipilimumab therapy in a retrospective analysis of 487 patients treated across three multicenter phase II clinical trials, the immune-related response criteria (irRC) were proposed to better characterize the response pattern (Wolchok et al. 2009). According to this irRC, new lesions are included in the determination of the overall tumor burden and do not automatically indicate progressive disease. In addition, evidence of disease progression requires confirmation with a subsequent radio- graphic assessment at least 4 weeks later.

3.1.1.4 The Biomarkers for Responses to Ipilimumab

Because only a subset of patients treated with CTLA-4 blockade exhibit a long-term benefit, it would be advantageous to predict clinical responses. Several biomarkers that appear to reflect immune activation and correlate with clinical response have been identified, including absolute lymphocyte count, amplification of preexisting humoral immune response to NY-ESO-1 antigen, and increases of ICOS- expressing CD4+ T cells and IFN-γ-producing antigen-specific CD4+ T cells in peripheral blood (Postow et al. 2012; Hamid et al. 2011; Carthon et al. 2010). 1 Antibody Therapies in Cancer 33

Genetic analyses of melanoma tumors revealed that higher numbers of mutations, termed “mutational load,” and creation of new antigens that can be recognized by T cells as a result of these mutations, termed “neoantigens,” correlated with clinical responses to anti-CTLA-4 therapy (Snyder et al. 2014). These studies highlight the complex interplay between cancer cells and the immune system, which will need to be further elucidated to guide rational development of combination therapies.

3.1.1.5 Side Effects of CTLA-4 Blockade

Due to the nonspecific nature of disinhibited T cells, ipilimumab has been associ- ated with a new category of side effects called immune-related adverse events (irAEs), which appear to be autoimmune in nature. These are largely confined to the skin and gastrointestinal systems although hepatic and endocrine issues have also been observed. The initial phase III reported irAEs in nearly 60 % of patients receiving 3 mg/kg ipilimumab, and about 12 % of patients suffered grade 3 and 2.3 % grade 4 irAE. The most common irAEs following ipilimumab include rash, diarrhea, colitis, hepatotoxicity, and endocrinopathies. Typically, the majority of grade 2–5 events are manifest within the first 3 months of treatment with a median resolution time of between 5 and 7 weeks (Hodi et al. 2010). Their occurrence in individuals with no prior history of autoimmunity validates the mechanism of action of anti-CTLA-4 in “releasing the brakes” on immune responses and under- scores the precarious balance that normally exists between self-tolerance and autoimmunity. Analyses and biopsies indicate that irAEs are mediated by infiltra- tion of highly activated CD4 and CD8 T cells, as well as increased serum inflam- matory cytokines (Hodi et al. 2003). With early recognition, these events are generally manageable with corticosteroids or infliximab. Any grade 3 or 4 irAEs are contraindication for further ipilimumab treatment.

3.1.2 PD-1

3.1.2.1 Biology of PD-1 Pathway

Programmed cell death 1 (PD-1, CD279), a homologue of CTLA-4, is another negative regulatory receptor mainly expressed on activated T cells. Similarly, PD-1binds to two ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), both of which are also members of the B7 immunoglobulin superfamily expressed by antigen-presenting cells. Similar to CTLA-4, PD-1 is absent on resting naı¨ve and memory T cells and is expressed upon TCR engagement. The two ligands PD-L1 and PD-L2 share 37 % sequence homology. However, their expression is highly divergent. PD-L1 is inducibly expressed on both hematopoietic cells and non-hematopoietic cells by inflammatory cytokine IFN-γ, while PD-L2 has much more selective expression on activated dendritic cells and some macrophages. The critical role of PD-1 signaling in immunoregulatory function has been demonstrated 34 S. Wang and M. Jia by inhibition of the effector T cells primarily in tumor and chronic viral infection (Wang and Chen 2004).

3.1.2.2 Expression of PD-1 and PD-L1 in Tumors

PD-1 is highly expressed on a large proportion of tumor-infiltrating lymphocytes (TILs) (Ahmadzadeh et al. 2009). Persistent expression of PD-1 on CD8+ TILs may reflect an anergic or exhausted state, as has been suggested by decreased cytokine production by PD-1+ compared with PD-1- TILs. This phenotype has been observed in TILs from various types of tumors and linked to poor prognosis and tumor recurrence (Thompson et al. 2007), highlighting PD-1 as an important molecule in regulating antitumor activity. The PD-1 ligands are commonly upregulated on the tumor cell from many different human tumors and also possess prognostic capacities (Hamanishi et al. 2007; Frigola et al. 2011; Thompson et al. 2006; Gao et al. 2009). PD-L2 has also been reported to be highly upregulated on cells from certain B-cell lymphomas. Forced expression of PD-L1 on mouse tumor cells inhibits local antitumor T-cell-mediated responses (Hirano et al. 2005). This combination of findings provides the basis for PD-1 pathway blockade to enhance antitumor effector functions in the tumor microenvironment. PD-L1 can be constitutively expressed by tumors cells, which endow tumors with intrinsic resistance to the elimination by endogenous tumor-specific T cells. The constitutive expression of PD-L1 can be driven by genetic alterations or activation of certain signaling pathways, such as the AKT pathway and STAT3, in tumor cells. For example, the expression of PD-L1 on glioblastomas is enhanced on deletion or silencing of PTEN, which implicates the involvement of the PI3K- AKT pathway (Parsa et al. 2007). Similarly, constitutive anaplastic lymphoma kinase (ALK) signaling has been reported to drive PD-L1 expression through signal transducer and activator of transcription 3 (STAT3) signaling (Marzec et al. 2008). In addition, primary mediastinal lymphomas commonly display gene fusions between MHC class II transactivator (CIITA) and PD-L1 or PD-L2, which place PD-1 ligands under the transcriptional control of the CIITA promoter (Steidl et al. 2011). A significant subset of Hodgkin lymphoma has amplification of chromosome 9p23-24, where PD-L1 and PD-L2 reside, resulting in overexpression of both ligands. A subset of Epstein-Barr virus-induced gastric cancers also display gene amplification with consequent induction of PD-L1 and PD-L2. Alternatively, PD-L1 expression can be induced on tumor cells in response to specific cytokines, in particular IFN-γ. This mechanism represents an adaptive immune resistance of tumor cells reflecting the adaptation of tumors to endogenous tumor-specific immune responses. Indeed, it was demonstrated that PD-L1 expres- sion in human tumors is strongly correlated with both T-cell infiltration and IFN-γ expression tumor microenvironment (Taube et al. 2014; Spranger et al. 2013). This suggests a negative feedback loop in which tumor cell uses the natural physiology of PD-1 ligand induction that normally occurs to protect a tissue from infection- 1 Antibody Therapies in Cancer 35 induced immune-mediated damage to protect itself from an antitumor immune response.

3.1.2.3 Clinical Impact of Drugs Blocking PD-1

The general findings of increased PD-1 and PD-L1 expression on TILs and tumor microenvironment, respectively, provided an important rationale for the capacity of blockade of PD-1/PD-L1 pathway to enhance intratumoral immune responses. Multiple anti-PD-1/anti-PD-L1 antibodies have been evaluated and shown clinical responses in multiple cancer types. The first clinical trials of specific mAbs against the receptor PD-1 and its ligand PD-L1 included subjects with late-stage, heavily pretreated kidney, lung, prostate, and colon cancer. The data are quite encouraging and go beyond typical expectations. The first anti-PD-1 antibody tested in patients with melanoma was nivolumab (formerly BMS-936558, MDX-1106, and ONO-4538), a fully human IgG4 antibody. This antibody blocks the interaction between PD-1 and PD-L1 and also the interaction between PD-1 and CD80. In the initial phase I trial with nivolumab, 39 patients with refractory or relapsed meta- static melanoma, CRC, castrate-resistant prostate cancer, NSCLC, or RCC received a single intravenous infusion of nivolumab at 0.3, 1, 3, or 10 mg/kg. Partial responses were observed in two patients with melanoma and RCC, and complete response was documented in one colon cancer. Two patients with melanoma and NSCLC had significant tumor regression, but didn’t meet partial response criteria (Brahmer et al. 2010). Given the favorable safety profile and preliminary clinical activity of nivolumab, a larger multiple-dose trial involving 296 patients with diverse cancers was launched (Topalian et al. 2012). Patients with advanced melanoma, NSCLC, castration-resistant prostate cancer, or RCC were enrolled to receive nivolumab at a dose of 0.1–10 mg/kg every 2 weeks. Objective responses were observed in patients with melanoma (28 %), RCC (27 %), and NSCLC (18 %). Many responses were durable with 20 of 31 responses lasting 1 year or more. The use of nivolumab in unresected or metastatic melanoma has advanced greatly since those initial studies. In a phase III trial, nivolumab was compared with the alkylating agent dacarbazine in 418 patients with previously untreated metastatic melanoma without BRAF mutation. The objective response rate was 40 %, and overall survival rate at 1 year was 72.9 % for patients treated with nivolumab as compared to an objective response rate of 13.9 % and overall survival rate of 42.1 % for patients treated with dacarbazine chemotherapy. Although the objective response rate with nivolumab was greater in the patients with PD-L1+ tumors (52.7 %) than the patients with PD-L1 tumors (33.1 %), a survival benefit compared with dacarbazine was seen in both subgroups. Pembrolizumab (formerly MK-3475) is a very high-affinity humanized IgG4 isotype antibody against PD-1. Pembrolizumab was explored in a phase I trial for its activity and safety in 135 patients with advanced melanoma, including patients with progression on prior treatment of ipilimumab. Doses ranged from 2 mg/kg every 3 weeks to 10 mg/kg every 2 weeks. The response rate across all dose cohorts was about 36 S. Wang and M. Jia

37–38 %, with the highest response rate (52 %) observed in the cohort given 10 mg/ kg every 2 weeks. Responses were durable and overall progression-free survival was longer than 7 months (Hamid et al. 2013). The efficacy of pembrolizumab in ipilimumab-refractory melanoma patients was confirmed in a subsequent random- ized dose-comparison cohort of a phase I trial, whereby 173 patients with progres- sive disease after at least two doses of ipilimumab were given pembrolizumab at either 2 mg/kg or 10 mg/kg every 3 weeks (Robert et al. 2014). The objective response rate was 26 % at both dose levels with a median time to response of 12 weeks. Survival at 1 year was similar in the two treatment groups (58 % and 63 %). These studies led to a first FDA approval for pembrolizumab in September of 2014, followed by nivolumab in December 2014 as a treatment for patients with metastatic melanoma. Lung cancer has always been considered to be nonimmunogenic. However, there is accumulating evidence that the interaction between lung cancer and the immune system is clinically relevant (Dasanu et al. 2012). In a randomized, open-label phase III study (Borghaei et al. 2015), nivolumab was compared with docetaxel in patients with nonsquamous NSCLC that had progressed during or after platinum- based doublet chemotherapy. Two hundred ninety-two patients received nivolumab at a dose of 3 mg/kg every 2 weeks, and 290 patients were administrated with docetaxel at a dose of 75 mg per square meter of body surface area every 3 weeks. The response rate was 19 % with nivolumab versus 12 % with docetaxel ( p ¼ 0.02). The median overall survival was 12.2 months in the nivolumab group and 9.4 months in the docetaxel group. At 1 year, the overall survival rate was 51 % with nivolumab versus 39 % with docetaxel. Although progression-free survival did not favor nivolumab over docetaxel (median, 2.3 months and 4.2 months, respec- tively), the rate of progression-free survival at 1 year was higher with nivolumab (19 %) than with docetaxel (8 %). Based on the result of this phase III trial, nivolumab was approved in March 2015 for patients with previously treated advanced or metastatic non-small cell lung cancer. Pidilizumab (formerly CD-011), a humanized anti-PD-1 IgG1 antibody, was explored in a phase II trial with metastatic melanoma patients, who were random- ized to receive either 1.5 or 6 mg/kg every 2 weeks for up to 54 weeks and were stratified by ipilimumab-experience status. The overall response rate is 6 %, which is much lower than that in the trials with nivolumab or pembrolizumab. However, the overall survival at 1 year was 64.5 %, similar to that reported in studies of nivolumab (62 %). Several anti-PD-L1 monoclonal antibodies have been developed, including MPDL3280A, BMS-986559, and MEDI4736. MPDL3280A (Roche), a humanized IgG1 antibody with mutated Fc domain that is completely lack of FcγR binding, inhibits the interaction of PD-L1 with PD-1 and CD80. The promising results from the initial phase I study in metastatic bladder cancer led to a breakthrough therapy designation of MPDL3280A granted from FDA. Several phase I and II trials and one phase III trial are ongoing to further define its role in both advanced refractory solid tumors and hematological malignancies. BMS-936559 (MDX-1105, BMS), a fully humanized IgG4 antibody, inhibits the binding of PD-L1 to both PD-1 and 1 Antibody Therapies in Cancer 37

CD80. Phase I studies have assessed its safety (Brahmer et al. 2012). Some active phase I trials are underway investigating its use in both patients with advanced refractory solid tumors and HIV infection. MEDI4736 (MedImmune/AstraZeneca) is a similarly engineered anti-PD-L1 antibody. Various phase I/Ib and II trials are underway investigating its use as a single agent and also in combination with other therapeutic modalities.

3.1.2.4 Predictive Biomarkers for PD-1 Inhibitors

With the success of the PD-1/PD-L1 blockade, it has become a top priority to identify and characterize the factors that predict which patients are likely to respond to this therapy. Early studies revealed a correlation between PD-L1 expression on tumor cells, measured on pretreatment archival samples by immunohistochemical (IHC) methods, and the likelihood of response to anti-PD-1. In an initial phase I trial with nivolumab, it was reported that patients with PD-L1-positive tumors (5 % staining for PD-L1 on tumor cells) had an objective response rate of 36 %, whereas patients with PD-L1-negative tumors did not show any objective clinical responses (Topalian et al. 2012). With the advent of several new automated PD-L1 IHC tests and interrogation of hundreds of patients with a variety of cancer types, a significant but not absolute relationship between PD-L1 expression in the TME and responsiveness to PD-1pathway blockade has been confirmed. Some patients with PD-L1-negative tumors had clinical responses to anti-PD-1 and anti-PD-L1 treat- ment. For example, on a phase I trial with nivolumab, patients with PD-L1-positive tumors had an objective response rate of 44 %, and patients with PD-L1-negative tumors had an objective response rate of 17 %. In a phase I study of MPDL3280A in multiple tumor types, objective response rates were reported as 46 % in patients with the highest PD-L1 expression, 17 % in patients with moderate PD-L1 expres- sion, 21 % in patients with minimal PD-L1 expression, and 13 % in the patients with no detectable level of PD-L1 expression in tumors. Although PD-L1 expression in tumor tissues does correlate with higher response rates, it should not be used as a predictive biomarker for selection or exclusion of patients for treatment with either anti-PD-1 or anti-PD-L1 antibodies. The potential importance of PD-L1 expression by infiltrating immune cells, the presence and location of CD8+ tumor-infiltrating lymphocytes, and other factors are currently under intense study individually and in combination to discern more sensitive and specific predictors of clinical outcomes. In a study of primary and metastatic melanoma samples, the patients whose tumor tissues were positive for both PD-L1 expression and infiltration of T cells were found to have improved overall disease-specific survival as compared to patients who had only one of the two features or lacked both features. A study of pembrolizumab in patients with metastatic melanoma showed that preexisting CD8+ T cells distinctly located at the invasive tumor margin are associated with expression of PD-1 and PD-L1 and are more predictive of clinical response to antibody than PD-L1 expression in tumor tissues (Tumeh et al. 2014). These data suggest that PD-L1expression in the tumor 38 S. Wang and M. Jia is most compelling when it is observed in the context of an active T-cell response and that the ongoing T-cell response is the key factor. In the tumors that expression of PD-L1 is constitutive and is neither associated with T-cell infiltration nor induced by IFN-γ, assessment of PD-L1 expression may be very useful in guiding treatment. In Hodgkin lymphoma, Reed-Sternberg cells are known to harbor ampli- fication of chromosome 9p24.1, which encodes PD-L1 and PD-L2 and leads to their constitutive expression. Nivolumab was shown to elicit an objective response rate of 87 % in a cohort of 20 patients (Ansell et al. 2015). Given the dynamic nature of immune responses to tumors and the complexity of regulation of expression of multiple immune checkpoints and their ligands, it may be difficult to rely on any single immunologic biomarker to select patients for treatment. It may be necessary to determine the patterns of expression of multiple biomarkers.

3.1.2.5 The Side Effects of PD-1 Blockade

In general, PD-1 and PD-L1 inhibitors are fairly well tolerated with less severe irAEs at a wide range of therapeutic doses. For example, a phase I study of nivolumab in melanoma reported 54 % of patients suffered irAEs but only 5 % of patients developed grade 3–4 irAEs (Topalian et al. 2014). The most common adverse effects include mild fatigue, rash, pruritus, diarrhea, decreased appetite, and nausea, with specific irAEs relating to skin and gastrointestinal disorders including pneumonitis, vitiligo, and colitis within the first 6 months of therapy. Asymptomatic increases in transaminases (especially increase in ALT) as well as grade 1–2 thyroiditis are also relatively common (10–20 %). Immune-related adverse events of special interests, including pneumonitis, vitiligo, colitis, and hypophysitis, have also been reported. In fact, in the initial phase I study of nivolumab, there were three reported deaths (1 % of the total treatment population) related to pneumonitis in patients with lung cancer. Predictably, the incidence and severity of these adverse effects are amplified when used in combination with other agents (specifically other immunotherapeutic agents) as seen in the preliminary results of the combination trials presented so far (Wolchok et al. 2013). Whether toxicity is predictive of a better immune response remains unknown. The blockade of PD-1 pathway appears to result in lower numbers of patients suffering severe irAEs than with CTLA-4 blockade. The treatment of the severe irAEs generally includes withholding the treatment and the use of immunosuppressants (Gangadhar and Vonderheide 2014). Infliximab (anti-TNF-α) and mycophenolate mofetil have been used especially in those patients who are refractory to corticosteroids. Most patients are able to restart the treatment after resolution of clinical symptoms. Ongoing trials will further outline the incidence and characteristics of the toxicity profile with these agents used alone and in combination in various malignancies. 1 Antibody Therapies in Cancer 39

Fig. 1.4 CTLA-4 and PD-1 checkpoints regulate different components in antitumor immune responses. (a) CTLA-4-mediated immune checkpoint is induced in T cells at the time of their initial response to antigen. CTLA-4 functions as a signal dampener to maintain a consistent level of T-cell activation in the face of widely varying concentrations and affinities of ligand for the TCR. (b) By contrast, the major role of PD-1 pathway is to regulate inflammatory responses in tissues by effector T cells recognizing antigen in peripheral tissues. Excessive induction of PD-1 on T cells in the setting of chronic antigen exposure can induce an exhausted or anergic state in T cells

3.1.2.6 Antitumor Mechanism of Checkpoint Blockade

The suppression of CTLA-4 in antitumor immunity has been viewed to reside primarily in secondary lymphoid organs where T-cell activation occurs, because its ligands CD80 and CD86 are expressed on antigen-presenting cells (e.g., den- dritic cells and monocytes) but not on non-hematologic tumor cells. In addition, in considering the important role of CTLA-4 in driving the suppressive function of T regulatory (Treg) cells, inhibition of Treg cell-mediated immunosuppression is probably an important mechanism for anti-CTLA-4 antibody therapy, whereas PD-1 blockade is viewed to work predominantly in tumor microenvironment, where its ligands are commonly overexpressed by tumor cells as well as infiltrating leukocytes. In addition, tumor-infiltrating lymphocytes (TILs) commonly express heightened levels of PD-1 and are thought to be “exhausted” because of persistent stimulation by tumor antigens, analogous to the exhausted phenotype seen in murine models of chronic viral infection, which is partially reversible by PD-1 pathway blockade (Barber et al. 2006). The drugs blocking PD-1 or its major ligand, PD-L1, have heightened tumor selectivity and reduced toxicity compared with anti-CTLA-4. They also appear to have a much broader spectrum of antitumor activity than anti-CTLA-4 (Fig. 1.4). Circumstantial evidence supports the notion that neoantigens created by the multiple somatic mutations in cancers provide the dominant antigenic targets that 40 S. Wang and M. Jia

T cells recognize when checkpoints are blocked. A recent study has demonstrated that melanomas with higher mutational loads were more responsive to anti-CTLA-4 therapy (Schumacher and Schreiber 2015; Snyder et al. 2014). Similarly, higher numbers of mutations, including mutations in DNA repair pathways, with subse- quent increase in numbers of neoantigens, was found to correlate with clinical responses in patients with NSCLC who received treatment with anti-PD-1 anti- bodies (Rizvi et al. 2015). In a recently published phase II study in patients with progressive metastatic colorectal cancers with or without mismatch repair defi- ciency (Le et al. 2015), the immune-related objective response rate and progression-free survival rate were 40 % and 78 %, respectively, for mismatch repair-deficient cancers and 0 % and 11 % for mismatch repair-proficient cancers. Mismatch repair-deficient tumors are associated with a much higher neoantigen load than tumors with proficient mismatch repair, and high somatic mutation loads were associated with prolonged progression-free survival. These studies provide a strong rationale to integrate genetic analyses of the tumor with immune profiling for a more comprehensive evaluation of mechanisms that contribute to clinical responses with immune checkpoint blockade.

3.1.3 Other Potential Checkpoints

3.1.3.1 LAG-3

Lymphocyte activation gene-3 (LAG-3, CD223) is another surface molecule of the immunoglobulin superfamily, expressed on activated T cells, NK cells, B cells, and plasmacytoid dendritic cells, which play an important role in negative regulation of T-cell proliferation. In addition, LAG-3 is highly expressed on Treg cells and required for optimal function of Treg cells. The only known ligand for LAG-3 is MHC class II molecules. Its co-expression with PD-1 in a significant fraction of tumor-infiltrating lymphocytes in certain malignancies correlates with impaired CD8+ effector T-cell function (Matsuzaki et al. 2010). Although LAG3 inhibition alone was not sufficient to restore antigen-specific T-cell responsiveness, the combined blockade of LAG3 and PD-1 was more effective than PD-1 blockade alone. Combined antibody-mediated blockade of LAG-3 and PD-1 resulted in tumor rejection in several models without any short-term evidence of autoimmune side effects. Multiple companies have developed anti-LAG-3 antibodies. A LAG3- specific antibody BMS986016 (Bristol-Myers Squibb) has recently entered clinical testing in cancer in a phase I trial that includes cohorts receiving anti-LAG-3 monotherapy or combination therapy with anti-PD-1. The bispecific antibodies targeting LAG-3 and PD-1 (Tesaro/AnaptysBio) are in preclinical development. In addition, a LAG-3/Fc fusion protein, IMP321 (Immutep), has shown biological activity and clinical responses in renal cell carcinoma, metastatic breast cancer, and advanced pancreatic cancer (Brignone et al. 2009; Wang-Gillam et al. 2013). 1 Antibody Therapies in Cancer 41

3.1.3.2 TIM-3

T-cell immunoglobulin and mucin-containing protein-3 (TIM-3) were a molecule expressed on T cells, NK cells, and monocytes. TIM-3 binds to galectin-9 as well as several other ligands, including HMGB1, phosphatidylserine, and carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1). Galectin-9 is mainly expressed on Treg cells, upregulated in various types of cancers. Administration of galectin-9 in vitro causes death of Th1 cells in a TIM-3-dependent manner. TIM-3 is believed to play a critical role in inhibiting Th1 responses through Treg cells expressing galectin-9. The role of TIM-3 immune checkpoint was studied in several murine tumor models. TIM-3 was nearly univer- sally co-expressed with PD-1 on the majority of TILs from mouse and human tumors. Co-expression of both checkpoint molecules reflected a more exhausted phenotype, functionally defined by a T cell’s reduced ability to proliferate and secrete IFN-γ, IL-2, and tumor necrosis factor a (TNF-α). TIM-3 blockade in animal models has demonstrated similar antitumor activity compared to PD-1 pathway blockade, with greater efficacy by combing PD-1 blockade (Sakuishi et al. 2010). TIM-3 blockade restored IFN-γ and TNF-α production as well as the proliferation of NY-ESO-1-specific CD8+ T cells from melanoma patients in response to antigenic stimulation (Fourcade et al. 2010). TIM-3 has now emerged as an immune checkpoint receptor with its selective expression in tumor tissue as well as its critical role in multiple immunosuppressive mechanisms, which strongly supports TIM-3-targeted immunotherapies as single or combined modalities. Although there are no TIM-3 antibodies in clinical trials, several are under preclin- ical development by several companies.

3.1.3.3 B7-H3 and B7-H4

B7-H3 (B7 homologue 3, CD276) and B7-H4 (B7 homologue 4, known as B7S1, B7x, and Vtcn1) are both member of B7 immunoglobulin superfamily. Most studies supported immune-inhibitory functions for these molecules. However, the biology of B7-H3 and B7-H4 is incompletely understood, and their ligands are unidentified. B7-H3 and B7-H4 are upregulated on a variety of human tumors, and their expression has been associated with a poor outcome (Jiang et al. 2010; Krambeck et al. 2006; Zang et al. 2007). MGA271, a fully human monoclonal antibody against B7-H3, has demonstrated potent antitumor activity in xenograft models of renal cell and bladder carcinoma (Loo et al. 2012). Clinical trials with MGA271 for multiple refractory cancers that express B7-H3 are ongoing.

3.1.3.4 Killer Inhibitory Receptors

NK cells are a population of innate immune cells with documented roles in infectious and tumor immunity. NK function is controlled by the complex interplay 42 S. Wang and M. Jia of a series of activating receptors and killer inhibitory receptors. Killer inhibitory receptors are a broad category of inhibitory receptors that can be divided into two classes: killer cell immunoglobulin-like receptors (KIRs) and C-type lectin recep- tors. KIRs interact with cell surface HLA to inhibit NK cell functions. So, in a sense, KIRs can be thought of as immune checkpoint molecules on NK cells, and blocking KIRs could be exploited to augment antitumor immunity. To that end, a fully human anti-KIR mAb, lirilumab, has entered clinical testing. Lirilumab (initially IPH-2101, Innate Pharma/Bristol-Myers Squibb) is an antagonist antibody that specifically binds to the human KIR molecules KIR2DL1, KIR2DL2, and KIR2DL3, as well as to KIR2DS-1 and KIR2DA-2, and prevents inhibitory signal- ing triggered by their binding to HLA-C molecules, thereby increasing NK cell- mediated killing of HLA-C-expressing tumor cells. A phase I trial of anti-KIR in acute myelogenous leukemia has been completed (Vey et al. 2012). Several studies are going in hematologic and solid cancers, but of particular interest are trials in which lirilumab is being combined.

3.1.3.5 The Mechanisms of Evading Phagocytosis by Tumor Cells

CD47 is a transmembrane protein ubiquitously expressed on normal cells to mark “self” and has increased expression in circulating hematopoietic stem cells, red blood cells, and a high proportion of malignant cells. Although CD47 has multiple functions in normal cell physiology, it acts primarily as a dominant “don’t eat me” signal. Tumor cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype. CD47 expressed on tumor cells can bind to signal regulatory protein-a (SIRP-a) on phagocytic immune cells to prevent engulf- ment (Chao et al. 2012). This inhibitory mechanism of CD47 expression is seen in a broad range of malignancies and is therefore an attractive therapeutic target for all tumors expressing CD47. Notably, cancer stem cells also utilize CD47 to escape the attention of macrophages. To date, several academic and industry laboratories have anti-CD47 or SIRP-a blocking antibodies under development with open enrollment or planned clinical trials in targeting both hematologic and solid tumors. The approach is effective in models of human tumors in mouse models, and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma (Chao et al. 2010). In the United States, two phase I dose-escalation trials are currently underway, with anti-CD47 antibodies as a monotherapy for the treatment of advanced solid tumors and hematologic cancers. The potential benefit of thera- pies with CD47-specific antibody is that they should allow engagement of activated macrophages that will engulf tumor cells and present tumor antigens, thereby stimulating an adaptive antitumor immune response. 1 Antibody Therapies in Cancer 43

3.2 Immunostimulating Antibody Therapy

Another potential mechanism that may be exploited to generate therapeutic tumor immunity is the ability of some mAbs to behave as surrogate ligands, providing agonistic signals to immunostimulatory receptors. Monoclonal antibodies that act as agonists of stimulatory receptors can directly augment antitumor immune responses. Several such antibodies have been developed to target the costimulatory receptors of TNFR family, including glucocorticoid-induced tumor necrosis factor receptor (GITR), CD134 (OX40), CD137(4-1BB), and CD40. In addition, anti- bodies can be used to target to immune effector cells to enhance the responses of these immune cells, such as bispecific anti-CD3 antibodies and immunocytokines.

3.2.1 TNFR Superfamily

3.2.1.1 4-1BB

4-1BB (CD137 or TNFRSF9) is a costimulatory receptor mainly expressed on T cells and natural killer cells, and 4-1BBL is expressed by activated DCs, B cells, macrophages, as well as neutrophils. Engagement of 4-1BB with 4-1BBL or an agonist antibody provides costimulation in a CD28-independent way for CD4+ and CD8+ T-cell responses. 4-1BB signaling enhances T-cell proliferation and Th1 cytokine production and provides protection to CD8+ T cells from activation- induced cell death through NF-kB-mediated activation and upregulation of the anti-apoptotic molecules Bcl2 and BCL-xl. Ligation of 4-1BB with an agonist antibody can reverse tolerance of CD8+ T cells and promote tumor regression of established tumors primarily via CD8 CTL activity and NK cell function (Melero et al. 1997; Shuford et al. 1997). Two agonistic 4-1BB-specific antibodies have been developed. BMS-663513, a fully humanized IgG4 anti-CD137 antibody developed by Bristol-Meyers Squibb, was tested in phase I dose-escalation study in patients with advanced cancer. Three partial responses and four stable disease cases occurred at all three doses tested in expansion cohorts. Preliminary biomarker analysis revealed an increased percentage of circulating activated CD8+and CD4+ T cells following a single treatment. Based on the phase I study, a multidose phase II randomized trial of BMS-663513 as a second-line monotherapy was designed in patients with previously treated unresectable stage III or IV melanoma. Unfortu- nately, the study was terminated early due to a high incidence of grade 4 hepatitis (Ascierto et al. 2010). New trials with BMS-663513 are underway to establish a safe and efficacious dose, including a monotherapy trial in patients with advanced or metastatic solid tumors or with relapsed or refractory non-Hodgkin lymphoma. PF-05082566 is a fully humanized IgG2 anti-CD137 antibody developed by Pfizer and currently tested in clinical trial as either a single agent in patients with solid tumors or in combination with rituximab in patients with CD20-positive B-cell non-Hodgkin lymphoma. Clinical efficacy was observed in 9 out of 24 evaluable 44 S. Wang and M. Jia patients, and, notably, toxicity was generally mild. A phase I trial of the combina- tion of PF-05082566 plus rituximab in non-Hodgkin lymphoma reported efficacy in rituximab-refractory non-Hodgkin lymphoma and no grade 3 toxicity. An interest- ing recent study showed that tumor-depleting antibody therapies such as cetuximab can upregulate 4-1BB on natural killer cells. This provides a clear rationale for the combination of 4-1BB-specific agonist antibody therapy with tumor-depleting antibodies, which function through antibody-dependent cell-mediated cytotoxicity (ADCC) that can be mediated by natural killer cells.

3.2.1.2 OX40

OX40 (also known as CD134/TNFRSF5) is expressed on diverse T-cell subsets, NK cells, NKT cells, and neutrophils, whereas its ligand OX40L (also known as CD252/TNFSF4) is found on DCs, B cells, and macrophages. OX40 signaling can promote costimulatory signals to T cells leading to enhanced cell proliferation, survival, effector function, and migration. OX40 agonist antibody treatment can reactivate the memory T-cell population. Initial experiments showed that injection of OX40 agonists into tumor-bearing mice early after tumor inoculation cured 20–80 % of the animals depending on the tumor model (Weinberg et al. 2000). Additional data suggest that OX40 engagement deactivates Treg cell population within tumors, which would further sustain effector T-cell function. In some murine models, an agonist OX40-specific antibody can deplete Treg cells. This activity is antibody specific and requires Fc receptor-mediated ADCC activation. OX40+ T cells were found in a wide variety of human malignancies, which increased the rationale for translating anti-OX40 therapy to the clinic. There are several OX40 agonist antibodies in clinical development. Results from a prostate cancer trial of a mouse IgG1 antihuman OX40 mAb (9B12, developed by AgonOx) in combination with standard of care showed an increase in the number of circulating CD4+ T cells, CD8+ T cells, and natural killer cells, supporting the hypothesis that OX40 agonist promotes the proliferation and survival of activated T cells (Weinberg et al. 2011).

3.2.1.3 CD40

CD40 (also known as TNFRSF5), in contrast to most of the receptors discussed above, which are mostly expressed on T cells and in some cases natural killer cells, is constitutively expressed on DCs, B cells, monocytes, and macrophages. The ligand for CD40, CD40L (also known as CD154/TNFSF5), is expressed on acti- vated T cells, platelets, and several other cell types. Engagement of CD40 on antigen-presenting cells leads to the upregulation of costimulatory molecules, production of pro-inflammatory cytokines, and facilitation of cross-presentation of antigens. CD40 plays a central role in the cross talk between CD4+ T cells, DCs, and B cells. Bidirectional CD40-CD40L interactions are central to the generation of 1 Antibody Therapies in Cancer 45 both T-cell-dependent, humoral immune responses and cytotoxic T-cell responses, licensing APCs to present antigen to and activate responding CD8+ cytotoxic T-cell precursors. The expression pattern of CD40 on a broad range of malignancies and the important immunostimulatory role of CD40 in vivo make it an attractive target for agonist antibody therapy. Targeting CD40 with an agonist antibody allowed T cells to overcome tolerance and promote tumor eradication in mouse models (Diehl et al. 1999; French et al. 1999). Several humanized anti-CD40 antibodies have completed phase I clinical trial and are currently being assessed in phase II trials. Lucatumumab (formerly HCD122) was used in two phase I clinical trials in patients with chronic lympho- cytic leukemia and multiple myeloma (Bensinger et al. 2012; Yu et al. 2013). Immunologically, there was minimal response, promoting the design of combina- tion therapy studies. Another CD40-specific antibody, dacetuzumab (SGN-40, Seattle Genetics), is a humanized IgG1 that can induce tumor cell apoptosis as well as ADCC and was recently shown to exert its antitumor effects by inducing Fc-mediated phagocytosis of tumor cells by macrophages. Dacetuzumab was tested in a phase I trial of patients with CLL and MM and showed minimal clinical activity (Furman et al. 2010; Hussein et al. 2010). Combination trials are currently evalu- ating bortezomib with dacetuzumab in patients with MM. CP-870893 is the only anti-CD40 antibody tested in patients with solid malignancies. The first single-dose trial showed an objective partial response in 14 % of patients with melanoma (Vonderheide et al. 2007). In a subsequent multiple doses of CP-870893, 26 % of patients had stable disease (Ruter et al. 2010). In a phase I trial combining CP-870893 with tremelimumab in patients with metastatic melanoma, the objective response rate was 27.3 %. The most common toxicity was grade 1–2 cytokine release syndrome, which occurred within 24 h of administration of anti-CD40 antibody in 79.2 % patients. In an elegant study in both mice and humans, CP-870893 in combination with gemcitabine was shown to be active. The antitumor activity was independent of dendritic cell and T cells, instead, acting directly on CD40-expressing macrophages (Beatty et al. 2011). These results demonstrate and highlight the importance of both innate and adaptive immune responses in mediating tumor regression.

3.2.2 Bispecific Antibodies

A promising approach to directly stimulate T-cell immunity with antibodies is the development of bispecific T-cell engager (BiTE) molecules that bind to CD3 on T cells with one arm and to an antigen on cancer cells, such as CD19, EpCAM, or EGFR, with another arm. BiTEs are recombinant single polypeptide chains consisting of two scFv joined together by a flexible linker. The most clinically advanced BiTE is blinatumomab. It was recently approved by the FDA for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL). Blinatumomab is a bispecific antibody targeting CD19 designed to direct T cells to target B cells. In a single-arm phase II trial of blinatumomab in adult 46 S. Wang and M. Jia patients with relapsed or refractory B-cell precursor ALL, regression (defined as CR or CR with partial hematologic recovery) occurred in 69 % of patients, and persistence or minimal residual disease (MRD) response occurred in 88 % of patients with regression. The most adverse events were pyrexia (Topp et al. 2015). Catumaxomab (tri, a bispecific antibody targeting CD3 and EpCAM) was approved in 2009 for treatment of EpCAM-positive cancers in Europe based on antitumor activity in an open-label phase II/III clinical trial in the treatment of malignant ascites (Heiss et al. 2010). Catumaxomab consists of two half antibodies that originate from parental mouse IgG2a and rat IgG2b isotypes. As expected, most patients developed a human anti-mouse or rat antibody response to catumaxomab. Surprisingly, this anti-drug antibody response correlated with more favorable clinical outcome including an increase in median overall survival (Ott et al. 2012). MT111 is the third BiTE to enter clinical trials. It targets CEA, an immunoglobulin superfamily glycoprotein that is expressed on a variety of solid tumors and on the gastrointestinal track. MT111 is currently in a phase I clinical trial against gastrointestinal adenocarcinoma.

3.2.3 Immunocytokines

Cytokines are key players in stimulating and regulating immune responses in physiological and pathological processes. Various cytokines have been used for therapy of cancers and other diseases. However, the therapeutic efficacies of the cytokines are often hampered by severe side effects and poor pharmacokinetic properties. Fusion of cytokines with antibodies or antibody fragments allows for a targeted delivery and should improve therapeutic efficacy and pharmacokinetics. A plethora of different antibody-cytokine fusion protein has been established during the last two decades. The most notable is hu14.18/IL-2 immunocytokine, a fusion protein of anti-ganglioside GD2 chimeric IgG1 antibody and IL-2. Ganglioside GD2 is a tumor-associated surface antigen found in a broad spectrum of human cancers and stem cells, including pediatric embryonal tumors (neuroblastoma, retinoblastoma, brain tumors, osteosarcoma, Ewing’s sarcoma, rhabdomyosar- coma), as well as adult cancers (small cell lung cancer, melanoma, soft tissue sarcomas). In phase I study of hu14.18/IL-2 in children with refractory neuroblas- toma and melanoma, no objective responses were observed (Osenga et al. 2006). In a phase II study of children with refractory neuroblastoma, 3 out of 13 patients with measurable disease had stable disease, and 5 out of 23 patients with evaluable disease achieved marrow complete response. Toxicity profile was similar to that observed with IL-2 alone (Shusterman et al. 2010). 1 Antibody Therapies in Cancer 47

4 Combination Therapy of Antibodies

Combining anticancer therapies has been essential to achieve complete remission and cures for patients with cancer. Immunochemotherapy, the combination of immunotherapy and chemotherapy, has become the standard of care for many tumors. For example, rituximab has become a standard part of treatment for B-cell lymphomas, for example, R-CHOP. The expression of PD-L1 is considered to be one of the key mechanisms for tumor to resistant to antitumor immunity. It is impressive that even as single agents checkpoint blockade can produce durable responses in some patients. The promising results obtained with such immunomo- dulating antibodies in early phase clinical trials open many perspectives for syner- gistic combination strategies.

4.1 Antibody Therapeutics in Combination with Conventional Therapies

Although tumor-targeted antibodies may be used as single agents, most clinical scenarios use these antibodies in conjunction with radiotherapy and/or chemother- apy and demonstrate enhancement of clinical activity as compared with conven- tional therapy when given without the antibodies.

4.1.1 Combination of Tumor-Targeting Antibodies with Cytotoxic Chemotherapies

The primary combination strategies of bevacizumab with two-drug combination of fluorouracil (plus leucovorin) and either irinotecan (FOLFIRI) or oxaliplatin (FOLFOX) have been widely adopted for treatment for metastatic colorectal cancer (Hurwitz et al. 2004; Saltz et al. 2008). In a recent phase III, randomized study of 508 patients with untreated metastatic colorectal cancer, the combination of bevacizumab with a triple-drug combination of fluorouracil (plus leucovorin), oxaliplatin, and irinotecan (FOLFOXIRI) was conducted as compared with bevacizumab plus FOLFIRI. The study showed improved progression-free survival among patients treated with the combination of FOLFOXIRI plus bevacizumab as compared with FOLFIRI plus bevacizumab. The median progression-free survival is prolonged by 2.4 months, reaching 12.1 months in the experimental group (Loupakis et al. 2014). Inclusion of anti-CD20 antibody, such as rituximab, in standard chemotherapy regimens significantly improved patients’ outcome with or without pretreatment and is accepted as a standard first-line therapy for CD20+ lymphomas (Feugier et al. 2005; Herold et al. 2007; van Meerten and Hagenbeek 2010). Similarly, addition of trastuzumab to first-line chemotherapy resulted in significantly longer time to disease progression (7.4 versus 4.6 months, 48 S. Wang and M. Jia respectively), higher objective response rate (50 % versus 32 %), and longer overall survival (median, 25.1 versus 20.3 months) compared with chemotherapy alone (Slamon et al. 2001). Continuation of trastuzumab in combination with chemother- apy has also proven to be beneficial in patients with breast cancer who have progressed on previous trastuzumab (von Minckwitz et al. 2009). Although chemotherapy has been traditionally thought to be immunosuppres- sive, this view has been changed in recent years. In a small study of treatment of HER2-overexpressing malignancies with trastuzumab and chemotherapy in the high-risk adjuvant breast cancer and stage IV setting, enhanced endogenous HER2-specific immune responses were seen in 44 % of treated patients and, interestingly, were of greater magnitude and more frequently observed in clinically responding patients. In addition, augmented HER2-specific CD4+ T-cell responses were also observed in six of ten evaluable individuals (Taylor et al. 2007). These results indicated that an adaptive immune response against HER2 was induced by this treatment. The choice of chemotherapeutic agent and timing of these combinations will be important, because many cytotoxic chemotherapeutics target rapidly dividing cells. Our result from animal experiments demonstrated that paclitaxel or cyclophospha- mide administrated shortly after anti-HER2 antibody may delete the proliferating T cells and dramatically interfere with the tumor-specific memory generated by the antibody. However, the sequential administration of anti-HER2 antibody after chemotherapy may allow chemotherapy to enhance the antibody-mediated antitumor effect (Park et al. 2010).

4.1.2 Combination of Tumor-Targeting Antibodies with Radiation Therapy

Radiotherapy has long been used for its powerful antiproliferative and death- inducing capacities. However, recent preclinical and clinical data indicate that immunogenic cell death may also be an important consequence of ionizing radia- tion and that localized radiotherapy can evoke and/or modulate tumor-associated immune responses. Based on this understanding, investigators have begun to examine the therapeutic impact of combined radiotherapy and immunotherapies. Cetuximab has been combinated with radiation therapy for treatment of HNSCC and NSCLC. In a phase III clinical trial of patients with locoregionally advanced HNSCC treated with high-dose radiation plus cetuximab or radiation alone, cetuximab plus radiotherapy extended the median duration of locoregional control from 14.9 to 24.4 months and also significantly improved median overall survival (49.0 versus 29.3 months) and progression-free survival (17.1 versus 12.4 months) compared to radiation therapy alone (Bonner et al. 2006). In a multicenter phase Ib/II study of patients with resectable, locally advanced esophageal cancer treated with cetuximab plus radiation and chemotherapy, objective responses were achieved in 62 % patients, including 24 patients with complete responses. Median overall survival was 22.7 months and 2-year overall survival rate was 49.3 %. 1 Antibody Therapies in Cancer 49

4.1.3 Combination of Immune Checkpoint Blockade with Conventional Therapies

Conventional cancer therapies may lead to tumor cell death to reduce the tumor burden and expose neoantigens to initiate activation of T cells. Therefore, combi- nation with conventional agents should create an “immunogenic” tumor microen- vironment with clinical benefit for immunotherapy. There are multiple ongoing trials with anti-CTLA-4 or anti-PD-1/anti-PD-L1 antibodies in combination with cytotoxic chemotherapy, radiation therapy, or small-molecule inhibitors. Although the trial combining ipilimumab with vemurafenib was terminated early due to hepatic toxicity, a phase I trial of bevacizumab plus ipilimumab revealed an impressive number of clinical responses with manageable toxicity. The study of 46 metastatic melanoma patients treated with a combination of ipilimumab and bevacizumab reported a disease control rate of 67.4 % (Hodi et al. 2014). A phase I trial of combination of nivolumab with either sunitinib or pazopanib as second-line therapy also showed greater response rates than expected for each agent individu- ally in patients with kidney cancer. There are multiple ongoing trials with radiation therapy in combination with anti-CTLA-4 or anti-PD-1/anti-PD-L1 antibodies. A case report highlighted radio- therapy as an attractive partner for combining with ipilimumab in a patient with melanoma (Postow et al. 2012). Ipilimumab plus radiation has been evaluated in a phase I/II study in prostate cancer, with demonstrated clinical activity and tolerable adverse effects (Slovin et al. 2013). The therapeutic effects of combining ipilimumab with chemotherapy are unclear, perhaps reflecting the limited activity of the standard chemotherapies or the side effects of chemotherapies on the immune system. Chemotherapy regimens that deplete proliferating lymphocytes may negatively affect the efficacy of thera- peutics such as ipilimumab and nivolumab, which act by facilitating the activation and proliferation of tumor-infiltrating lymphocytes. An open-label, randomized phase II study reported a nonsignificant trend favoring ipilimumab combined with dacarbazine compared with ipilimumab alone, with disease control rates of 37.1 % versus 21.6 %, respectively (Hersh et al. 2011). In a phase III trial evaluating a similar combination of ipilimumab and dacarbazine in untreated patients with metastatic melanoma, a benefit in overall survival (11.2 versus 9.1 months) was reported. Survival rates for patients treated with ipilimumab and dacarbazine were higher than for patients treated with dacarbazine and placebo at 1 year (47.3 % versus 36.3 %), 2 years (28.5 % versus 17.9 %), and 3 years (20.8 % versus 12.2 %). Grade 3 or 4 adverse events occurred in 56.3 % of patients treated with ipilimumab plus dacarbazine, as compared with 27.5 % of patients treated with dacarbazine and placebo (Robert et al. 2011). The combination of nivolumab with erlotinib (tyrosine kinase inhibitor) as first- line therapy resulted in 19 % of objective response rate in patients with advanced EGFR-mutant NSCLC. Combination treatment with nivolumab plus pazopanib or sunitinib in patients with mRCC resulted in promising clinical responses, with 50 S. Wang and M. Jia response rates that were similar across all patients regardless of PD-L1 expression in pretreatment tumor tissues. The clinical trials evaluating combinations of PD-1 pathway inhibitors with BRAF inhibitors such as vemurafenib or dabrafenib and with MEK inhibitors such as trametinib are now underway in melanoma. These studies will provide valuable information regarding schedule, safety, and efficacy of these combinations for future studies.

4.2 Antibody Therapeutics in Combination with Immunotherapies

A single-agent immune checkpoint blockade is only effective for a subset of patients with advanced cancers, but most patients do not respond to such single- agent therapy. Combining immunological agents may improve the therapeutic efficacy by stimulating or regulating the different components of antitumor immune responses.

4.2.1 Combination of Multiple Checkpoint Blockades

There are currently many trials pairing PD-1 pathway blockade with novel and approved agents to improve the response rates relative to monotherapy. CTLA-4 and PD-1 pathways have different mechanisms for regulating T-cell responses. The combination of CTLA-4 and PD-1 blockade has reported synergistic antitumor effects in the preclinical and clinical studies. In a phase I study that investigates sequential and concurrent administration of ipilimumab and nivolumab in patients with advanced melanoma, the group with concomitant therapy had a better objec- tive response rate than the sequenced-regimen group (40 % versus 20 %, respec- tively). Notably, the concurrent-regimen caused tumor regression in 53 % patients, most with tumor regression of 80 % or more (Wolchok et al. 2013; Weber et al. 2013). Similar data were reported for a combination study with ipilimumab plus nivolumab in patients with metastatic renal cell carcinoma. In a phase III trial comparing nivolumab and ipilimumab to the combination in patients with mela- noma, nivolumab in combination with ipilimumab had better objective response rates than ipilimumab alone (43.7 %, 57.6 %, and 19 %, respectively). The patients with PD-L1-negative tumors can also achieve a response, albeit at a lower rate compared with the patients with PD-L1-positive tumors. The response rate was independent of tumor BRAF mutation status. This combinatorial treatment is also being tested in other cancers. The combination of nivolumab concurrently with ipilimumab followed by maintenance nivolumab was tested in 46 patients with previously untreated NSCLC. In this setting, the ORR was 22 % with an additional 33 % of the patient population achieving stable disease at 24 weeks. Grade 3/4 toxicities occurred in 48 % of the patients, with 36 % of patients discontinuing 1 Antibody Therapies in Cancer 51 treatment due to treatment-related adverse events. There were also three deaths reported that were related to the treatment regimen. Other checkpoint blockade combinations are in the early clinical developmental stage, such as combining PD-1 pathway inhibitors with antibodies against LAG-3 or TIM-3. LAG-3 and TIM-3 are being considered as markers of T-cell exhaustion, and the combinations of blockade of PD-1 pathway and LAG-3 or TIM-3 were supported by preclinical models which demonstrated promising antitumor activity. Phase I study comparing LAG- 3 vs. LAG-3 plus PD-1 blockade among patients with advanced solid tumors has just launched.

4.2.2 Combination of Checkpoint Blockade with Other Immunotherapies

Immunosuppression is dominant in tumor microenvironment. It is believed that immunotherapy regimen should begin with immune checkpoint blockade (e.g., a PD-1 or CTLA-4 antagonist) rather than a direct immune stimulator. Release from immunoinhibition will open the door for combination with a large number of immunotherapies that directly stimulate the immune response. Various combination strategies for immunotherapy have been widely studied in preclinical studies. A number of them have been explored in clinical trials to date. Combinations of ipilimumab with tumor vaccines have been the most common for combination therapies with antibodies, including peptide vaccines, cellular vac- cines, and DNA/RNA vaccines. The combination of ipilimumab with gp100 pep- tide vaccine was tested in a randomized phase III study of patients with metastatic melanoma, but failed to show superior activity to ipilimumab alone (Hodi et al. 2010). However, ipilimumab in combination with GM-CSF-producing allo- geneic pancreatic tumor cells has shown promising results in patients with previ- ously treated pancreatic cancer, which has been consistently viewed as a nonimmunogenic tumor type (Le et al. 2013). A regimen combining ipilimumab and IL-2 was tested in a single-arm phase I/II study, which showed an overall response rate of 22 %, with three complete responses, but it is unclear if this regimen is superior to monotherapy (Maker et al. 2005). A randomized trial of ipilimumab with and without GM-CSF reported an overall survival benefit to adding GM-CSF and showed a trend toward improved tolerability in the GM-CSF arm over ipilimumab alone. Combination treatments are also being developed to enable blockade of an inhibitory pathway while providing an agonistic signal through a stimulatory pathway, such as ICOS, OX40, 4-1BB, vaccines, cytokines, and oncolytic virus. 52 S. Wang and M. Jia

5 Future Prospects

The use of antibodies for cancer therapy is one of the great success stories of the past decade. Tumor-targeting antibodies currently provide clinical benefit to cancer patients and have been established as agents of standard of care for several highly prevalent human cancers. In recent years, it is more excited to witness how immunotherapy, specifically treatment with checkpoint blockade monoclonal anti- bodies, is becoming one of the main armamentarium for cancer therapy. Unprec- edented durable response among patients with advanced melanoma, Hodgkin disease, renal cell carcinoma, and lung and bladder cancers, among others, with anti-PD-1 or anti-PD-L1 antibody monotherapy, has set the stage to revolutionize treatment approaches for patients with advanced cancer and opened the doors to develop new generation of immunomodulators that may be most effective when employed in combination treatments.

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