Monoclonal Antibodies for the Treatment of Cancer

Figure 50-1 Structure of IgG.

Currently, fully human antibodies are being produced using two common approaches: screening recombinant antibody libraries and engineering transgenic animals to express human immunoglobulin genes. A library of antibody fragments can be generated from human B cells, or by using cloning techniques, and can be used to construct phage or microbial display libraries. These libraries are subjected to multiple rounds of screening against the antigen of interest, and eventually high-affinity antibody fragments can be isolated. ¹⁰ These fragments can be used to generate full-length human antibodies.

More recently, transgenic mice expressing various human antibody gene sequences have been used to develop high-affinity, highly specific, fully human antibodies. ¹¹ Panitumumab, an antibody targeting the epidermal growth factor receptor (EGFR), is an example of an antibody generated in transgenic animals and is currently approved for the treatment of refractory metastatic colon cancer. ¹²

Mechanisms of Action of Anti-Cancer Antibodies

Signaling Perturbation

Solid tumors often upregulate expression of growth factor receptors or depend on these receptors for their proliferation or survival, with an enhanced capacity to invade and metastasize. Members of the epidermal growth factor (EGF) family of receptors, including EGFR1, HER2/neu (EGFR2), EGFR3, and EGFR4, are often overexpressed on solid tumors and thus serve as attractive targets for antibody therapy. ¹³ Most antibodies targeting EGFR family members work in part by inhibiting ligand binding and subsequent downstream oncogenic signaling (Figure 50-3 ). Notable exceptions are antibodies that target HER2/neu. Because this receptor has no known ligand, anti-HER2/neu antibodies such as trastuzumab and pertuzumab perturb signaling by preventing dimerization with other EGFR family members. ¹⁴

ADCC

Several tumor-targeted monoclonal antibodies rely on activation of host immune responses, such as ADCC, in order to exert their full therapeutic benefit. Both IgG1 and IgG3 subclasses can bind with high affinity to FcγRs and initiate ADCC-mediated killing of tumor cell targets. Antibody-coated tumor cells bind to FcγRs on innate effector cells, primarily NK cells, macrophages, and neutrophils, leading to targeted release of cytoplasmic granules containing perforin and granzyme, resulting in tumor cell apoptosis (see Figure 50-3). Preclinical studies demonstrated that FcγRs are critically important for the antitumor activities of trastuzumab and the anti-CD20 antibody rituximab. ¹⁵ Furthermore, expression of inhibitory FcγRIIB limits the efficacy of trastuzumab and rituximab in vivo, suggesting that the balance between activating and inhibitory FcγRs is a critical determinant of the antitumor activity of these antibodies. Positive correlations between FcγRIIA-131H and FcγRIIIA-158V polymorphisms and clinical outcomes have been demonstrated in patients with follicular lymphoma treated with rituximab^16,17and patients with refractory colorectal cancer treated with cetuximab. ¹⁸ Similarly, patients with metastatic breast cancer harboring FcγRIIA-131H and/or FcγRIIIA-158V polymorphisms show a greater response to trastuzumab-based treatment regimens compared to patients with the 131R and/or 158F polymorphisms. ¹⁹ The FcγRIIIA-158V receptor has a higher affinity for human IgG1 and a greater capacity to mediate ADCC compared to the 158F receptor.^20,21

Figure 50-2 Nomenclature of monoclonal antibodies.

CDC

In addition to binding FcγRs, IgG1 and IgG3 subclasses are potent activators of the classical complement cascade. The cascade is initiated with the binding of the C1q complex to the Fc domain of antibodies bound to tumor antigens. The C1q complex is composed of the hexameric C1q protein bound to the zymogens C1r and C1s. Binding of multiple C1q molecules activates its enzymatic activity, resulting in cleavage and activation of the C1s serine protease, which in turn activates downstream complement proteins culminating in the formation of the membrane attack complex (MAC). The MAC forms a pore in the lipid bilayer that compromises membrane integrity and causes target cell death. Certain complement protein fragments, such as C5a, induce a local inflammatory response by functioning as chemoattractants for neutrophils, monocytes, and lymphocytes. Rituximab is capable of mediating CDC against various B-cell malignancies in vitro in part because of its ability to translocate CD20 onto lipid raft microdomains, resulting in more efficient complement fixation. ²² The antitumor activity of rituximab is abolished in C1q knockout animals, supporting CDC as an important mechanism underlying the therapeutic efficacy of rituximab. ²³ Clinically, however, the importance of CDC in the context of rituximab therapy is still undetermined. Patients with follicular lymphoma harboring the C1qA276 allele, which results in reduced serum levels of C1q compared to the C1qG276 allele, had prolonged remission following rituximab monotherapy compared to patients with the G allele. ²⁴ Efforts to optimize CDC led to the development of ofatumumab, which also binds to CD20 but at a distinct epitope from rituximab. ²⁵ Ofatumumab has been demonstrated to induce more CDC in vitro compared to rituximab, even in the setting of low CD20 expression where rituximab is not efficacious. ²⁶ Unlike rituximab, ofatumumab does not directly induce apoptosis in B-cell lines, suggesting that ADCC and CDC are the primary mechanisms of action of ofatumumab. ²⁵ However, preliminary clinical data show that ofatumumab has limited efficacy in the setting of rituximab-resistant follicular lymphoma ²⁷ and comparable efficacy in the setting of non-Hodgkin lymphoma (NHL), although data from trials directly comparing ofatumumab to rituximab are lacking. ²⁵ In the setting of refractory chronic lymphocytic leukemia (CLL), patients demonstrated a 47% to 58% response rate to ofatumumab, leading to its approval by the U.S. Food and Drug Administration (FDA) in 2009 for the treatment of refractory CLL. ²⁸

Figure 50-3 Mechanisms of action of monoclonal antibodies. ADCC, Antibody-dependent cell-mediated cytotoxicity; MAC, membrane attack complex.

Induction of Adaptive Immunity

There is growing evidence to suggest that tumor-targeted antibodies are capable of inducing a therapeutically relevant, tumor-targeted adaptive immune response. ADCC, CDC, and tumor cell signaling perturbation result in target cell apoptosis, which creates tumor cell fragments that can be phagocytosed by antigen-presenting cells (APCs), such as macrophages and dendritic cells. Antibodies can also serve as opsonins by coating tumor cells and engaging Fc receptors on APCs to induce phagocytosis. These engulfed antigens can be processed through the endocytic pathway and presented on MHCII molecules to prime tumor-specific CD4⁺ T-cell responses. Alternatively, tumor antigens can be loaded on to MHCI molecules to prime CD8⁺ cytotoxic T cell (CTL) responses, in a process called cross presentation ²⁹ (see Figure 50-3). Activated CTLs are capable of directly killing tumor cells that express cognate antigen on MHCI and have powerful prognostic significance in a wide range of human cancers. ³⁰ In vitro, myeloma cells coated with an anti-syndecan1 antibody are capable of activating dendritic cells and inducing the cross presentation of cancer-testis antigen NY-Eso-1, generating NY-Eso-1–specific CTLs capable of lysing myeloma targets. ³¹ Similarly, antibody-coated melanoma and ovarian cancer cells were capable of inducing cross presentation of various tumor cell antigens, resulting in the generation of melanoma- and ovarian cancer–specific CTLs capable of lysing tumor cell targets. ³² Blockade of inhibitory FcγRIIB enhances dendritic cell activation and cross presentation, suggesting a potential therapeutic approach to boost antibody-initiated adaptive immunity. ³³ Recent work in the setting of colorectal cancer shows that cetuximab in combination with chemotherapy induces a potent CTL response and thus may contribute to the in vivo activity of cetuximab. ³⁴ Animal models demonstrate that the therapeutic efficacy of HER2/neu-directed antibodies is due in part to induction of an adaptive immune response, as depletion of T cells abrogates antitumor activity.^35,36Although the preclinical data implicating the induction of adaptive immunity as an important effector mechanism of tumor-targeted antibodies are provocative, more clinical evidence is needed to determine the clinical relevance of this mechanism of action.

Table 50-1

FDA-Approved Antibodies Used in Oncology

EGF, Epidermal growth factor; FDA, U.S. Food and Drug Administration; VEGF, vascular endothelial growth factor.

Antibodies Targeting Solid Tumors

As mentioned earlier, EGFR family members are overexpressed in a multitude of solid tumors, including colorectal, lung, head and neck, ovarian, and malignant gliomas. The most extensively studied anti-EGFR1 antibody (Table 50-1 ) is cetuximab, which binds to EGFR1 and blocks ligand binding, receptor dimerization and induces EGFR internalization and degradation, culminating in inhibition of receptor-mediated phosphorylation.^37,38Preclinical data suggest that ADCC and CDC may be important to the efficacy of cetuximab; however, more clinical data are needed to validate these claims. Cetuximab monotherapy has limited clinical activity but significantly improves outcomes when combined with chemotherapy. ³⁹ First-line therapy with cetuximab added to a regimen of FOLFIRI (leucovorin, fluorouracil, and irinotecan) chemotherapy significantly improves overall survival compared to FOLFIRI alone in the setting of metastatic colorectal cancer. ⁴⁰ It is important to note that the mutational status of KRAS, which is downstream of EGFR, is critical in predicting response to cetuximab: patients harboring activating KRAS mutations generally do not benefit from cetuximab therapy. ³⁹

Panitumumab is another antibody targeting EGFR, but unlike cetuximab, panitumumab possesses an IgG2 isotype and thus is unable to induce high levels of ADCC or CDC. Panitumumab significantly improves progression-free survival (PFS) and overall response rates in patients with refractory metastatic colon cancer compared to best supportive care (BSC). ¹² Like cetuximab, use of panitumumab is relegated to patients with wild-type KRAS. ⁴¹

Three new anti-EGFR antibodies are currently being evaluated in Phase I and Phase II clinical trials: necitumumab, zalutumumab, and nimotuzumab. Necitumumab is a fully human IgG1 that binds to a similar epitope of EGFR compared to cetuximab and has antitumor activity that is comparable or superior to cetuximab in preclinical models.^42,43No hypersensitivity reactions have been reported so far, whereas for cetuximab, approximately 3% of patients develop hypersensitivity reactions requiring treatment, mostly during the first infusion of antibody. Phase III studies are ongoing in the setting of non–small-cell lung cancer (NSCLC) (NCT00982111).

Zalutumumab is capable of inducing ADCC in addition to inhibiting receptor phosphorylation. Results from a Phase III trial showed modest improvement in PFS compared to BSC in patients with squamous carcinoma of the head and neck, although no improvement in overall survival was demonstrated. ⁴⁴

Nimotuzumab has been shown to sensitize NSCLC cell lines with high EGFR expression to radiation therapy while having no effect on EGFR low-expressing lines. ⁴⁵ Nimotuzumab has a highly favorable toxicity profile with virtually no grade 3 or 4 skin toxicities observed, often a dose-limiting toxicity seen with other anti-EGFR antibodies. Unlike cetuximab and panitumumab, nimotuzumab has a 10-fold lower affinity for EGFR and binds to EGFR in a manner that still allows the receptor to adopt an active confirmation and mediate ligand independent signaling. ⁴⁶ This basal level of signaling may be enough to sustain growth of normal cells and explain nimotuzumab’s favorable toxicity profile. An alternate hypothesis is that nimotuzumab requires bivalent binding for stable attachment to EGFR, and that normal cells do not have sufficient EGFR expression to mediate enough bivalent binding to be adversely affected. ⁴⁷ Based on favorable Phase II studies, nimotuzumab is approved for use in more than 20 countries in patients with head and neck cancer and/or gliomas. ⁴⁸

HER2/neu is overexpressed and gene amplified in 25% to 30% of breast cancer, and these properties confer a negative prognosis in patients. It is also overexpressed in some adenocarcinomas of the lung, ovary, and gastrointestinal tract. ⁴⁹ Trastuzumab monotherapy showed a 26% response rate in patients with untreated metastatic breast cancer ⁵⁰ and significantly improved PFS and OS when combined with chemotherapy, although some patients developed serious cardiotoxicity. ⁵¹ The exact mechanism of action of trastuzumab is still unclear but is thought to involve inhibition of receptor dimerization, inhibition of receptor shedding, and activation of immune effector mechanisms such as ADCC. ¹⁴

Trastuzumab emtansine, commonly referred to as trastuzumab-DM1 (T-DM1), is an immunoconjugate in which the cytotoxic agent DM1 is covalently attached to trastuzumab. DM1, a derivative of maytansine, is a highly potent antimitotic but has limited clinical utility due to a poor therapeutic window. Phase I and II studies have demonstrated that targeted delivery of DM1 with T-DM1 significantly improves toxicity and has antitumor activity in patients with metastatic breast cancer previously treated with HER2 targeted agents.^52,53In addition to the cytotoxic effects of DM1, T-DM1 maintains trastuzumab’s mechanisms of action, including signaling perturbation and induction of ADCC. ⁵⁴ Results from a pivotal Phase III trial demonstrate that T-DM1 improved PFS without significant cardiotoxicity in breast cancer patients whose disease had progressed following trastuzumab therapy. ⁵⁵

Pertuzumab is another approved HER2-directed antibody that binds to a distinct epitope from trastuzumab, but has complementary mechanisms of action including inhibition of receptor dimerization and induction of ADCC.^56,57Results from the CLEOPATRA trial demonstrated that adding pertuzumab to a combination of trastuzumab plus chemotherapy significantly increased PFS in patients with metastatic breast cancer compared to trastuzumab plus chemotherapy alone. ⁵⁸ Notably, no enhanced cardiotoxicity was seen in patients receiving pertuzumab and trastuzumab combination therapy. ⁵⁸ These results formed the basis for the FDA approval of pertuzumab in 2012.

Antibodies Targeting Hematological Malignancies

Rituximab is a chimeric antibody that targets CD20 and is used clinically for the treatment of a wide range of B-cell malignancies. A pivotal study leading to FDA approval showed a 48% response rate to rituximab in patients with relapsed low-grade or follicular lymphoma. ⁵⁹ Combination therapy with rituximab and cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy improved OS in elderly patients with large B-cell lymphoma. ⁶⁰ In the setting of CLL, rituximab seems to be more effective in patients who have not received prior chemotherapy. ⁶¹ This antibody continues to be an indispensable backbone for the therapy of patients with B-cell malignancies.

Alemtuzumab is approved for the treatment of fludarabine-refractory CLL and binds to CD52, which is highly expressed on various B- and T-cell malignancies. CD52 is a GPI-linked protein with unknown function that is normally expressed in parts of the male reproductive tract and on a wide range of leukocytes, including lymphocytes and monocytes. The exact mechanism of action of alemtuzumab is unclear but is thought to involve CDC, ADCC, and induction of apoptosis. In addition to chemoresistant CLL, alemtuzumab has activity in the setting of prolymphocytic leukemia (PLL) and low-grade NHL.^62–64Significant toxicities attributed to alemtuzumab therapy include myelosuppression and opportunistic infections, sometimes leading to death. These toxicities are presumably a result of CD52 expression on normal leukocytes.

CD30 is a member of the TNF superfamily and is expressed by the malignant Reed-Sternberg cells characteristic of Hodgkin’s lymphoma. Brentuximab vedotin is an antibody-drug conjugate composed of the anti-CD30 antibody brentuximab linked to the anti-microtubule agent monomethyl auristatin E (MMAE). On binding to CD30, the antibody-CD30 complex is internalized and trafficked to the lysosome, where MMAE is released by proteolysis. MMAE binds to tubulin, resulting in cell cycle arrest and apoptosis. Brentuximab vedotin is generally well tolerated, with neutropenia and peripheral neuropathy being the most common serious adverse events. Results of Phase II studies show a 75% overall response rate in patients with relapsed or refractory Hodgkin’s lymphoma after stem cell transplant and an 86% response rate in patients with anaplastic large-cell lymphoma.^65,66

CD74 is another interesting target for non–CD20-expressing malignancies such as multiple myeloma. CD74 was originally described as the invariant chain of MHC Class II and was shown to be critical for proper loading of peptide and trafficking of MHCII molecules to the cell surface. More recently, non-MHC Class II–related functions of CD74 have been described, including its role in cell survival and proliferation. The ligand for CD74 is macrophage inhibitory factor (MIF), and because CD74 is not capable of initiating signaling on its own, CD44 is required as a co-receptor. CD74 is expressed on 85% of NHL, CLL, and various multiple myeloma cell lines and has limited cell surface expression on normal cells. ⁶⁷ Milatuzumab is a chimeric antibody that targets CD74 and has shown efficacy in preclinical studies of multiple myeloma and NHL.^68,69Milatuzumab does not induce ADCC or CDC and is rapidly internalized on binding to CD74. This rapid internalization of antibody-CD74 complexes makes milatuzumab a prime candidate for the development of an antibody drug conjugate. Preclinical studies on a milatuzumab-doxorubicin conjugate are encouraging. ⁷⁰ Currently, milatuzumab is in multiple Phase I and II studies for multiple myeloma (NCT00421525), CLL (NCT00603668), and NHL (NCT00989586).

Antibodies Targeting Immune Cells

Ipilimumab, an antibody that targets cytotoxic T-lymphocyte–associated antigen (CTLA-4), is the first anti-cancer antibody to gain FDA approval that specifically targets nonmalignant immune cells. CTLA-4 is expressed on T-cells and is a negative regulator of T-cell activation (Figure 50-4 ). T-cell activation is initiated by engagement of the T-cell receptor (TCR) with peptide-loaded MHC; however, co-stimulatory signals are needed to ensure proper activation. The most important of these signals is the interaction of CD28 on T cells with the B7 family members CD80 and CD86 on APCs. CTLA-4 binds to CD80 and CD86 with higher affinity than CD28 and serves to inhibit activation by outcompeting CD28 and transducing inhibitory signals. CTLA-4 suppresses antitumor immunity by downregulating CD4⁺ T-cell responses and enhancing the immunosuppressive effect of T-regulatory cells (Treg). ⁷¹ Targeting CTLA-4 with a monoclonal antibody demonstrated remarkable antitumor activity in preclinical models, ⁷² and ipilimumab showed promise in early clinical studies that demonstrated antitumor activity and activation of antitumor immune responses. ⁷³ Notable adverse events included immune-related diarrhea and vitiligo, consistent with ipilimumab’s mechanism of action. A pivotal Phase III trial showed that ipilimumab therapy improved median survival by 3.6 months in patients with metastatic melanoma, making ipilimumab the only therapeutic demonstrated to improve survival in patients with metastatic melanoma. ⁷⁴

Another emerging target for antibody therapy is programmed cell death protein 1 (PD-1). PD-1 is upregulated on activated T cells and serves to dampen effector T-cell responses in peripheral tissues, including the tumor microenvironment. PD-1 is also expressed on Treg cells and has been demonstrated to promote the development and activity of peripherally induced Treg cells. ⁷⁵ PD-1 ligands, most notably PD-L1, are expressed on tumor cells and myeloid cells in the tumor microenvironment, providing further rationale for therapeutic targeting of this pathway. ⁷⁶ Data from two large Phase I trials of the anti-PD-1 antibody BMS-936558, a fully human IgG4, demonstrated safety and antitumor efficacy in the setting of metastatic melanoma, renal cancer, and NSCLC.^77,78Remarkably, monotherapy with BMS-936558 resulted in durable antitumor responses, even in the setting of NSCLC, which in contrast to melanoma and renal cancer had not been considered to be an immunotherapy-responsive tumor. In addition to multiple Phase I and II trials, BMS-936558 is also being evaluated in combination with ipilimumab for the treatment of metastatic melanoma (NCT01024231).

Whereas ipilimumab and BMS-936558 block signaling through inhibitory receptors, another approach to re-activate anti-tumor immunity is to activate stimulatory receptors. CD40 is a stimulatory receptor expressed by many different cell types including B cells, dendritic cells, monocytes, endothelial cells, and fibroblasts. The ligand for CD40, CD40L, is primarily expressed on activated T cells and platelets. Engagement of CD40 leads to B-cell proliferation and maturation of dendritic cells, resulting in enhancement of T-cell activation. Interestingly, CD40 is expressed on a wide range of malignancies, including nearly all B-cell malignancies and many solid tumors, and engagement of CD40 on some malignant cells leads to apoptosis. ⁷⁹ Three antibodies targeting CD40 are currently being evaluated for safety and efficacy. Dacetuzumab (SGN-40) is a humanized IgG1 with weak agonist activity that is capable of inducing ADCC in vitro and has antitumor activity against B-cell lymphoma xenografts in vivo. ⁸⁰ A Phase I study of dacetuzumab showed encouraging antitumor activity in NHL patients, with the most common adverse events being related to cytokine release syndrome, including fever, headache, and fatigue. ⁸¹

Figure 50-4 Potential targets to enhance T-cell activation. APC, Antigen-presenting cell; IDO, indoleamine 2,3-dioxygenase; MHC, major histocompatibility complex; TCR, T-cell receptor.

Lucatumumab (HCD122) is an IgG1 that is a pure antagonist of CD40 capable of inducing ADCC and has shown activity against B-cell CLL in preclinical models. ⁸² A recent Phase I study showed that lucatumumab is reasonably well tolerated; however, it shows minimal activity as a single agent in the setting of relapsed CLL. ⁸³

CP-870,813 is a fully human IgG2 that functions as a CD40 agonist and has antitumor and immunostimulatory activity in patients with melanoma. ⁸⁴ Remarkably, CP-870,813 in combination with gemcitabine has also demonstrated activity in a small cohort of patients with pancreatic cancer. ⁸⁵ In the same study, the authors used a mouse model of spontaneous pancreatic cancer to identify a potential mechanism of action of CP-870,813. Unexpectedly, the antitumor activity of CP-870,813 was independent of T cells and dependent on its capacity to alter the phenotype of tumor-associated macrophages to a more tumoricidal phenotype. ⁸⁵

Antibodies Targeting Angiogenesis

The tumor stroma is enriched with pro-angiogenic factors that facilitate recruitment and remodeling of blood vessels essential for tumor growth. A critical driver of vascular endothelial cell migration and proliferation is the vascular endothelial growth factor (VEGF) family, comprising six members (VEGF-A through -E and placental growth factor [PlGF]). VEGF has three known receptors, VEGFR1 through -3, with VEGFR-1 and -2 being most relevant to tumor biology. VEGF levels are increased in a wide range of malignancies and are often associated with a poor prognosis. ⁸⁶ Bevacizumab is an antibody targeting VEGF-A and has been shown to improve survival when combined with chemotherapy in patients with metastatic colon cancer. ⁸⁷ The most frequent adverse event was hypertension, with no increased incidence of hemorrhage, frequently observed in other trials of bevacizumab. ⁸⁷ The addition of bevacizumab to a regimen of carboplatin and paclitaxel improved response rates and survival in patients with NSCLC, although a significant increase in bleeding, specifically lethal pulmonary hemorrhage, was observed. ⁸⁸ Single-agent bevacizumab received accelerated approval for the treatment of glioblastoma multiforme (GBM) based on demonstrated durable response rates and manageable adverse events when administered as monotherapy.^89,90Larger clinical trials are needed to evaluate bevacizumab’s impact on the clinical course of GBM. The role of bevacizumab in the setting of HER2/neu–negative metastatic breast cancer is a subject of debate. In 2008, bevacizumab received accelerated approval for the treatment of HER2/neu–negative metastatic breast cancer based largely on results from the E2100 trial, which demonstrated that combination therapy with bevacizumab and paclitaxel improved PFS compared to paclitaxel alone, with no significant difference in OS between the two treatment arms. ⁹¹ However, subsequent trials showed that the addition of bevacizumab to chemotherapy resulted in only modest improvements in PFS, with no impact on overall survival.^92–94Based on the results of these trials, the FDA revoked approval of bevacizumab for the treatment of metastatic breast cancer in 2011, citing an unfavorable risk/benefit profile. Debate continues on the utility of using PFS or pathological complete response (pCR) versus overall survival to evaluate the clinical efficacy of new drugs and treatment regimens.

VEGFR-2 is upregulated in the tumor vasculature. Signaling through VEGFR-2, as opposed to VEGF-1, is sufficient to mediate the pro-angiogenic functions of VEGF. ⁹⁵ The VEGF/VEGFR-2 axis is also detrimental to the antitumor immune response, whereas signaling through VEGF-1 may promote lymphoid development. ⁹⁶ Ramucirumab is a fully human IgG1 that specifically targets VEGFR-2 and inhibits VEGF binding. A Phase I study demonstrated antitumor efficacy. However, significant toxicities were observed including hypertension, abdominal pain, vomiting, and deep-vein thromboses. ⁹⁷ Several Phase II and III studies in the setting of renal, breast, colon, and NSCLC are ongoing. ⁹⁵

Recent work suggests that cancer-associated fibroblasts (CAFs) are an important source of pro-angiogenic stimuli, such as VEGF and fibroblast growth factor (FGF). CAFs from tumors resistant to anti-VEGF therapy upregulate expression of other pro-angiogenic factors and may contribute to resistance of VEGF targeted therapy. ⁹⁸ A potential therapeutic target for CAFs is fibroblast activated protein-alpha (FAP), which is highly expressed on CAFs compared to normal fibroblasts. ⁹⁹ Depletion of FAP⁺ stromal cells results in inhibition of tumor growth and activation of antitumor immune responses. ¹⁰⁰ However, clinical efforts to target FAP using the anti-FAP antibody sibrotuzumab have been disappointing. ¹⁰¹ One possible explanation for this lack of efficacy is that sibrotuzumab does not inhibit the enzymatic activity of FAP. ¹⁰² Conjugation of sibrituzumab to the antimitotic DM1 has shown promise in a preclinical study. However, further studies are needed to determine the clinical utility of this antibody-drug conjugate. ¹⁰³

Antibody Engineering

Advancements in protein engineering have allowed for the modification of antibody structure in an attempt to improve targeting and enhance antitumor activity. Bispecific antibodies represent an important class of engineered antibodies that are capable of recognizing two distinct antigens. The most common bispecific antibodies target a tumor antigen and CD3, which is a component of the T-cell receptor. Bispecific T-cell engagers (BiTEs) are a class of bispecific antibodies that link the variable domains of an antitumor antibody with the variable region of an anti-CD3 antibody. BiTEs induce robust polyclonal T-cell activation, resulting in targeted release of perforin and granzyme and tumor cell apoptosis. Importantly, BiTES are capable of stimulating T-cells and inducing tumor cell lysis in the absence of co-stimulatory signals and MHC expression. Blinatumomab is a BiTE that binds to CD19 and CD3 that has clinical activity in patients with minimal residual disease (MRD) positive, B-cell acute lymphoblastic leukemia (ALL). ¹⁰⁴ Of the 20 patients treated, 16 became MRD negative, and 12 of the 16 responders had never turned MRD negative with any previous treatment. ¹⁰⁴

Bispecific antibodies that have an intact Fc domain are termed trifunctional antibodies, because of their capacity to bind and activate FcγR⁺ immune effectors. Trifunctional antibodies targeting a tumor antigen and CD3 are capable of inducing T-cell–mediated lysis in addition to ADCC and phagocytosis. Catumaxomab is an example of a trifunctional antibody that has dual specificity for CD3 and epithelial cell adhesion molecule (EpCAM), which is ubiquitously expressed on tumors of epithelial origin. ¹⁰⁵ Catumaxomab contains one light chain and one heavy chain from an anti-EpCAM mouse IgG2, and one light chain and one heavy chain from an anti-CD3 rat IgG2b. The Fc domain generated from mouse and rat constant regions binds preferentially to activating FcγRs, with low binding to FcγRIIB, resulting in potent ADCC and phagocytosis of EpCAM⁺ tumor cells. ¹⁰⁵ Interestingly, preclinical studies demonstrated that the related antibody, BiLU (anti-human EpCAM × anti-mouse CD3), was capable of eliciting a protective adaptive immune response against syngeneic tumors. ¹⁰⁶ A small clinical study showed that catumaxomab was capable of eliciting tumor-specific T cells, suggesting that induction of adaptive immunity may contribute to catumaxomab’s mechanisms of action. ¹⁰⁷ Catumaxomab is approved in the European Union for the treatment of malignant ascites and been shown to improve quality of life and increase time to next paracentesis. ¹⁰⁸ The main adverse events associated with catumaxomab are related to severe cytokine release syndrome, necessitating careful dosing and patient monitoring.

Modifications to the Fc domain are capable of enhancing effector functions and improving the pharmacokinetic profile of monoclonal antibodies. Fc mutational analyses have identified optimal amino acid sequences that result in greater binding to activating FcγRs and C1q to enhance ADCC and CDC. An anti-CD19 antibody engineered to have greater FcγR binding demonstrated more potent ADCC, phagocytosis, and antitumor activity compared to its parent antibody. ¹⁰⁹ Improving interactions with neonatal FcRs can extend serum-half life and potentially reduce the cost of treatment. ¹¹⁰ However, modulation of oligosaccharide residues within the CH2 domain is the modification that has been most widely used to date.

Human IgG molecules contain two N-linked, complex-type oligosaccharides within the CH2 domain. These oligosaccharides contain a fucose residue, and removal of this residue dramatically enhances the capacity of antibodies to mediate ADCC, even at low antigen densities. ¹¹¹ Obinutuzumab is a defucosylated anti-CD20 antibody that displays greater ADCC in vitro and anti-lymphoma activity in vivo compared to rituximab. ¹¹² Two Phase I studies of obinutuzumab demonstrated activity in the setting of relapsed NHL with manageable adverse events, mostly associated with infusion-related toxicity.^113,114Interestingly, both studies showed activity (13% to 22% response rate) in a subset of patients refractory to rituximab.^113,114

Summary and Future Directions

Monoclonal antibodies have revolutionized the treatment of cancer and will continue to serve as mainstays of cancer therapy for the foreseeable future. Antibodies offer the unique capability to specifically engage a tumor cell and stimulate a multifaceted program of cell death involving perturbation of homeostatic signaling and activation of the host immune system, without overt toxicity. Advances in antibody therapy are contingent on the identification and validation of new tumor antigens, manipulation of the tumor microenvironment, and optimization of antibody structure to improve effector functions and cytotoxic drug delivery.

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