Trial Watch

Abstract

Since the advent of hybridoma technology, dating back to 1975, monoclonal antibodies have become an irreplaceable diagnostic and therapeutic tool for a wide array of human diseases. During the last 15 years, several monoclonal antibodies (mAbs) have been approved by FDA for cancer therapy. These mAbs are designed to (1) activate the immune system against tumor cells, (2) inhibit cancer cell-intrinsic signaling pathways, (3) bring toxins in the close proximity of cancer cells, or (4) interfere with the tumor-stroma interaction. More recently, major efforts have been made for the development of immunostimulatory mAbs that either enhance cancer-directed immune responses or limit tumor- (or therapy-) driven immunosuppression. Some of these antibodies, which are thought to facilitate tumor eradication by initiating or sustaining a tumor-specific immune response, have already entered clinical trials. In this Trial Watch, we will review and discuss the clinical progress of the most important mAbs that are have entered clinical trials after January 2008.

Introduction

In 1975, Georges Köhler and César Milstein published in Nature the procedure to generate hybridomas, which are hybrid cell lines resulting from the fusion between a specific antibody-producing B cell (which normally cannot be maintained in culture) and a myeloma cell (which, as a cancer cell, is immortalized and hence can grow in culture, but does not synthesize specific antibody chains).¹ In doing so, Köhler and Milstein provided a method to generate high amounts of antibodies with the same specificity, that is monoclonal, thereby de facto revolutionizing an incredible number of research procedures and clinical applications.² In 1984, thanks to this breakthrough discovery, Köhler and Milstein shared the Nobel Prize for Medicine or Physiology with Niels Jerne, who made other contributions to immunology.³

Antibodies are composed of three functional units, two antigen-binding fragments (Fabs) and one constant fragment (Fc). Each Fab is made up by the association of one heavy and one light immunoglobulin chain, and at the level of the Fab three hypervariable complementarity-determining regions (CDRs) form the antigen-binding sites and confer antigen specificity. The Fc results from the association of two heavy chains, and links antibodies to immune effector functions.^4-7 Based on the sequence of their heavy chains, antibodies can be subdivided into five classes (i.e., IgM, IgG, IgD, IgA and IgE), which are characterized by distinct functional profiles.⁸ The profound impact that monoclonal antibodies (mAbs) had on medical research, diagnostics, and clinical applications derives, for the most part, from (1) their ability to selectively bind two antigens of the same type with high affinity, and (2) from their elevated stability, in vitro and in vivo. Compared with their polyclonal counterparts, which are obtained from the serum of animals upon immunization, mAbs are advantageous in that they exhibit a much higher specificity and are less prone to contamination by pathogens.²

Thus, the monoclonal antibody technology has generated or considerably improved uncountable diagnostic applications, including (but not limited to) epitope-specific immunoblotting, immunofluorescence and immunohistochemistry. Moreover, mAbs have already been successfully employed in vivo (in animal models of disease or patients) to: (1) neutralize circulating factors, (2) activate immune effector mechanisms against disease-sustaining cell populations, (3) antagonize disease-specific molecules or molecular cascades, (4) crosslink plasma membrane receptors and hence activate therapeutic (diseased cell-autonomous or not) signaling pathways, (5) bring radionuclides, prodrugs, toxins or drug-filled liposomes in the proximity of target cells.⁴

Initially, mAbs were entirely murine, implying that they were relatively immunogenic and hence that their usefulness in humans was limited by the rapid elicitation of a neutralizing anti-mAb immune response. This issue was progressively solved with the ever increasing substitution of murine sequences with human ones, which eventually led to the development of fully humanized mAbs.⁹ Of note, even fully human mAbs do not entirely evade the control by the host immune system, as they are normally capable of eliciting an anti-idiotype response.¹⁰ Additional strategies that have been implemented to improve the pharmacological and pharmacodynamic properties of mAbs include (1) reduced or absent fucosylation and/or site specific mutations of the Fc, to enhance antibody-dependent cellular cytotoxicity (ADCC, see below),¹¹ complement-dependent cytotoxicity (CDC, see below),¹² and/or antibody-dependent cellular phagocytosis (ADCP, see below),¹³ or to increase serum half-life (by favoring the binding to the neonatal Fc receptor, which prevents IgG degradation),¹⁴ (2) addition of sialylated glycans, to increase anti-inflammatory properties,¹¹ and (3) isotype chimerism, to boost ADCP¹⁵ and CDC.¹⁶

Irrespective of these considerations, antibodies of all types (murine, chimeric and human) have been approved by FDA and by other international regulatory agencies for the treatment of several pathologies, including (but not limited to) autoimmune diseases and cancer.^2,4 Just to mention a few examples: muromonab—the first mAb ever approved for use in humans (for the therapy of transplant rejection)—is murine,¹⁷ infliximab—an anti-TNFα mAb that is currently employed against rheumatoid arthritis and Crohn's disease - is chimeric,^18,19 and ofatumumab - an anti-CD20 mAb that is used for the therapy of chronic lymphocytic leukemia - is entirely human.²⁰ Interestingly, the suffix of the international non-proprietary names for mAbs denotes the antibody format, as follows: -omab, mouse IgG2; -ximab, chimeric IgG1; -zumab, humanized IgG1; -umab, human antibody from phage display or transgenic mice technology; -axomab, trifunctional (bispecific) mouse-rat hybrid mAb; -cept, Fc fusion protein; -stim, Fc fusion peptide (Table 1). Most mAbs that are currently approved for use in humans (irrespective of their indication) or under clinical evaluation belong to the IgG isotype.^2,4

Table 1. International suffixes for mAbs and mAb-related therapeutics.

Suffix	Type of Molecule
-axomab	Bispecific mouse-rate hybrid mAb
-cept	FC fusion protein
-omab	Mouse IgG2
-stim	Fc fusion peptide
-umab	Human antibody from phage display or transgenic mice technology
-ximab	Chimeric IgG1
-zumab	Humanized IgG1

Abbreviations: Fc, constant fragment.

There are at least six classes of mAbs that are relevant for cancer therapy: (1) mAbs that directly inhibit tumor cell-autonomous pro-survival cascades (e.g., cetuximab and panitumumab, which inhibit the epidermal growth factor receptor, EGFR, and are currently approved for the treatment of colorectal cancer);²¹ (2) mAbs that interfere with the tumor-stroma interaction, thereby indirectly inhibiting tumor growth (e.g., bevacizumab, which blocks the vascular endothelial growth factor, VEGF, and is currently employed in the for the therapy of colorectal, breast, renal and lung cancer);^22-24 (3) mAbs that binds to antigens expressed on the surface of tumor cells and function by selectively engaging immune effector mechanisms such as ADCC,^4,5 ADCP,²⁵ and CDC^6,7 (e.g., rituximab, a naked anti-CD20 in use for the therapy of lymphoma);^26,27 (4) trifunctional (bispecific) mAbs, which can bind two different antigens while remaining capable of engaging immune effector functions (e.g., catumaxomab, an anti-CD3, anti-EpCAM chimeric mAb currently approved for the treatment of malignant ascites in patient with EpCAM⁺ neoplasms);²⁸ (5) immunoconjugates (e.g., ⁹⁰Y-ibritumomab tiuxetan and ¹³¹I-tositumomab, radionuclide-coupled anti-CD20 mAbs that are used for the therapy of lymphoma);^29,30 and (6) immunostimulatory mAbs, i.e., mAbs that facilitate the development of a tumor-specific immune response by targeting the cancer cell/immune system crosstalk and the signaling pathways that this crosstalk elicits. An interesting sub-group encompassing these latter two categories is represented by immunoconjugates between putative tumor antigens and mAbs that target receptors expressed on the surface of dendritic cells (DCs), including CLEC9A, DC-SIGN, DEC205.^31-33 By delivering tumor proteins (or protein epitopes) to DCs, these molecules stimulate antigen processing and cross-presentation, thus driving the establishment of a cancer-specific immune response involving CD4⁺ and CD8⁺ cells that—at least in preclinical models—often results in vaccination.³⁴ This approach has also provided promising results in the field of infectiology.^35,36

Table 2 summarizes the mAbs that are currently approved for use in cancer therapy. In this Trial Watch, we will discuss the clinical progress of mAbs that entered clinical trials for this indication after January 2008.

Table 2. Monoclonal antibodies currently approved for cancer therapy*.

mAb	Target	Approved	Type	Indication(s)
Alemtuzumab	CD52	2001	Hzed IgG1	Chronic lymphocytic leukemia
Bevacizumab	VEGF	2004	Hzed IgG1	Colorectal, breast, renal and lung cancer
Catumaxomab	CD3 and EpCAM	2009	M-R hybrid	Malignant ascites in patients with EpCAM-positive cancer
Cetuximab	EGFR	2004	C IgG1	Colorectal cancer
Gemtuzumab	CD33	2000	Hzed IgG4	Acute myeloid leukemia (coupled with calicheamicin)
Ibritumomab tiuxetan	CD20	2002	M IgG1	Non-Hodgkin lymphoma (coupled with 90Y or 111In)
Ipilimumab	CTLA-4	2011	H IgG1	Melanoma
Panitumumab	EGFR	2006	H IgG2	Colorectal cancer
Ofatumumab	CD20	2009	H IgG1	Chronic lymphocytic leukemia
Rituximab	CD20	1997	C IgG1	Non-Hodgkin lymphoma
Tositumomab	CD20	2003	H IgG1	Non-Hodgkin lymphoma (naked or coupled with 131I)
Trastuzumab	ERBB2	1998	Hzed IgG1	Breast cancer

Abbreviations: C, chimeric; CTLA-4, cytotoxic T lymphocyte antigen 4; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; H, human; Hzed, humanized; M, murine; R, rat; VEGF, vascular endothelial growth factor. * by FDA or European Medicines Agency (EMA) at the day of submission.

Monoclonal Antibodies Under Advanced (Phase III-IV) Clinical Evaluation

More than 75% of the ongoing clinical trials that test mAbs as anticancer agents aim at assessing the safety and efficacy of mAbs that have already been commercialized (Table 2), either as off-label medications (i.e., for diseases other than those for which they were initially approved) or combined with various chemo- and radiotherapeutic regimens (source www.clinicaltrials.gov). In spite of the fact that these mAbs are approved for cancer therapy in humans, only a relatively low percentage of these studies (around 12%) is in the late phases (III-IV) of evaluation. Moreover, bevacizumab-based (31) and trastuzumab-based (18) trials account alone for more than half of phase III and IV studies involving FDA-approved mAbs.

As few as 13 phase III-IV clinical studies based on experimental (not yet FDA approved) mAbs were being performed on August 2011 (when this manuscript has been prepared). These trials involve a total of 8 distinct agents and 7 cancer-specific molecular targets (Table 3).

Table 3. Monoclonal antibodies currently under evaluation for cancer therapy in phase III-IV trials*.

mAb	Target	Type	Identifier	Phase	Indication(s)
Brentuximab vedotin**	CD30	MMAE-conjugated C IgG1	NCT01100502	III	Hodgkin lymphoma
Ch14.18	GD2	C IgG1	NCT01041638	III	Neuroblastoma
Denosumab***	RANKL	H IgG2	NCT01077154	III	High risk early breast cancer receiving neoadjuvant or adjuvant therapy
Ganitumab	IGF1R	H IgG1	NCT01231347	III	Metastatic adenocarcinoma of the pancreas
Necitumumab	EGFR	H IgG1	NCT00981058	III	NSCLC
			NCT00982111	III	NSCLC
Nimotuzumab	EGFR	Hzed IgG1	NCT01074021	III	Nasopharyngeal cancer
			NCT00957086	III	HNC
			NCT01249352	III	Locally advanced esophageal cancer
			NCT01402180	III	Esophageal squamous cell carcinoma
Ramucirumab	VEGFR2	H IgG1	NCT00917384	III	Metastatic gastric or gastresophageal junction adenocarcinoma.
			NCT01140347	III	Hepatocellular carcinoma after 1st line therapy with sorafenib
Siltuximab	IL-6	C IgG1	NCT01266811	III	Relapsed or refractory multiple myeloma

Abbreviations: C, chimeric; EGFR, epidermal growth factor receptor; H, human; HNC, head and neck cancer; Hzed, humanized; IGF1R, insulin-like growth factor 1 receptor; IL, interleukin; MMAE, monometil auristatin E; NSCLC, non-small cell lung cancer; RANKL, RANK ligand; VEGFR2, vascular endothelial growth factor receptor 2. *started after January, 1st 2008 and not completed or terminated at the day of submission; **currently reviewed by FDA and EMA; ***approved by both FDA and EMA for the therapy of bone loss during osteoporosis.

Brentuximab vedotin

Brentuximab vedotin (SGN-35) is a chimeric IgG1 mAb coupled to 3–5 units of the antimitotic compound monometil auristatin E (MMAE).³⁷ The mAb moiety of brentuximab targets CD30 (TNFRSF8), a cell membrane protein of the tumor necrosis factor receptor (TNFR) family that is overexpressed by anaplastic large-cell and Hodgkin lymphoma.³⁸ Contrarily to previous CD30-targeting approaches, brentuximab vedotin was found to induce durable objective responses and resulted in tumor regression in most patients with relapsed or refractory CD30-positive lymphomas.³⁹ Today, brentuximab vedotin is being investigated in patients at high risk of residual Hodgkin lymphoma following stem cell transplant (Table 3, NCT01100502).

Ch14.18

Ch14.18 is a chimeric IgG1 mAb that targets disialoganglioside GD2, which often is present in high amounts at the surface of tumor cells of neuroendocrine origin.⁴⁰ Characterized by an improved capacity to trigger ADCC (in particular by neutrophils)⁴¹ compared with other GD2-targeting mAbs,⁴⁰ Ch14.18 entered phase I clinical trials for the treatment of melanoma and neuroblastoma in the early 1990s,^42,43 with rather unsatisfactory results. Driven by preclinical data suggesting that granulocyte macrophage-colony stimulating factor (GM-CSF) exacerbates neuroblastoma cell killing by Ch14.18,⁴¹ several clinical trials were initiated later on to test Ch14.18 in combination with GM-CSF alone or GM-CSF + interleukin-2 (IL-2)^44-46 Promising results have only been obtained when Ch14.18 was associated with GM-CSF, IL-1 and isotretinoin (a retinoid), leading to significantly improved outcome as compared with standard therapy in patients with high-risk neuroblastoma.⁴⁶ As an alternative strategy, which never entered clinical tests, a Ch14.18-GM-CSF fusion protein was created, displaying comparable activity against neuroblastoma, in vitro, than Ch14.18 plus GM-CSF, and a good profile of ADCC and CDC induction.⁴⁷ Now, Ch14.18 is being tested in combination with GM-CSF, IL-2, and isotretinoin after autologous stem cell transplant in neuroblastoma patients (Table 3, NCT01041638).

Denosumab

Denosumab (AMG 162) is a fully human IgG2 mAb currently approved by FDA for use in postmenopausal women with risk of osteoporosis. Denosumab specifically targets the receptor activator of NFκB ligand (RANKL), thus inhibiting the development and activity of osteoclasts, decreasing bone resorption and increasing bone density.⁴⁸ As bone destruction is also an oncological problem (bones are a common site for metastases), it has been hypothesized that the anti-resorptive properties of denosumab might also be beneficial to cancer patients.⁴⁹ Results from recent clinical trials suggest that denosumab can help prevent bone metastases in prostate cancer better than zoledronate, a bisphosphonate that had previously been shown to be superior to denosumab in the therapy of multiple myeloma.⁵⁰ Today, denosumab is under evaluation for the prevention of bone metastasis in women with high risk early breast cancer receiving neoadjuvant or adjuvant therapy (Table 3, NCT01077154).

Ganitumab

Also known as AMG 479, ganitumab is a fully human IgG1 mAb directed against the insulin-like growth factor 1 receptor (IGF1R), a tyrosine kinase receptor that plays a critical role in oncogenic transformation.⁵¹ The IGF1R is highly overexpressed in most, if not all, malignant tissues, where it functions as an anti-apoptotic signal transducer.⁵² In vitro, ganitumab has been shown to virtually abolish ligand-induced IGF1R activation, thus limiting the viability of pancreatic cancer cells, in particular in conditions of serum deprivation. Ganitumab also inhibited tumor growth in vivo, in immunodeficient mice xenografted with human pancreatic cancer cells, suggesting that the antineoplastic effect of this mAb mostly rely on the inhibition of cell-autonomous signaling pathways.⁵¹ According to phase I clinical tests, there seem to be no major safety concerns for the use of ganitumab,⁵³ which is now being tested in several phase I-II studies as well in a phase III trial (coupled with gemcitabine) for the therapy of metastatic adenocarcinomas of the pancreas (Table 3, NCT01231347).

Necitumumab

Necitumumab (IMC-11F8) is a fully human IgG1 mAb targeting the epidermal growth factor receptor (EGFR). A number of distinct tumors including colorectal and non small-cell lung cancer (NSCLC) exhibit activating mutations of the EGFR and several agents that inhibit the EGFR are approved as anticancer agents in the US and Europe. These include small cell-permeant chemicals that inhibit the tyrosine kinase activity of the receptor, like erlotinib and gefinitib,^54-56 as well as the anti-EGFR mAbs cetuximab and panitumumab (Table 2), which block ligand-induced EGFR activation.^57,58 Necitumumab shares this latter mechanism of action with cetuximab and panitumumab, and it has been shown to be well tolerated by patients and to achieve biologically relevant concentrations throughout the dosing period.⁵⁹ Now, necitumumab is being evaluated in two distinct phase III trials for its efficiency against NSCLC, in combination with cisplatin (a DNA-damaging agent) and either gemcitabine or pemetrexed (an antimetabolite) (Table 3, NCT00981058 and NCT00982111, respectively).

Nimotuzumab

Similar to necitumumab, cetuximab and panitumumab, nimotuzumab is an anti-EGFR mAb. Developed in the early 2000s, nimotuzumab (a humanized IgG1 mAb also known as h-R3) soon demonstrated consistent in vitro and in vivo anticancer properties,⁶⁰ boosting an intense wave of clinical test, often relying on the combination of nimotuzumab with radiotherapy.^61-64 In parallel, nimotuzumab was conjugated to radionuclides including ¹⁸⁸Os, ⁹⁹mTc, ¹⁸⁸Re and ¹⁷⁷Lu, and some of the resulting immunoradioconjugates entered clinical tests as diagnostic or therapeutic tools for tumors of epidermal origin.^65-68 Now, approximately 20 phase I-II clinical studies are investigating the efficacy of nimotuzumab for indications as diverse as glioma, head and neck, colorectal and lung cancer. Moreover, nimotuzumab is being evaluated in phase III trials (1) for the therapy of nasopharyngeal and head and neck cancer (HNC), in combination with not better specified chemotherapy and radiation (Table 2, NCT01074021, NCT00957086); (2) for the treatment of locally advanced esophageal cancer, in combination with 5-fluorouracil (a pyrimidine analog) and radiotherapy (Table 2, NCT01249352); and (3) in esophageal cancer patients who suffer with recurrence in regional lymph nodes after esophagectomy, combined with specified chemotherapy and radiation (Table 2, NCT01402180).

Ramucirumab

Ramucirumab (IMC-1121B) is a fully human IgG1 that specifically binds the VEGF receptor 2 (VEGFR2), thus antagonizing endogenous VEGF signaling.⁶⁹ Similar to bevacizumab, which directly binds to and neutralizes circulating VEGF,⁷⁰ ramucirumab was developed to inhibit perhaps the most important tumor-stroma interaction of all, angiogenesis.⁷¹ Ramucirumab was well tolerated in phase I clinical trials in patients with advanced cancers, and its most frequent mechanism-related dose-limiting toxicities were found to be hypertension and deep venous thrombosis.^69,72 These studies also revealed promising rates of partial responses and stable disease, soliciting further clinical tests. Now, ramucirumab is under evaluation in multiple phase I-II trials as well as in two distinct phase II studies, one as a standalone intervention in patients with adenocarcinomas of the gastresophageal junction (Table 2, NCT00917384), and one as 2nd line treatment in patients with hepatocellular carcinoma after 1st line therapy with the chemical VEGFR2 inhibitor sorafenib (Table 2, NCT01140347).

Siltuximab

Siltuximab (CNTO-328) is a chimeric IgG1 mAb that binds to interleukin-6, a pleiotropic cytokine that is known to influence proliferation, apoptosis, and angiogenesis in cancer.⁷³ However, contrarily to VEGF-targeting antibodies, siltuximab not only inhibits tumor growth by blocking the tumor-stroma interaction but also by interfering with tumor cell-autonomous signaling pathways, and notably with an MCL-1-mediated IL-6-driven antiapoptotic autocrine loop.^74,75 Results from the first phase I clinical trial based on siltuximab were published as early as in 1998,⁷³ yet more advanced studies lagged for another decade, with results from phase II studies being reported in the early 2010s,^76-79 along with an ever more refined pre-clinical characterization of this mAb.^80-84 Siltuximab is currently being tested in a phase III study in combination with the proteasomal inhibitor bortezomib and dexamethasone (a synthetic glucocorticoid) for the treatment of subjects with relapsed or refractory multiple myeloma (Table 2, NCT01266811).

Monoclonal Antibodies Under Early (phase I-II) Clinical Evaluation

There are around 200 open phase I-II clinical trials that test investigational (non FDA-approved) mAbs for oncological indications. Approximately one third of these studies is based on mAbs that are also being evaluated in phase III-IV trials (Table 3). As for the rest, there is a great heterogeneity of approaches, with some prominent trends that we will discuss here (source www.clinicaltrials.gov).

Cixutumumab (fully human IgG1) and dalotuzumab (humanized IgG1), two anti-IGF1R mAbs,^85,86 are now being tested in 35 phase I-II clinical studies. By taking into account ganitumab (see above), the number of early clinical studies involving IGFR1-targeted therapies raises to more than 50, indicating that the expectations from this therapeutic approach are very high.

Bavituximab is a chimeric IgG1 specific for phosphatidylserine (PS), an anionic phospholipid that normally resides in the inner leaflet of the plasma membrane (from where it cannot be bound by mAbs).⁸⁷ PS get exposed on the cell surface in some instances of cell death,^88,89 cell activation and malignant transformation, and has been suggested to constitute a specific marker of tumor vasculature.^90,91 In preclinical studies, bavituximab retarded the growth of different human tumors xenotransplanted in immunodeficient mice while eliciting no evident toxicity in the host,⁸⁷ which supported its evaluation in the clinical setting. Now, the safety and efficiency of bavituximab, alone or in combination with conventional chemo-and radiotherapeutic regimens, are being investigated in 7 phase I-II trials, in a wide array of solid tumors (Table 4).

Table 4. Examples of monoclonal antibodies currently under evaluation for cancer therapy in phase I-II trials.

mAb	Target	Type	Trials*	Phase	Main indication(s)
AHN-12	CD45	90Y-conjugated Hzed IgG1	1	I	Advanced leukemia.
Bavituximab	PS	C IgG1	7	I-II	Breast cancer; liver cancer; NSCLC; pancreatic cancer; prostate cancer.
BC8	CD45	90Y- or 131I-conjugated M IgG1	4	I-II	Acute lymphoblastic leukemia; AML; Hodgkin and non-Hodgkin lymphoma; myelodysplastic syndrome.
Blinatumomab	CD3 and CD19	na	2	II	B-precursor acute lymphoblastic leukemia.
Claudiximab	GC128	C IgG1	1	II	Advanced gastresophageal cancer
Conatumumab	TRAILR2	H IgG1	2	I-II	Colorectal cancer; lymphoma; NSCLC; pancreatic cancer; solid tumors.
Cixutumumab	IGF1R	H IgG1	26	I-II	Adrenocortical carcinoma; breast cancer; colorectal cancer; HNC; islet cell cancer; liver cancer; neuroendocrine cancer; NSCLC; pancreatic cancer; prostate cancer; sarcoma; SCLC; solid tumors.
c.T84.66	CEA	90Y-conjugated C IgG1	2	I-II	Colorectal cancer; NSCLC.
Dalotuzumab	IGF1R	Hzed IgG1	9	I-II	Advanced solid tumors; breast cancer; colorectal cancer; NSCLC; pancreatic cancer; SCLC.
Ensituximab	NPC-1C	C IgG1	1	I	Colorectal cancer; pancreatic cancer.
Fresolimumab	TGFβ	H IgG4	3	I-II	Breast cancer; kidney cancer; melanoma; mesothelioma.
J591	PSMA	177Lu-conjugated Hzed IgG1	2	II	Prostate cancer; solid tumors.
KB004	EphA	Hzed IgG1	1	I	Hematologic malignancies.
Lintuzumab	CD33	225Ac-conjugated or naked Hzed IgG1	2	I-II	Leukemia; myelodysplastic syndrome.
M5A	CEA	90Y-conjugated Hzed IgG1	3	I-II	Colorectal cancer; solid tumors.
MDX-1106	PD1	Hzed IgG4	7	I-II	Melanoma; renal cell carcinoma; solid tumors.
MT110	CD3 and EpCAM	na	1	I	Advanced solid tumors
Olaratumab	PDGFRα	H IgG1	7	I-II	GIST; glioblastoma multiforme; NSCLC; prostate cancer; ovarian cancer; soft tissue sarcoma; solid tumors.
RAV12	RAAG12	C IgG1	1	II	Pancreatic cancer.
SAR650984	CD38	Hzed IgG1	1	I	CD38+ malignancies.
TF2	CEA and IMP288	na	4	I-II	Colorectal cancer; SCLC.
Tigatuzumab	TRAILR2	Hzed IgG1	1	II	Triple negative breast cancer.
Tremelimumab (Ticilimumab)	CTLA-4	H IgG2	4	I-II	Advanced hepatocellular carcinoma; prostate cancer; recurrent melanoma.

Abbreviations: AML, acute myeloid leukemia; C, chimeric; CEA, carcinoembryonic antigen; CTLA-4, cytotoxic T lymphocyte antigen 4; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; GIST, gastrointestinal stromal tumors; H, human; HNC, head and neck cancer; Hzed, humanized; IGF1R, insulin-like growth factor 1 receptor; M, murine; na, not applicable; NSCLC, non-small cell lung carcinoma; PD1, programmed death 1; PDGFR, platelet-derived growth factor receptor; PS, phosphatidylserine; PSMA, prostate-specific membrane antigen; SCLC, small cell lung carcinoma; TGFβ, transforming growth factor β; TRAILR2, tumor necrosis factor-related apoptosis-inducing ligand receptor 2. *total n° of trials started after January, 1st 2008 and not completed or terminated at the day of submission.

Olaratumab (IMC-3G3) is a fully human IgG1 antibody that specifically binds the α subunit of the platelet-derived growth factor receptor (PDGFRα), thus antagonizing endogenous PDGF signaling.⁹² PDGF receptors constitute promising targets for anticancer therapies as they are involved in the proliferation and survival of different tumors as well as in the regulation of the growth of tumor stroma and blood vessels.⁹³ Blocking PDGFα with olaratumab on tumor (but not stromal) cells delayed tumor progression and reduced the size of skeletal metastases in preclinical models of pancreatic cancer.⁹⁴ Now, olaratumab is being evaluated, either alone or combined with conventional chemotherapeutics, in 7 phase I-II trials against a variety of solid tumors (Table 4).

Similar to ipilimumab, a fully human IgG1 mAb currently approved for the therapy of melanoma (Table 2), tremelimumab (a human IgG2 also known as ticilimumab and CP-675,206) antagonizes the cytotoxic T lymphocyte antigen 4 (CTLA-4) on the surface of helper T cells, inhibiting the development of peripheral immune tolerance. Thus, tremelimumab belongs to the group of immunostimulatory mAbs, i.e., mAbs that do not directly target cancer cells but rather activate the immune system against them. Early clinical trials suggested that efficient doses of tremelimumab can be safely administered to humans, with major side-effects being autoimmune phenomena such as diarrhea, dermatitis, vitiligo, panhypopituitarism and hyperthyroidism.⁹⁵ Several other phase I-II clinical studies have been conducted to evaluate the pharmacological profile of in tremelimumab in a wide array of solid tumors, all corroborating the encouraging results of preliminary tests.^96-98 In spite of this, there appears to be no currently ongoing phase III-IV study based on tremelimumab, and only a few phase I-II studies are active (Table 4). Perhaps, this is not entirely unrelated to the success and commercialization of ipilimumab, which is now being tested in more than 30 clinical studies, all phases confounded.

Both conatumumab (human IgG1 also known as AMG 655) and tigatuzumab (humanized IgG1 also known as CS-1008) target the tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAILR2), a member of the death receptor protein family also known as death receptor 5 (DR5).⁹⁹ Conatumumab and tigatuzumab operate as agonists, de facto activating TRAILR2 signaling and inducing the apoptotic demise of TRAILR2-expressing cancer (but not normal) cells.^100,101 The exact mechanisms underlying the cancer-selective toxicity of TRAILR agonists remain to be fully elucidated, but may be related to the differential expression of TRAILRs and downstream signaling proteins in transformed vs. normal cells.¹⁰² Irrespective of these considerations, both conatumumab and tigatuzumab exhibited promising safety and efficacy profiles in preclinical tests and in initial phase I-II studies,^100,103-105 which are now being extended to several oncological indications (Table 4).

MDX-1106 (ONO-4538) is a fully human IgG4 that specifically targets programmed death 1 (PD1), a transmembrane receptor that mediates immunosuppressive functions in activated T cells.¹⁰⁶ Preclinical models suggested that the interruption of PD1 signaling leads to improved antitumor T cell responses and disease control,¹⁰⁷ and stimulated a first wave of clinical tests, yielding promising results.¹⁰⁸ MDX-1106 is now being investigated in 7 phase I-II studies, either alone or combined with vaccination strategies, for the therapy of solid tumors including melanoma and renal cell carcinoma (Table 4).

There are 14 distinct phase I-II trials currently assessing the safety and efficacy of bifunctional mAbs, including (but not limited to) the so-called BiTEs (bispecific T-cell engagers). BiTEs, such as the FDA-approved molecule catumaxomab (Table 2) and the investigational drugs blinatumomab and MT110 (Table 4), always target CD3 (a T lymphocyte transmembrane protein) plus one tumor-specific antigen (e.g., EpCAM in the case of catumaxomab and MT110, CD19 in the case of blinatumomab) and act as immunostimulatory agents by taking T cells in the close proximity of tumor cells.^28,109,110 Another interesting approach that is being tested in phase I-II trials is known as pre-targeted radioimmunotherapy and is exemplified by TF2, a bispecific mAb that simultaneously targets the tumor-associated carcinoembryonic antigen (CEA) and a heterologous hapten peptide (IMP-288).¹¹¹ In the context of preclinical studies that yielded encouraging results, TF2 was pre-administered to mice and allowed to localize to CEA-expressing tumors, followed by the injection of ¹⁷⁷Lu-coupled IMP-288.¹¹² Similar approaches are now being evaluated in patients affected by CEA-expressing malignancies, including colorectal and small cell lung cancer (Table 4).

Several distinct immunoconjugates, most of which carry radioactive isotopes, are the object of phase I-II studies (Table 4). These include (but are not limited to) CD45-targeting molecules (e.g., AHN-12 and BC8),^113-115 which are being studied for the therapy of CD45-expressing hematopoietic cancers; immunoconjugates that specifically bind to CEA (e.g., c.T84.66 and M5A),^116,117 which are being evaluated as a treatment for colorectal cancer and other solid tumors; and molecules that target the cell surface myelomonocytic differentiation antigen CD33 (e.g., lantuzumab, which—coupled to ²²⁵Ac—is under investigation in the context of hematopoietic malignancies).¹¹⁸ Of note, naked lantuzumab has previously been tested in phase III clinical trials, but failed to yield statistically significant improvements in response rates and patient survival.^119,120 Another CD33-targeting immunoconjugate, gemtuzumab calicheamicin, is approved since 2000 for the treatment of acute myeloid leukemia (Table 2), possibly explaining why the interest in lantuzumab appears to be decreasing.

Some further examples of mAbs that are currently being investigated in phase I-II clinical trials for oncological indications are reported in Table 4.

Concluding Remarks

At the end of the 19th century, the immunologist Paul Ehrlich (Strehlen 1854; Bad Homburg 1915) hypothesized that several pathologies would be cured if “magic bullets” were available for selectively targeting the cause of disease. Thanks to the invention of mAbs,¹ the “magic bullet” concept has evolved until becoming an ever more refined clinical reality. It can be anticipated that (at least) three major approaches will drive the development of the next generation of mAb-based therapies. First, novel cancer-specific targets will be identified, leading to the creation of mAbs with new specificities. Second, mAbs with known specificities will be engineered to improve their biological functions. Third, new mAbs that do not directly target cancer cells (including mAbs that interfere with the tumor-stroma interaction as well as immunostimulatory mAbs) will be discovered, along with the increasing knowledge that is being generated on the host-tumor crosstalk.

As discussed above, mAbs can function as anticancer agents by modulating tumor cell-intrinsic signaling cascades, by interfering with the tumor-stroma interaction, or by acutely activating the innate immune system (via ADCC, ADCP and CDC) against tumor cells. Intriguingly enough, emerging evidence indicates that—at least in some instances—mAbs exert therapeutic effects by triggering or enhancing a T cell-based response, aside from any effector mechanisms from innate immunity.^121-123 In preclinical models, such an adaptive immune response appears to be required for the therapeutic effect of mAbs.^124,125 The cellular and molecular circuitries underlying the induction of adaptive immunity by mAbs remain poorly understood.¹²⁶ As a possibility, mAbs may enhance antigen uptake or cross-presentation by DCs,^127,128 or facilitate immunogenic cell death.¹²⁹ The precise elucidation of these aspects is eagerly awaited, as it could have profound implications for the development of novel mAbs.

Two additional elements that will have to be taken into consideration for the development of novel mAb-based therapies relate to the Fc receptor, and in particular to the facts that (1) some cancer cell types express functional Fc receptors, which can trigger unconventional (and hence presumably unwanted) signaling pathways,^130-132 and that (2) the genes coding for human Fc receptors are very heterogeneous (there are 3 Fcγ receptor classes, specifying at least 12 receptor isoforms) and all of them present multiple polymorphisms that affect IgG binding and/or signal transduction efficiency.¹³³ These polymorphisms have been shown to influence the patient response to FDA-approved mAbs that elicit immune effector functions, such as cetuximab and rituximab,^134-136 calling for strategies to circumvent this issue. As mentioned above, approaches whereby the Fc fragment is subjected to specific mutations or to changes in its glycosylation status (i.e., reduced or absent fucosylation)¹³⁷ are underway for resolving this problem.¹¹ Future will tell which one among all these strategies will eventually lead to the generation of the next wave of FDA-approved mAbs.

Glossary

Abbreviations:

ADCC

antibody-dependent cellular cytotoxicity

ADCP

antibody-dependent cellular phagocytosis

BiTEs

bispecific T-cell engagers

CDC

complement-dependent cytotoxicity

CDR

complementarity-determining region

CEA

carcinoembryonic antigen

DCs

dendritic cells

DR5

death receptor 5

EGFR

epidermal growth factor receptor

Fab

antigen-binding fragment

Fc

constant fragment

GM-CSF

granulocyte macrophage-colony stimulating factor

mAb

monoclonal antibody

MMAE

monometil auristatin E

NSCLC

non small-cell lung cancer

PDGFRα

plateled-derived growth factor receptor α

RANKL

receptor activator of NF-κB ligand

TNFR

tumor necrosis factor receptor

TRAILR2

tumor necrosis factor-related apoptosis-inducing ligand receptor 2

VEGFR

vascular endothelial growth factor receptor