Monoclonal Antibodies and Cancer

  • Kewal K. Jain
Chapter

Abstract

A monoclonal antibody (MAb) is an antibody made from a single clone (hybridoma) of white blood cells. MAbs are used to block key receptors on tumor cell surfaces, compromising their function. MAbs may also be used to recruit the cellular arm of the immune system, planting a homing beacon on the transformed cell. MAbs, although produced in the laboratory, mimic the antibodies naturally produced by the body as part of immune system’s response to disease. The remarkable specificity of MAbs is also being harnessed in other ways, e.g., they are being paired with powerful toxins to create specific agents that seek out cancer cells and kill them. MAbs are also being employed for diagnosis, helping to identify the source of a tumor and provide a possibility for the treatment. MAbs enable the combination of diagnostics with therapeutics.

8.1 Introduction

A monoclonal antibody (MAb) is an antibody made from a single clone (hybridoma) of white blood cells. MAbs are used to block key receptors on tumor cell surfaces, compromising their function. MAbs may also be used to recruit the cellular arm of the immune system, planting a homing beacon on the transformed cell. MAbs, although produced in the laboratory, mimic the antibodies naturally produced by the body as part of immune system’s response to disease. The remarkable specificity of MAbs is also being harnessed in other ways, e.g., they are being paired with powerful toxins to create specific agents that seek out cancer cells and kill them. MAbs are also being employed for diagnosis, helping to identify the source of a tumor and provide a possibility for the treatment. MAbs enable the combination of diagnostics with therapeutics.

8.1.1 Murine MAbs

Murine MAbs against tumor-specific antigens were initially envisioned as therapeutic agents capable of directly attacking cancer cells. Once thought to be “magic bullets,” it was hoped that murine MAbs would effectively target disease but not affect healthy cells. This approach, however, proved to be disappointing. Since tumors are not well perfused, relatively large doses of the MAbs were required, which caused problems relating primarily to adverse immunological reactions against the antibodies that the body recognized as large foreign proteins. The MAbs of the first generation caused toxicity with little efficacy. Adverse effects of murine MAbs, in combination with poor target selection due to the lack of data on tumor antigens, eventually led to their abandonment as therapeutic agents. Many of the murine MAbs in use now are humanized.

8.1.2 Humanized MAbs

The ability to make MAbs against antigens expressed by human cells has facilitated the identification not only of lymphocytes with different subset functions but also of different antigens on tumor cells. MAbs have several potential targets for cancer therapy, including the CD20 antigen, the IL-2 receptor (CD25), the epidermal growth factor receptor (EGFR), and the VEGF. The FDA has approved several MAbs against cancer listed in Table 8.1.
Table 8.1

Monoclonal antibodies for cancer approved by the FDA

Drug/year of approval

Company

Type

Target

Indication

Alemtuzumab (Campath) 2001

Millennium and Ilex Partners

Humanized MAb

CD52

B-cell chronic lymphocytic leukemia

Bevacizumab (Avastin)/2004

Genentech

Humanized MAb

VEGF

Metastatic colorectal cancer Combined with 5-FU

Cetuximab (Erbitux)/2004

ImClone Systems/Bristol-Myers Squibb

IgG1 chimeric MAb

EGFR

Metastatic colorectal cancer Combined with irinotecan

Daclizumab (Zenapax)/2002

Protein Design Labs

Chimeric MAb

IL-2R

Leukemia

Gemtuzumab (Mylotarg)/2000

Wyeth

Humanized MAb

CD33

Acute myeloid leukemia

Ibritumomab (Zevalin)/2002

Cell Therapeutics Inc.

Murine MAb/90Y

CD20

Non-Hodgkin lymphoma

Ipilimumab (Yervoy)/2011

Bristol-Myers Squibb

Humanized MAb

CTLA-4

Melanoma

Ofatumumab (Arzerra)/2009

Genmab

Humanized MAb

CD20

B-cell chronic lymphocytic leukemia

Panitumumab (Vectibix) 2006

Amgen Inc.

Human XenoMouse®

EGFR

EGFR-expressing metastatic colorectal cancer

Rituximab (Rituxan)/1997

Genentech/Biogen Idec

Chimeric MAb

CD20

Non-Hodgkin lymphoma, chronic lymphocytic leukemia

Tositumomab (Bexxar)/2003

Corixa/GlaxoSmithKline

Murine MAb/131I

CD20

Non-Hodgkin lymphoma, chronic lymphocytic leukemia

Trastuzumab (Herceptin)/1998

Genentech/Roche

Humanized MAb

HER-2/neu

Breast cancer, NSCLC, pancreatic cancer

© JainPharmaBiotech

CTLA-4 cytotoxic T lymphocyte-associated antigen 4, EGFR epidermal growth factor receptor, VEGF vascular endothelial growth factor, NSCLC non-small-cell lung cancer

8.2 Actions and Uses of Monoclonal Antibodies in Cancer

MAbs have been used in the treatment of cancer for the following purposes:
  • Direct modulation of tumor function

  • Promotion of tumor lysis by immune effector cells

  • Immunization of patients against tumor antigens

  • Induction of apoptosis

  • Antiangiogenic effect

  • Disruption of signaling

  • Targeted radiolysis

There are multiple mechanisms for explaining anticancer effects of MAbs, and more than one mechanism may be involved in the action of a product. Anti-EGFR MAbs promote a slow endocytic process distinct from the rapid EGF-induced receptor internalization. Combining MAbs that engage distinct epitopes significantly accelerates receptor degradation. In addition, MAb combinations have been found to be more effective than the use of single antibodies in inhibiting HER2 signaling in vitro and tumorigenesis in animals. The use of MAbs for the therapy of cancer is one of the major advances in cancer immunology. Modulation of immune system by interplay with tumor cells through targeting of T-cell receptors (TCRs) has emerged as a powerful new therapeutic strategy for tumor therapy and to enhance cancer vaccine efficacy (Scott et al. 2012). One of the major challenges now is to combine two major immune-based treatments—MAbs and vaccines. A series of clinical trials is exploring this approach.

8.2.1 Targeted Antibody-Based Cancer Therapy

8.2.1.1 Antibody–Cytokine Fusion Proteins

Antibody–cytokine fusion proteins consist of cytokines fused to an antibody to improve antibody-targeted cancer immunotherapy. These molecules have the capacity to enhance the tumoricidal activity of the antibodies and/or activate a secondary antitumor immune response. A review of studies of multiple antibody–cytokine fusion proteins, including preclinical and clinical studies focusing on IL-2, IL-12, and GM-CSF, has demonstrated significant antitumor activity as direct therapeutics or as adjuvants of cancer vaccines (Ortiz-Sanchez et al. 2008). Tumor-targeting ability of scFv(L19) in patients with cancer has been demonstrated using scintigraphic methods, and scFv(L19)-based antibody–cytokine fusion proteins are currently in clinical trials.

8.2.1.2 Antibody J591 for Targeted Delivery of Anticancer Therapy

Since the prostate-specific membrane antigen (PSMA) exists only on tumors and not other tissues, J591 armed with a drug or radiation offers a way to selectively target cancer while leaving healthy tissues unharmed, thereby resulting in very low levels of toxicity and fewer side effects for patients. A clinical trial has for the first time proven that J591 could exclusively target tumors without hitting normal tissues. Although it does not reduce tumor size, it provides a vehicle for selectively transporting drugs or a radioactive isotope to destroy the blood vessels that feed tumors, thereby cutting off the tumor’s blood supply. Acceptable toxicity and excellent targeting of known sites of metastases were demonstrated in patients with multiple solid tumor types, highlighting a potential role for the anti-PSMA antibody J591 as a vascular-targeting agent (Milowsky et al. 2007). The trial involved cancer patients with a wide range of solid tumors including kidney, bladder, lung, breast, colorectal, pancreas, and melanoma. A radioactive tracer, attached to the antibody, was used to follow J591’s progress throughout the body. The next step will be to arm the J591 antibody with drugs or radioactivity and then will assess tumor response. Such armed antibodies have been used in patients with prostate cancer and show significant antitumor activity has been demonstrated. Antiangiogenic agents are less effective against more advanced tumors with established blood vessels. By directly targeting tumor blood vessels, however, J591 treatments could destroy the tumor’s blood supply and shrink even advanced tumors.

8.2.1.3 Anti-Thomsen–Friedenreich Antigen MAb

Thomsen–Friedenreich antigen (TF-Ag) is expressed in many cancers, including those of the breast, colon, bladder, and prostate. TF-Ag is important in adhesion and metastasis and as a potential immunotherapy target. Passive transfer of JAA-F11, an anti-TF-Ag MAb, was demonstrated to bind with a carbohydrate on the tumor cell surface that is involved in adhesion of the cell during the metastatic process and blocked metastases in murine lung vasculature in an in vivo metastatic deposit formation assay (Heimberger et al. 2009). JAA-F11 significantly extended the median survival time of animals bearing metastatic 4T1 breast tumors and caused a >50 % inhibition of lung metastasis. Not only would drugs attached to the antibody JAA-F11 bind to the tumor cell surface to direct their cytotoxic effect, but the binding of the antibody itself would block the cell from metastasizing. Currently research is determining if JAA-F11 could increase the effectiveness of existing cancer drugs as well as studying the possibility of using the antibody as a vehicle for the targeted delivery of drugs to aid cancer diagnosis and therapy.

8.2.1.4 Bavituximab

Bavituximab (Peregrine Pharmaceuticals) is an MAb that targets phosphatidylserine (PS) and represents a new approach to the treatment of cancer. PS is a highly immunosuppressive molecule usually located inside the membrane of healthy cells but gets displaced to be exposed on the exterior of cells that line tumor blood vessels, enabling a tumor to evade immune detection. Cancer therapies increase PS exposure on the cell surface, further increasing immune suppression in the tumor environment, whereas bavituximab targets PS and blocks this immunosuppressive signal, resulting in the maturation of dendritic cells and cancer-fighting (M1) macrophages leading to the development of cytotoxic T cells for fighting solid cancers.

Bavituximab and other PS-targeting antibodies have shown anticancer activity in several preclinical studies including models of lung, pancreatic, breast, prostate, and brain cancer. Experimental cancer vaccine regimens using bavituximab have demonstrated the potential of generating protective immune responses against specific tumor challenges. In a phase I clinical trial, bavituximab was well tolerated and pharmacokinetic studies support a weekly dosing regimen (Gerber et al. 2011). MAbs directed against exposed PS have potential as a powerful adjunct to postoperative chemotherapy in preventing relapses after cancer surgery (Judy et al. 2012). Phase I and II clinical trials are in progress to investigate bavituximab in combination with chemotherapy and other molecularly targeted agents.

8.2.1.5 Combining MAbs with Anti-CD55 Antibody

Efficacy of many therapeutic products, including MAbs, may be limited by the presence of certain proteins on tumor surfaces that protect the tumor from attack by the immune system. One of these proteins is CD55, which is expressed on most tumor cells at far higher levels than on normal cells, and its function helps prevent the destruction of tumor cells. The anti-CD55 antibody (Viragen) was shown to effectively remove this protective effect and significantly enhance the activity of rituximab (Biogen Idec’s Rituxan®), when both drugs were used together in a cell-based evaluation study. The anti-CD55 antibody binds to a specific target expressed on the surface of tumor cells and removes one of the tumor’s most important protective mechanisms, thereby making cancer cells vulnerable to attack by the immune system or other anticancer products. Results of the study showed that the combination of the anti-CD55 antibody and rituximab led to a significant increase in the destruction of cancer cells as compared to rituximab alone. The anti-CD55 antibody is not approved for sale in any market and human clinical trials will be required prior to seeking approval from any international regulatory agency.

8.2.1.6 MAbs Targeted to Alpha-Fetoprotein Receptor

ProtoKinetix has generated antibodies to the RECAF™ (receptor for alpha-fetoprotein), which is a biomarker for cancer. The presence of alpha-fetoprotein in the human body (other than in pregnant women) is a nonspecific indicator of the existence of cancer in the system. MAbs specific for the RECAF site do not have an affinity to healthy cells. Anti-RECAF antibodies, modified according to ProtoKinetix’s Super–Antibody proprietary technology and the Peregrine Pharmaceuticals’ catalytic antibody technology, improve the contrast between cancer cells and the surrounding nonmalignant tissue. Preliminary results of a study carried out by the Georges Pompidou Hospital in France, which was designed to validate the Histo-RECAF version 2.0, are positive and indicate that labeling of the malignant cells is strong with a nice delineation between malignant and normal cells. This technology has the potential for targeted cancer therapy and is being commercialized by BioCurex Inc.

8.2.1.7 MAbs Targeted to Tumor Blood Vessels

DX-2240 (Dyax Corporation) is a fully human MAb that targets the Tie-1 receptor on tumor blood vessels and has therapeutic potential in numerous oncology indications. In preclinical animal models, DX-2240 has demonstrated activity against a broad range of solid cancers. The antibody works by altering tumor vascular morphology, thereby increasing hypoxia and necrosis. In addition, DX-2240 in vivo increases the anticancer activity of other therapies such as VEGF pathway inhibitors and chemotherapeutic agents when used in combination. Sanofi-Aventis has licensed DX-2240 from Dyax for further development.

8.2.1.8 Volociximab

Volociximab is a first-in-class chimeric MAb that targets α5β1 integrin. Preclinical studies have shown the ability of volociximab to inhibit tumor neoangiogenesis by blocking the interaction between α5β1 and fibronectin. Volociximab’s safety profile, pharmacokinetics, and pharmacodynamics have been established. Ongoing clinical trials are evaluating its efficacy in the treatment of different types of solid tumors as a single agent or in combination with chemotherapy. It has shown promising activity in different types of cancer (Almokadem and Belani 2012).

8.2.2 MAbs for Immune Activation

The mechanism by which low doses of MAbs activate immune responses to tumor-specific antigens is, in part, analogous to the mechanism of a classic technique in experimental immunology used to produce antibodies against molecules that usually do not elicit an immune response. In this classic technique, the molecule of interest is attached to foreign antibody that is highly immunogenic by itself. In the process of attacking the foreign antibody, the body is also “tricked” into mounting an immunological reaction against the targeted molecule (tumor-associated antigen [TAA]) now attached to the protein.

MAbs in development by AltaRex Corporation serve as large highly immunogenic proteins that bind to tumor-specific antigens. Very low doses of MAbs, administered intravenously, effectively induce this potentially therapeutic immune response. The lead product, OvaRex® MAb, has shown promise for the treatment of ovarian cancer patients in both remission and recurrent stages of the disease. OvaRex® MAb is documented to induce cellular and humoral immune responses against the tumor-specific antigen CA-125, which is the most thoroughly studied serum marker for ovarian cancer. There is a correlation between the extent of the immunogenic response against CA-125 and progression-free and/or survival time of patients. The antibodies generated in response to the administration of OvaRex MAb are directed against multiple epitopes of the CA-125 molecule, indicating a highly effective immune induction in response to the product.

8.2.3 Delivery of Cancer Therapy with MAbs

MAbs can be used for the delivery of cytotoxin payloads such as radionucleotides, toxins, and chemotherapeutic agents to the tumors as shown in Table 8.2.
Table 8.2

Anticancer agents linked to monoclonal antibodies

Drugs

Anthracyclines: doxorubicin, daunorubicin, idarubicin, nemorubicin

Antimetabolites: aminopterin, methotrexate, fluorouracil, floxuridine, cytarabine

Alkylating agents: melphalan, chlorambucil, mitomycin, cisplatin, trenimon

Antimitotic drugs: vinca alkaloids, podophyllotoxin, colchicine

DNA intercalations: ethidium dimer neocarzinostatin, tyrosine kinase inhibitor genistein

Toxins

Pseudomonas exotoxin

Ricin

Saporin

Diphtheria toxin

Isotopes

131Iodine

99Technetium

90Yttrium

Enzymes

β-Lactamase

Alkaline phosphatase

Antibody-directed enzyme prodrug therapy (ADEPT)

Carboxypeptidase

Cytidine deaminase

Glucose oxidase

Cytokines

TNF-α

Interferon

Interleukin-2

Sensitizers

Radiosensitizers

Photosensitizers

Miscellaneous agents

MRI contrast agents

© JainPharmaBiotech

8.2.4 Antibody-Directed Enzyme Prodrug Therapy

Antibody-directed enzyme prodrug therapy (ADEPT) involves both an antibody–enzyme conjugate and a low-toxicity prodrug. First the antibody–enzyme conjugate is delivered to the target cells that express tumor antigens on their surface. The unbound antibody is allowed to clear before the prodrug is administered. The enzyme part of the conjugate then cleaves the prodrug to unleash the active form of the drug at the tumor site. Not all tumor cells need to be targeted because activated drug accumulates and diffuses at the tumor site, killing nearby cells.

Previous clinical trials have shown evidence of tumor response, but the activated drug had a long half-life, which resulted in dose-limiting myelosuppression. Also, the targeting system, although giving high tumor-to-blood ratios of antibody–enzyme conjugate (10,000:1), required the administration of a clearing antibody in addition to the antibody–enzyme conjugate. Because ADEPT therapy uses an enzyme carboxypeptidase G2 (CP) not naturally found in humans, a patient’s immune system can recognize and reject the enzyme before it can do its job. This has been addressed by identification and disguising clinically important immunogenic sites on recombinant fusion proteins to enable them to repeatedly elude the immune system. The advance could allow patients to safely receive multiple courses of ADEPT to treat their cancer.

8.2.5 Chemically Programmed Antibodies

Studies at the interface of chemistry and biology have enabled the development of an immunotherapeutic approach called chemically programmed antibodies (cpAbs), which combines the merits of traditional small-molecule drug design with immunotherapy and breaks the “one antibody–one target axiom” of immunochemistry. Small-cell-targeting molecules used with a nontargeting catalytic aldolase MAb 38C2 create a novel compound for retargeting it to breast cancer cell lines expressing integrin αvβ3. This approach enables the effective assembly of cpAbs in vitro as well as in vivo and intracellular delivery of cpAbs into cells. A substantial enhancement of the therapeutic effect over the peptidomimetic itself has been demonstrated in animal models of breast cancer metastases.

This technology possesses potential for the diagnosis as well as treatment of disease. An imaging agent could be attached to the small molecule, enabling a physician to monitor the localization of a drug before arming the agent with the antibody molecule. Whereas treatment with MAbs requires a different antibody for each specific target, cpAbs approach enables different small-molecule-targeting agents, called programming agents or adapters, to selectively direct the same MAb to different sites for different uses so that only a single antibody is required for multiple tasks. Among the potential therapeutic advantages is a dramatically increased circulatory half-life of the compound, which could give patients greater exposure to the benefits of any treatment. The new hybrid compound remained in circulation for a week as compared to a small-molecule drug that is cleared in a matter of minutes. cpAB approach has been used in a melanoma model, dramatically enhancing the effectiveness of a small-molecule drug (Popkov et al. 2006).

8.2.6 Combination of Diagnostics with Therapeutics Based on MAbs

Two tests—Poteligeo Test IHC (immunohistochemistry) and Poteligeo Test FCM (Kyowa Medex)—were approved in Japan in March 2012 as companion diagnostics for mogamulizumab (Kyowa Hakko Kirin’s Poteligeo) injection, a therapeutic MAb for the treatment of adult T-cell leukemia (ATL). Poteligeo binds to CCR4, which is expressed on the surface of ATL cells which are killed by MAb-dependent cell-mediated cytotoxicity. The companion diagnostic tests detect the presence of CCR4 expressed by ATL cells before treatment with Poteligeo to enable the identification of patients who would benefit from the drug. Poteligeo Test IHC is for use on tissue samples, such as lymph nodes, whereas Poteligeo Test FCM uses flow cytometry to analyze blood samples from patients.

8.2.7 Radiolabeled Antibodies for Detection and Targeted Therapy of Cancer

Radiolabeled MAbs are widely used in the detection and treatment of cancer. There are several preparations and a few examples will be described here.

8.2.7.1 Ibritumomab Tiuxetan

Ibritumomab tiuxetan (Zevalin), the first MAb conjugated with a radionucleotide to be approved, is a radioimmunotherapy marketed for certain types of B-cell non-Hodgkin lymphoma (NHL) in the USA. It is a chimeric MAb of murine origin, but over 95 % of it is humanized. It binds to the same target as Rituxan but complements Rituxan by killing NHL cells through a different mechanism. Binding of Rituxan to CD20 on the tumor cell surface recruits the immune system to destroy the cell. It destroys tumor with the radionuclide yttrium-90 that is tightly bound to the antibody by means of a covalently linked chelator. Yttrium-90 releases a beta particle that can penetrate surrounding tumor cells over a 5-mm radius, leaving cellular free radicals followed by cell death, in its wake. As a radiolabeled antibody, murine origin is an advantage. A shorter half-life decreases yttrium-90 circulation time, and risk of provoking human antibodies is low, possibly due to its dose being orders of magnitude smaller than that of Rituxan. The vastly lower required dose reflects the enhancement of potency by the addition of the radionuclide.

8.2.7.2 huHMFG1

huHMFG1 (formerly Therex) is a humanized version of the mouse MAb HMFG1 (human milk fat globule-1). It binds to MUC1, a cell membrane protein present in a variety of tumors of epithelial origin, including breast, ovarian, pancreatic, gastric, and colon cancers. R1550 acts as a “marker flag,” attaching itself to tumor cells and helping components of the immune system, including natural killer cells, to find and destroy them. Humanization is intended to make the antibody less immunogenic (i.e., to make the antibody less likely to be recognized as foreign material by the patient’s immune system) and thus increase its suitability for repeat intravenous administration. It has undergone a phase I trial in metastatic breast cancer and pharmacokinetics was characterized (Royer et al. 2010).

8.2.7.3 KW-2871

KW-2871 (Kyowa Hakko Kirin Ltd.). KW-2871, a chimeric antibody made by POTELLIGENT™ technology, is under development for stage IV malignant melanoma and is in phase I/IIa clinical trials in the USA. POTELLIGENT technology boosts the efficacy of antibodies. When an antibody had a reduced amount of fucose in its sugar chains, it exhibits much higher antibody-dependent cellular cytotoxicity (ADCC) activity as compared to a highly fucosylated conventional antibody. The mechanism behind the enhanced ADCC of a low-/no-fucose antibody is its increased affinity to FcγRIIIa (CD16), the major Fc receptor for ADCC in humans. Knockout of a gene called FUT8 that is responsible for the fucose addition to sugar chains created a new production method for fucose-free antibodies in Chinese Hamster Ovary (CHO) cells. Finally the FUT8-knockout CHO cells (POTELLIGENT® Cells) were created, which are shown to produce fucose-free, thereby ADCC-enhanced antibodies (POTELLIGENT® MAbs), with unchanged basic properties (stability, growth rate, productivity, and scalability) compared to parental CHO cells.

8.2.7.4 Ferritarg

Ferritarg (MAT Biopharma), a rabbit polyclonal antibody that is coupled with either yttrium-90 or indium-111 and is directed against acidic ferritin, is used for radioimmunotherapy. Ferritin is produced by most cells in the human body and the protein’s function is to bind and store iron. Elevated levels of different variants of ferritin (isoferritins) have been measured on the surface of various types of cancer cells including those from cancers of the liver, lung, pancreas, head and neck, kidney, ovaries, intestines, stomach, and breast, as well as NHL and Hodgkin lymphoma. This upregulation of ferritin in cancer cells differentiates these cells from normal cells and ferritin is thus a cancer cell antigen. Ferritarg is being developed initially for the treatment of refractory Hodgkin disease. Ferritarg has been granted orphan drug status in the EU and the USA for the treatment of Hodgkin disease. It is phase I/II clinical trials.

8.2.7.5 Cotara

Cotara (Peregrine Pharmaceuticals) is a TNF-targeting chimeric MAb, labeled with 131I, which is in phase III clinical trials for glioblastoma multiforme. Cotara links a radioactive isotope to a targeted MAb designed to bind to the DNA histone complex that is exposed by dead and dying cells found at the center of solid tumors. Cotara’s targeting mechanism enables it to bind to the dying tumor cells, delivering its radioactive payload to the adjacent living tumor cells and essentially destroying the tumor from the inside out, with minimal radiation exposure to healthy tissue. Cotara is delivered in a single dose using convection-enhanced delivery, a method that targets the specific tumor site in the brain.

8.2.7.6 Clivatuzumab

Clivatuzumab (Immunomedics) is a humanized MAb targeting a mucin antigen expressed in most pancreatic cancers, but not pancreatitis, normal pancreas, or most other normal tissues. The yttrium-90-labeled MAb has orphan drug status in both the USA and the EU and fast track status in the USA for the treatment of pancreatic cancer. Preclinical studies in mice with human pancreatic cancer xenografts given the murine version of yttrium-90 clivatuzumab tetraxetan demonstrated favorable tumor responses, which could be further improved when given in combination with gemcitabine. A prior phase I single dose-escalation study of yttrium-90 clivatuzumab tetraxetan in treatment-relapsed pancreatic cancer patients has also produced encouraging results, with evidence of objective responses. A phase III clinical trial of yttrium-90 clivatuzumab in combination with gemcitabine is ongoing for the treatment of patients with newly diagnosed, inoperable, untreated stage III or stage IV cancer of the pancreas.

8.2.8 Methods for Increasing MAb Selectivity for Tumor Tissue

MAb selectivity for tumor tissue can be increased. Some of the improvements are as follows:

Bispecific Antibodies. Using either hybridoma fusion, chemical methods, or genetic engineering technology, antibodies with dual specificity can be constructed. These so-called bispecific antibodies have been used to redirect the cytolytic activity of a variety of immune effector cells such as cytotoxic T lymphocytes (CTLs), natural killer cells, neutrophils, and monocytes/macrophages to tumor cells.

Immunomedics has designed a bispecific fusion protein by linking portions of genes encoding two distinct antibodies to yield a single protein molecule with two binding sites: one to the tumor and the other to a small molecule carrying a therapeutic agent. The fusion protein is half the size of a typical antibody and is substantially humanized by molecular engineering. In the first step, the bispecific antibody fusion protein is injected intravenously and subsequently binds to cancer cells, while unbound material is cleared rapidly from the body. In the second step, a small drug carrier is injected and shows specific uptake into the tumors that have been bound by the fusion protein. As early as 4 h after injection of the small-molecule drug carrier, researchers measured a 13:1 ratio of uptake of the therapeutic drug in the tumor as compared with the blood. This is an eightfold higher tumor selectivity ratio than that achieved with the directly antibody-bound drug. In experiments with chemically conjugated fusion proteins, drug targeting resulted in almost 100 times more uptake in the tumor than in normal tissues. It is in phase II clinical trials with a carcinoembryonic antigen (CEA) bispecific antibody in patients with CEA-expressing tumors, such as colorectal and medullary thyroid cancers.

Currently, systemic application of bispecific antibodies is suitable only for adjuvant treatment of minimal residual disease because of poor tumor cell accessibility. As an alternative, attacking the tumor’s blood supply by delivering coagulation factors or toxins or by bispecific antibody-directed immunotherapies holds great promise for anticancer therapy.

Trifunctional Antibodies. These antibodies are called trifunctional because they bind to cancer cells and also to two different cells of the immune system: T cells and macrophages. Through the creation of this cell complex, the trifunctional antibodies initiate an especially efficient eradication of tumor cells. The goal is to eliminate those tumor cells that may still be present in the body, e.g., following surgical resection of a tumor—to prevent relapse or the development of metastases. The trifunctional antibodies were developed by TRION Pharma, a partner of Fresenius Biotech, which successfully completed a phase I/II study of the treatment of ascites in ovarian cancer using the trifunctional antibody Removab®. The antibody was shown to be well tolerated and demonstrated the first significant indications of efficacy. The following additional studies with Removab® and the other trifunctional antibody Rexomun™ have been done:
  • Two phase I studies have investigated the use of Rexomun™ in breast cancer.

  • Phase II/III studies have investigated Removab® in NSCLC, malignant pleural effusion, stomach cancer, pancreatic cancer, and ovarian cancer.

Immunotoxins. MAbs can also be coupled with cellular toxins. One of the most potent ones is Pseudomonas exotoxin. Proxinium (Viventia Biotech) is a protein engineered from the fusion of a truncated form of Pseudomonas exotoxin A to the humanized scFv, which is specific for the epithelial cell adhesion molecule (Ep-CAM). It is in phase II clinical trials for head and neck cancer. SS1P (Enzon Pharmaceuticals) is a recombinant immunotoxin consisting of anti-mesothelin MAb fragment and Pseudomonas exotoxin. It is in phase II clinical trials for pancreatic and ovarian cancers.

Local Injection of MAbs or MAb Complexes. Such injections can be made directly into the tumors. This method is useful for chemotherapy of breast cancer nodes in the adjoining lymph system.

Immunoliposomes (Antibody-Coupled Liposomes). Attempts have been made at targeting MAbs to the tumor site by binding them to liposomes. Some of the problems regarding immunoliposome preparation and application such as antibody coupling and immunoliposome stability and pharmacokinetics have been overcome during the last decade. Some of the challenges that still need further work are:
  • Difficulty of maintaining the biological activity of therapeutic proteins when they are attached to polyethylene glycol (PEG)-liposomes

  • Difficulty of releasing the drug from the immunoliposome complex

  • Limited capacity of the immunoliposomes to carry drugs

Fab type C showed low reticuloendothelial system (RES) uptake and a long circulation time and enhanced accumulation of the liposomes in the solid tumor. The small Fab type C (PEG-immunoliposomes) predominantly passes through the leaky tumor endothelium by passive convective transport and provides an important insight into the potential of type C liposomes for target-specific drug delivery.

Immunoliposomes that target EGFR and/or its truncated variant EGFRvIII can be constructed to provide efficient intracellular drug delivery in tumor cells overexpressing these receptors. MAb fragments can be covalently linked to liposomes containing various reporters or drugs such as doxorubicin, vinorelbine, or methotrexate. In each case, the immunoliposome agent is significantly more cytotoxic than the corresponding nontargeted liposomal drug in target cells, whereas it is equivalent in cells lacking EGFR/EGFRvIII overexpression. Anti-HER2 immunoliposomes containing HERMES Bioscience’s proprietary antibody fragment F5 and the chemotherapy drug doxorubicin are in development toward clinical trials.

Combined Use of MAbs and Cytokines. The potent antitumor activity of certain cytokines is often achieved at the expense of unacceptable toxicity. One avenue to improve the therapeutic index of cytokines in cancer therapy consists of fusing them to MAbs capable of a selective localization at the tumor site. Fusion proteins of IL-12 and TNF-α with L19, an antibody fragment specific to the extra domain B of fibronectin which has been shown to target tumors in animal models and in patients with cancer. These fusions display a potent antitumor activity in several immunocompetent murine models of cancer but do not lead to complete remissions of established aggressive tumors. They have further evaluated the tumor-targeting properties and the anticancer activities of combinations of the two antibody–cytokine fusion proteins, as well as of a triple fusion protein between IL-12, L19, and TNF-α. Although all fusion proteins were active in vitro, the triple fusion protein failed to localize to tumors in vivo and to show significant therapeutic effects. By contrast, the combination of IL-12–L19 and L19–TNF-α displayed potent synergistic anticancer activity and led to the eradication of F9 teratocarcinomas grafted in immunocompetent mice. When cured mice were rechallenged with tumor cells, a delayed onset of tumor growth was observed, indicating the induction of a partial antitumor vaccination effect. The combined administration of the two fusion proteins showed only a modest increase in toxicity, compared with treatments performed with the individual fusion proteins. These results show that the targeted delivery of cytokines to the tumor environment strongly potentiates their antitumor activity and that the combination treatment with IL-12–L19 and L19–TNF-α appears to be synergistic in vivo.

huHMFG1huDNase I. It is known that HMFG1 is internalized. In addition, DNase has been shown to be highly toxic when injected directly into isolated tumor cells. It kills the cells by inducing apoptosis, which causes the cells to break down into small particles that can be cleared by natural scavenger cells. This may avoid the potentially life-threatening inflammatory responses experienced by some cancer patients treated with conventional therapies. In vitro experiments have confirmed that, while neither the antibody nor the enzyme alone is toxic, the combination of a humanized MAb HMFG1 with the enzyme DNase demonstrates rapid cell killing. The gene for the DNase-based drug has been stably inserted into a human cell line, an important step toward the development of a reliable manufacturing process. In addition, it has been shown that cells containing this gene produce a substance that targets and kills tumor cells.

MAbs That Selectively Target Cancer. Micromet’s fully human MAb MT201 (adecatumumab) is directed against the Ep-CAM. The product is currently being tested in two multicenter phase II clinical trials for the treatment of prostate and metastatic breast cancer. Ep-CAM, the target antigen for MT201, is overexpressed with high frequency on most human carcinomas, suggesting that it may have therapeutic potential in the treatment of a broad range of cancers, including prostate, breast, colon, lung, stomach, pancreatic, head and neck, and ovarian cancer. MT201 has been specifically designed to selectively eliminate tumor cells while leaving healthy tissues largely unharmed. Phase I data have demonstrated an excellent safety profile of MT201, and no MT201-neutralizing antibodies have been observed so far in man. An Investigational New Drug (IND) for the product has been cleared by the FDA for the initiation of phase II studies in the USA.

MAb 806 (Life Science Pharmaceuticals) specifically targets EGFR on a wide range of tumor types but has no uptake by normal tissues. The 806 antigen is not exposed on inactive wild-type EGFR, but is exposed on a transitional form of the EGFR. The epitope studies are supported by IHC demonstrating that the 806 antibody binds to a broad range of epithelial cancers and to gliomas, but not to normal human tissues. Other preclinical data suggest that MAb 806 would not have the side effects observed with other EGFR-targeting MAbs. Results of a clinical trial confirmed the target specificity and safety of MAb 806 in human patients with a variety of cancers including squamous cell carcinomas of the lung, head and neck, and skin; colorectal cancer; mesothelioma; and glioma (Scott et al. 2006).

8.2.9 Advantages and Limitations of MAbs for Cancer Therapy

MAbs offer the following advantages for cancer therapy:
  • High specificity for tumor antigens

  • Long half-life which enables reduction of dose frequency

  • Low cross-reactivity with normal cells

  • Possibility of manufacture in large quantity and with a high degree of purity

  • Flexibility of design: customization with respect to immunoglobulin binding sites

  • Shorter development time compared to traditional small-molecule drugs

MAbs are likely to enhance, rather than replace, current cancer therapies by targeted destruction of cancer cells and possible recruitment of the body’s immune system. Although MAbs have been proposed as vehicles to target cancer cells specifically, several theoretical factors for their safety/efficacy have been a major concern. MAbs have the following limitations:
  • Cellular targets are restricted to surface antigens as their large size prevents direct cell penetration and important intracellular protein targets remain inaccessible. Since cancer cells express only 10–15 % of proteins on cell surface, the remaining 85–90 % cannot be targeted.

  • Slow elimination from the blood, poor vascular permeability, and low tumor uptake.

  • Tumor selectiveness rather than tumor specificity, i.e., they can bind to normal cells that have the same tumor receptors as tumors.

  • Antigenic heterogeneity.

  • Induction of human anti-mouse antibody responses.

Alternatives to MAbs to overcome some of the limitations include single-chain antibody-binding (SCA) protein technology, monoclonal TCR technology, and antibody–drug conjugates (ADCs). These are described in the following sections.

8.3 Single-Chain Antibody-Binding Protein Technology

SCA proteins, like MAbs, deliver therapeutic proteins to targeted disease sites. The advantages over MAbs are:
  • SCA proteins are easier to produce and do not need to go through the humanization process.

  • SCAs penetrate the tumor much better because their molecular weight is only a fraction of that of the usual antibodies.

  • Immunogenicity is reduced because protein that is not required for antigen binding is not included.

  • Flexibility to tailor half-life via PEG technology.

  • More cost-effective scale-up for manufacturing when compared with MAbs.

  • Better delivery opportunities offering potential for non-parenteral delivery.

Enzon is developing SCA technology for the delivery of cancer therapeutics. Using an 123iodine-labeled SCA selected from a combinatorial library, clinical evidence of efficient tumor targeting in patients with CEA-producing cancer has been demonstrated.

8.4 Monoclonal T-Cell Receptor Technology

T cells are a powerful defense against cancer cells. Every peptide antigen has a corresponding T cell, which can bind to its target via the TCR on its surface. T cell, thus activated, destroys the cancer cell. TCRs, isolated in a soluble form and engineered like antibodies, have the potential to become specific and sensitive tools for targeting cancer. They can overcome some of the limitations of conventional monoclonal antibodies. TCRs differ from MAbs in antigen recognition. MAbs recognize and target TAAs expressed as membrane-bound proteins on the surface of tumor cells. Some of these are tumor type specific, whereas others are expressed in a wide variety of tumors. Cancer cells may also present tumor-associated peptide antigens (TAPAs), some of which are derived from intracellular proteins and are tumor-specific and useful targets for therapeutic intervention. TAPAs are not recognized by antibodies but by TCRs. Monoclonal TCRs (mTCRs) thus represent a new approach to the targeted treatment of cancer. While retaining the benefits of MAbs, mTCRs provide additional benefits such as enhanced ability to target the breakdown products of intracellular proteins and to deliver cytotoxic agents to cancer cells.

There are, however, technical problems of using TCRs. Unlike antibodies, TCRs are not expressed in a soluble form, but are anchored to the T-cell surface by an insoluble transmembrane domain. Characterization and development of TCRs have been hampered by the lack of suitable methods for producing them as soluble and stable proteins. mTCR technology (Immunocore) enables the production of fully human, soluble TCRs and links them to an antibody fragment, anti-CD3, which can activate the immune system to kill the targeted cancer cells. ImmTACs (Immune mobilizing mTCR Against Cancer), which are bispecific biologics comprising a soluble, high-affinity TCR fused to a cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirect T cells to kill cancer cells expressing extremely low surface epitope densities (Liddy et al. 2012). ImmTACs potently suppress tumor growth in vivo and overcome immune tolerance to cancer. Lead product, IMCgp100, is currently in a phase I/II dose-finding clinical study in patients with late-stage malignant melanoma.

8.5 Antibody–Drug Conjugates

ADCs combine the high selectivity of MAbs with potency of small molecules to increase the anticancer effect. MAbs directed to TAAs or antigens differentially expressed on the tumor vasculature have been covalently linked to drugs that have different mechanisms of action and various levels of potency. The use of ADCs to selectively deliver drugs to tumors has the potential to both improve antitumor efficacy and reduce the systemic toxicity of therapy. Several ADCs, particularly those that incorporate internalizing antibodies and tumor-selective linkers, have demonstrated impressive activity in preclinical models. Gemtuzumab ozogamicin (Mylotarg, Pfizer), a calicheamicin conjugate that targets CD33, was approved by the FDA for the treatment of acute myelogenous leukemia but was withdrawn from the market later on.

ADCs, however, are not just a sum of their individual parts and several challenges need to be addressed. The target selection, the interaction of ADC with tumor and off-tumor targets, and the internalization of ADCs are critical for the effective maturation of ADC technology. Ongoing developments in attachment sites and linker chemistry can provide fine-tuning of drug loading, elements of ADC pharmacokinetics, and off-target ADC toxicity (Adair et al. 2012).

Sutro Biopharma is using its cell-free protein synthesis technology platform to design and develop new ADCs and bispecific antibodies for targeted cancer therapies of Celgene. Sutro’s biochemical synthesis technology enables rapid and systematic exploration of many protein-drug variants to identify drug candidates. The new treatments are aimed at significantly extending the clinical impact of current oncology therapeutic approaches beyond that of current cell-based expression technologies.

Spirogen’s ADC technology delivers extremely potent anticancer agents to cancer cells by attaching them to antibodies. Spirogen has developed highly potent warheads based on its proprietary pyrrolobenzodiazepines (PBDs) warheads joined to antibodies by linkers, which are stable in the bloodstream but release the PBD warhead once it is safely inside the targeted cancer cells. The naturally occurring PBDs, isolated from various Streptomyces species, bind covalently and sequence selectively to purine–guanine–purine motifs in the minor groove of DNA. The DNA-binding activity of the molecules interferes with DNA processes including transcription and replication, allowing them to act as antitumor and antibiotic agents. The PBD dimer SG2000 is currently undergoing phase II clinical trials for the treatment of cisplatin-resistant ovarian cancer.

Currently ~60 ADCs are in development for oncology including ~20 in clinical trials, most of which are tubulin inhibitor-based immunoconjugates (Sapra et al. 2011). Only two, Kadcyla and Adcetris, have been approved. Selected 15 clinical trials of ADC are shown in Table 8.3.
Table 8.3

Antibody–drug conjugates in clinical trials for cancer

Product

Target antigen/drug

Company

Indication

Phase

AGS-16M8F

AGS-16/auristatin

Agensys/Astellas Pharma

Renal cell cancer

Phase I

AGS-5ME

AGS-5/auristatin

Agensys/Astellas Pharma

Prostate, pancreatic, gastric cancers

Phase I

BAY 79-4620 (CAIX/ADC)

MN carbonic anhydrase IX (CAIX)/auristatin

Bayer Pharma/MorphoSys

Solid cancers expressing CAIX

Phase I

BAY 94-9343 (mesothelin-ADC)

Mesothelin/maytansinoid TAP

Bayer Pharma/ImmunoGen Inc.

Mesothelioma

Phase I orphan

BIIB015

Cripto/maytansinoid

Biogen/ImmunoGen Inc.

Cripto-positive solid tumors

Phase I

BT-062

CD138/maytansinoid

Biotest/ImmunoGen Inc.

Multiple myeloma

Phase I

Glembatumumab vedotin (CDX-011)

Glycoprotein NMB (GPNMB)/auristatin

Celldex Therapeutics

Breast cancer expressing GPNMB

Phase II

IMGN-388

Integrin αv/maytansinoid

ImmunoGen Inc.

Solid tumors

Phase I

Inotuzumab ozogamicin (CMC-544)

CD22/calicheamicin

Pfizer

Non-Hodgkin lymphoma

Phase III

Lorvotuzumab mertansine (IMGN-901)

CD56/maytansinoid

ImmunoGen Inc.

Multiple myeloma, Merkel cell carcinoma, ovarian cancer

Phase I/II

MDX-1203

CD70/duocarmycin

Medarex/Bristol-Myers Squibb

Renal cell cancer

Phase I

PSMA ADC

PSMA/auristatin

Progenics Pharmaceuticals Inc.

Prostate cancer

Phase I

SAR3419

CD19/maytansinoid

Sanofi-Aventis

Non-Hodgkin lymphoma

Phase II

SG2000

Antibodies/pyrrolobenzodiazepines

Spirogen Ltd.

Cisplatin-resistant ovarian cancer

Phase II

SGN-75

CD70/auristatin

Seattle Genetics

Renal cell cancer, non-Hodgkin lymphoma

Phase I

© JainPharmaBiotech

ADCs are predicted to become an important class of cancer therapeutics as evidenced by the promising objective response rates when administered to chemorefractory cancer patients. Further improvements are being made to ADC design, and a third generation of agents is already emerging. Mersana Therapeutics is focusing on developing ADCs using its Fleximer polymer backbone and customized linkers to optimally link MAb and drug.

8.5.1 Ado-Trastuzumab Emtansine

Ado-trastuzumab emtansine (Roche/Genentech’s Kadcyla) was the first FDA-approved ADC for treating HER2-positive metastatic breast cancer, an aggressive form of the disease. Ado-trastuzumab emtansine is made up of the antibody, trastuzumab, and the chemotherapeutic, mertansine (DM1), joined together using a stable linker. It combines the mechanisms of action of both trastuzumab and DM1. Genentech has studied ADC science for more than a decade and has several ADCs in clinical trials for different types of cancer as well as >25 candidates in pipeline. Results of a randomized clinical trial, EMILIA study, showed that T-DM1 significantly prolonged progression-free and overall survival (~6 m) with less toxicity than lapatinib plus capecitabine in patients with HER2-positive advanced breast cancer previously treated with trastuzumab and a taxane (Verma et al. 2012).

8.5.2 Brentuximab Vedotin

Brentuximab vedotin (Seattle Genetics’ Adcetris) consists of an anti-CD30 antibody, a cell membrane protein of the TNF family, conjugated to the antimitotic agent monomethyl auristatin E (Bradley et al. 2013). It was approved by the FDA for the treatment of Hodgkin lymphoma (HL) and systemic anaplastic large-cell lymphoma (ALCL) in 2011. Approval was based on two single-arm multicenter clinical trials of patients with CD30-positive HL after failure of autologous stem cell transplant and patients with CD30-positive systemic ALCL who had previously received chemotherapy. The objective response rates were 73 % and 86 %, respectively, while the complete remission rates were 32 % and 57 %, respectively, and the partial remission rates were 40 % and 29 %, respectively. Adcetris is also in development for a range of other CD30-expressing lymphoma and non-lymphoma malignancies, both as monotherapy and in combination with chemotherapy.

8.6 Current Status and Future Trends in MAb-Based Anticancer Drugs

An analysis of the current commercial clinical pipeline of MAb candidates for cancer revealed trends toward the development of a variety of noncanonical MAbs, including ADCs, bispecific antibodies, engineered antibodies, and antibody fragments and/or domains (Reichert and Dhimolea 2012). The authors found substantial diversity in the antibody sequence source, isotype, carbohydrate residues, targets, and mechanisms of action. Although well-validated targets, such as EGFR and CD20, continue to provide opportunities for companies, there were notable trends toward targeting less well-validated antigens and exploration of innovative mechanisms of action such as the generation of anticancer immune responses or recruitment of cytotoxic T cells.

References

  1. Adair JR, Howard PW, Hartley JA, et al. Antibody-drug conjugates – a perfect synergy. Expert Opinion on Biological Therapy 2012;12:1191-206.CrossRefGoogle Scholar
  2. Almokadem S, Belani CP. Volociximab in cancer. Expert Opin Biol Ther 2012;12:251-7.CrossRefGoogle Scholar
  3. Bradley AM, Devine M, DeRemer D. Brentuximab vedotin: an anti-CD30 antibody-drug conjugate. Am J Health Syst Pharm 2013;70:589-97.CrossRefGoogle Scholar
  4. Gerber DE, Stopeck AT, Wong L, et al. Phase I safety and pharmacokinetic study of bavituximab, a chimeric phosphatidylserine-targeting monoclonal antibody, in patients with advanced solid tumors. Clin Cancer Res 2011;17:6888-96.CrossRefGoogle Scholar
  5. Heimberger AB, Sampson JH. The PEPvIII-KLH (CDX-110) vaccine in glioblastoma multiforme patients. Expert Opin Biol Ther 2009;9:1087-98.Google Scholar
  6. Judy BF, Aliperti LA, Predina JD, et al. Vascular endothelial-targeted therapy combined with cytotoxic chemotherapy induces inflammatory intratumoral infiltrates and inhibits tumor relapses after surgery. Neoplasia 2012;14:352-9.Google Scholar
  7. Liddy N, Bossi G, Adams KJ, et al. Monoclonal TCR-redirected tumor cell killing. Nat Med 2012;18:980-7.CrossRefGoogle Scholar
  8. Milowsky MI, Nanus DM, Kostakoglu L, et al. Vascular targeted therapy with anti-prostate-specific membrane antigen monoclonal antibody J591 in advanced solid tumors. J Clin Oncol 2007;25:540-7.CrossRefGoogle Scholar
  9. Ortiz-Sanchez E, Helguera G, Daniels TR, Penichet ML. Antibody–cytokine fusion proteins: applications in cancer therapy. Expert Opin Biol Ther 2008;8:609-632.CrossRefGoogle Scholar
  10. Popkov M, Rader C, Gonzalez B, et al. Small molecule drug activity in melanoma models may be dramatically enhanced with an antibody effector. Int J Cancer 2006;119:1194-207.CrossRefGoogle Scholar
  11. Reichert JM, Dhimolea E. The future of antibodies as cancer drugs. Drug Discov Today 2012;17:954-63.CrossRefGoogle Scholar
  12. Royer B, Yin W, Pegram M, et al. Population pharmacokinetics of the humanised monoclonal antibody, HuHMFG1 (AS1402), derived from a phase I study on breast cancer. Br J Cancer 2010;102:827-32.CrossRefGoogle Scholar
  13. Sapra P, Hooper AT, O’Donnell CJ, Gerber HP. Investigational antibody drug conjugates for solid tumors. Expert Opin Invest Drugs 2011;20:1131-49.CrossRefGoogle Scholar
  14. Scott AM, Gill SS, Lee F, et al. Journal of Clinical Oncology, 2006 ASCO Annual Meeting Proceedings 2006;Vol 24 Part I (Supplement 18S): Abstract No.13028.Google Scholar
  15. Scott AM, Allison JP, Wolchok JD. Monoclonal antibodies in cancer therapy. Cancer Immun 2012;12:14.Google Scholar
  16. Verma S, Miles D, Gianni L, et al. Trastuzumab Emtansine for HER2-Positive Advanced Breast Cancer. N Engl J Med 2012;367:1783-91.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Kewal K. Jain
    • 1
  1. 1.Jain PharmaBiotechBaselSwitzerland

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