Monoclonal Antibody and Peptide-Targeted Radiotherapy of Cancer

Raymond M. Reilly

• Publisher: Wiley
• Year: 2010
• Print ISBN:
  9780470243725
• eBook ISBN:
  9781118035153
• Edition: 1
• Topic: Scienze fisiche
• Subtopic: Chimica industriale e tecnica

Oncology Book of 2011, British Medical Association's Medical Book Awards

Awarded first prize in the Oncology category at the 2011 BMA Medical Book Awards, Monoclonal Antibody and Peptide-Targeted Radiotherapy of Cancer helps readers understand this hot pharmaceutical field with up-to-date developments. Expert discussion covers a range of diverse topics associated with this field, including the optimization of design of biomolecules and radiochemistry, cell and animal models for preclinical evaluation, discoveries from key clinical trials, radiation biology and dosimetry, and considerations in regulatory approval. With chapters authored by internationally renowned experts, this book delivers a wealth of information to push future discovery.

Chapter 1

Antibody Engineering: Optimizing the Delivery Vehicle

Diane E. Milenic

1.1 Introduction

The progression of monoclonal antibodies (MAbs) for radioimmunotherapy (RIT) has been driven by the need to solve a series of problems. As variants of antibodies have been developed and evaluated in preclinical studies, opportunities and limitations have become evident. Recent advances in DNA technology have led to the ability to tailor and manipulate the immunoglobulin (Ig) molecule for specific functions and in vivo properties. This chapter discusses the use of monoclonal antibodies for radiotherapy with an emphasis on the problems that have been encountered and the subsequent solutions.

The exploration of monoclonal antibodies as vehicles for the delivery of radionuclides for therapy has been ongoing for almost 50 years [1]. In 1948, Pressman and Keighley reported the first in vivo use of a radiolabeled antibody for imaging [2]. Ten years later, the first report of radiolabeled tumor-specific antibodies was utilized for radioimmunodiagnosis, and in 1960, radiolabeled antibodies were used to selectively deliver a therapeutic dose of radiation to tumor tissue [1, 3]. Even at these early stages, investigators were quick to realize the obstacles associated with utilizing antibodies for radioimmunotherapy. Radiation doses delivered to tumors in patients were too low to have significant effects on tumor growth, and the prolonged retention of the radiolabeled antibodies in the blood led to toxicity complications [4]. The inherent heterogeneity in specificity and affinity of polyclonal antibodies resulted in in vivo variability. The advent of hybridoma technology and the ability to generate monospecific, monoclonal antibodies produced a resurgence in the use of antibodies as “magic bullets” [5, 6]. In the 1980s, the literature exploded with reports of radiolabeled MAbs being evaluated in the clinical setting, initially in radioimmunodiagnostic applications, confirming that MAbs against tumor-associated antigens could target tumors in patients. Subsequently, RIT clinical trials were initiated to deliver systemically administered radiation to tumors with a specificity that would spare normal tissues from damage [7]. This optimistic viewpoint was quickly tempered by the realization of the obstacles inherent to the use of a biological reagent, especially one of xenogeneic origin.

The preclinical and clinical RIT trials exposed the major constraints to the successful clinical use of radiolabeled MAbs: (i) development of human anti-murine immunoglobulin antibodies (HAMA); (ii) inadequate (low) therapeutic levels of radiation doses delivered to tumor lesions; (iii) slow clearance of the radiolabeled MAbs (radioimmunoconjugates) from the blood compartment; (iv) low MAb affinity and avidity; (v) trafficking to, or targeting of, the radioimmunoconjugates to normal organs; (vi) and insufficient penetration of tumor tissue [8, 9]. In addition, there were toxicities associated with conjugated radionuclides when the radioimmunoconjugates were metabolized or when the radionuclide dissociated from the immunoconjugate [9]. With these problems in mind, a primary focus has been to optimize RIT by manipulating the MAb molecule. As technology permitted, this was initially accomplished with chemical or biochemical techniques to generate a variety of immunoglobulin forms but is now predominated by genetic engineering.

1.2 Intact Murine Monoclonal Antibodies

In May 2008, a perspective on MAbs by Reichert and Valge-Archer [10] reported that in the periods 1980–1989, 1990–1999, and 2000–2005, 37, 25, and 8 murine MAbs, respectively, were evaluated in the clinic as cancer therapeutics. During this entire 25-year period, radiolabeled MAbs comprised 33% of the murine MAbs [10]. To date, only two radiolabeled murine (mu) MAbs, both targeting CD20, have received FDA approval. Zevalin, ⁹⁰Y-rituxan (ibritumomab-tiuxetan), was approved in 2002 and is indicated for relapsed or refractory low-grade follicular transformed non-Hodgkin's lymphoma (NHL). The overall response rate of patients is reported to be 80%; 46% for those with rituximab refractory disease [11]. Bexxar (¹³¹I-tositumomab) was approved in 2003 for the treatment of non-Hodgkin's B-cell lymphoma in rituximab refractory patients (see Chapter 6). Objective responses following ¹³¹I-tositumomab therapy have ranged from 54% to 71% in patients who have undergone previous therapies while for newly diagnosed patients the response rates are 97% with 63% of those experiencing a complete response [12].

In clinical trials using muMAbs for RIT of solid tumors, approximately 73% (ranging from 16% to 100%) of the patients developed HAMA following a single infusion of MAb [13]. In contrast, only about 42% of the patients in RIT trials for treatment of hematologic malignancies develop HAMA. When multiple doses of a radioimmunoconjugate have been administered, the amount of MAb that effectively targets tumor tissue is usually compromised after the second administration [13]. In general, the human antibody response, especially at earlier time points, is directed against the Fc portion of the MAb molecule (Fig. 1.1). With the passage of time and particularly after repeated infusions, the specificity of the human antibody response matures and becomes increasingly specific for the variable region of the MAb [13]. In some instances, anti-variable region antibodies develop after a single infusion of the MAb [13, 14]. This response has the potential of directly inhibiting the ability of the injected MAb from interacting with the targeted tumor [14]. As with any therapeutic regimen, for RIT to be effective, multiple treatment cycles will be necessary. Immunomodulatory drugs such as deoxyspergualin, cyclosporin A, or cyclophosphamide have been evaluated as a means of minimizing or suppressing a patient's immune response during RIT [15].

Figure 1.1 Schematic of an immunoglobulin structure. Enzymatic digestion of the intact IgG molecule yields F(ab′)₂ and Fab fragments.

To address these challenges of MAb-directed therapy, several strategies have been employed that center around modifying the MAb molecule. These alterations include reduction in the size of the MAb molecule, deglycosylation, or the addition of side groups. Reduction in size of the MAb molecule has been accomplished through methods such as enzymatic cleavage or genetic engineering [16–18]. Digestion of an antibody with pepsin removes the Fc region of the heavy chain on the carboxyl terminus of cysteamine producing F(ab′)₂ fragments that retain two antigen binding sites and have a molecular weight of ∼100 kDa (Fig. 1.1). Fab fragments are generated by digestion with papain, an enzyme with a specificity for the amino group of cysteines. In this case, the disulfide bridges between the heavy chains are removed with the Fc region, which results in a molecule (M_r ∼ 50 kDa) with one antigen binding site. Fab′ fragments are produced through reduction and alkylation of F(ab′)₂, which also yields a MAb molecule with a single antigen binding site and an M_r of ∼50 kDa [16–18]. Comparisons of intact MAbs and F(ab′)₂ fragments (Fig. 1.1) in RIT clinical trials have demonstrated that the F(ab′)₂ fragments do have a shorter serum half-life than intact MAbs. Patient antibody responses against F(ab′)₂ fragments appear to occur with lower frequency after a single administration of the radioimmunoconjugate. Furthermore, some objective responses to treatment with a radiolabeled F(ab′)₂ fragment have been observed [19, 20]. Autoradiographic studies of radiolabeled MAbs administered to athymic mice bearing human tumor xenografts have illustrated the ability of Fab′ and F(ab′)₂ fragments to penetrate tumor tissue with greater efficiency than intact MAbs [20, 21]. The pharmacokinetics of Fab or Fab′ fragments is even more rapid than F(ab′)₂ fragments (t_½α ∼ 10 min, t_½β ∼ 1.5 h for Fab′ fragments versus t_½α ∼ 30 min, t_½β ∼ 12 h for F(ab′)₂ fragments) [22]. In general, Fab and Fab′ fragments have proven to be less immunogenic than intact MAbs [23]. Their greatest disadvantage for RIT applications is their high and persistent renal localization, which appears to be a function of molecular size [22], which greatly increases the risk for renal toxicity. The degree to which the radiolabel is retained in the kidneys depends on the radionuclide and the radiolabeling chemistry (see Chapter 2). Radioiodinated MAbs are rapidly dehalogenated and the radioiodine excreted via the kidneys or into the stomach and intestines. Free radioiodine is trapped in the thyroid gland if there is inadequate blocking with stable iodine. Chelated radiometallonuclides, that is, ¹¹¹In, ⁹⁰Y, and ¹⁷⁷Lu, are not as readily eliminated from normal tissues when the radioimmunoconjugate is metabolized [24]. The retention of radiometals in the kidneys is due to the reabsorption of antibody fragments after their glomerular filtration followed by degradation of the radioimmunoconjugates with trapping of radioactive metabolites within the renal tubular cells [22, 24, 25]. Although they are readily eliminated from the body, radioiodines may also pose a concern for toxicity to renal tissue, depending on the dose of radioactivity administered. An effective means of enhancing renal excretion of the radioimmunoconjugates is the blocking of its readsorption from the luminal fluid in the proximal tubules by administering basic amino acids such as lysine or arginine, prior to or with the radiolabeled MAb fragment [26, 27].

Fragments of MAb that retain immunoreactivity, however, are often difficult to generate [22]. As mentioned, they are prepared by proteolytic digestion of intact MAb using enzymes, a procedure that must be optimized for each MAb and usually requires threefold or more MAbs to obtain the final desired quantity of the fragment. The process is inefficient and costly when producing the amounts necessitated by a RIT clinical trial.

1.3 Recombinant Immunoglobulin Molecules

Antibodies consist of four polypeptide chains, two heavy and two light chains, connected by disulfide bonds; the heavy chains are glycosylated (Fig. 1.1). Several criteria must be met to generate and produce genetically engineered antibodies. First, a host cell is needed that would produce and secrete a properly assembled functional antibody molecule with the appropriate carbohydrate side chains. Second, the DNA must be introduced into the recipient cell in an efficient manner. Finally, expression vectors must be available that permit the expression of the introduced genes as well as the isolation of the cells expressing the introduced antibody genes [28]. The vectors require a plasmid origin for replication, a gene encoding a selectable biochemical phenotype in bacteria and a gene encoding a selectable marker in eukaryotic cells. The creation of recombinant immunoglobulin molecules also requires the transfection of the host cell with two expression vectors, one containing the gene for the heavy chain and the other containing the gene that encodes the light chain.

1.3.1 Chimeric Monoclonal Antibodies

Chimeric MAbs are constructed by ligating the gene encoding the variable region of a murine MAb to the gene encoding the constant region of a human Ig (Fig. 1.2). There are a variety of vectors available into which the murine and human Ig gene sequences can be inserted. In turn, there are a number of expression systems, prokaryotic and eukaryotic, into which the recombinant Ig genes can be introduced and the protein expressed [28, 29]. The ability to tailor a MAb of a particular specificity for a certain function broadens the horizon for MAb-directed therapies.

Figure 1.2 The “humanization” of the murine IgG to generate forms with increasing percentages of human sequences.

The first clinical trial involving a recombinant/chimeric MAb employe...

Table of contents

• Cover
• Title Page
• Copyright
• Dedication
• Preface
• Contributors
• Chapter 1: Antibody Engineering: Optimizing the Delivery Vehicle
• Chapter 2: The Radiochemistry of Monoclonal Antibodies and Peptides
• Chapter 3: The Design of Radiolabeled Peptides for Targeting Malignancies
• Chapter 4: Peptide Receptor Radionuclide Therapy in Patients with Somatostatin Receptor-Positive Neuroendocrine Tumors
• Chapter 5: Targeted Radiotherapy of Central Nervous System Malignancies
• Chapter 6: Radioimmunotherapy for B-Cell Non-Hodgkin Lymphoma
• Chapter 7: Radioimmunotherapy of Acute Myeloid Leukemia
• Chapter 8: Pretargeted Radioimmunotherapy of Cancer
• Chapter 9: Targeted Auger Electron Radiotherapy of Malignancies
• Chapter 10: Viral Introduction of Receptors for Targeted Radiotherapy
• Chapter 11: Preclinical Cell and Tumor Models for Evaluating Radiopharmaceuticals in Oncology
• Chapter 12: Radiation Biology of Targeted Radiotherapy
• Chapter 13: Dosimetry for Targeted Radiotherapy
• Chapter 14: The Bystander Effect in Targeted Radiotherapy
• Chapter 15: The Role of Molecular Imaging in Evaluating Tumor Response to Targeted Radiotherapy
• Chapter 16: The Economic Attractiveness of Targeted Radiotherapy: Value for Money?
• Chapter 17: Selected Regulatory Elements in the Development of Protein and Peptide Targeted Radiotherapeutic Agents
• Color Plates
• Index