Antibodies also function as carriers of cytotoxic agents, such as radioisotopes, drugs, and toxins (Fig. 3). In most settings, antibody conjugates are found to be more potent to cancer cells, yet also more toxic to collateral normal tissues, than unconjugated antibodies. In lymphomas, anti-CD20 radioimmunoconjugates have superior anti-tumor activity compared to their unconjugated antibody counterparts, but there is increased concomitant, but manageable, hematological toxicity (Witzig et al. 2002; Davis et al. 2004). The development and prospects of cancer therapy with radiolabeled antibodies, termed radioimmunotherapy, or RAIT (Goldenberg 1978a, b, 1980), will be reviewed.

Radiolabeled antibodies were first investigated in the 1950s (Pressman and Keighley 1948; Pressman and Korngold 1953; Bale and Spar 1957; Bale et al. 1955), and laid the foundation for subsequent studies undertaken with human tumor xenografts and human tumor antigens in the 1970s (Bale and Spar 1957; Silverstein 2004). The first human cancer target for radiolabeled antibodies was carcinoembryonic antigen (CEA), which was first disclosed by Gold and Freeman as a specific antigen for gastrointestinal cancers (Gold and Freedman 1965), and resulted also in a serum diagnostic assay (Gold et al. 1978; Thomson et al. 1969). ¹³¹I-labeled antibody to CEA provided the first clinical evidence that radioactivity could be selectively delivered to human tumors for cancer detection by external imaging (Goldenberg et al. 1978), and later for human colonic cancer therapy in an experimental model (Goldenberg et al. 1981). Early therapy trials by Order et al. (Order et al. 1981; Ettinger et al. 1982) integrated a relatively low dose of a radiolabeled antibody into a standard chemotherapy and radiation regimen, but this did not disclose the activity of the radiolabeled antibody alone. While the emphasis in clinical studies in the early 1980s was for cancer detection by nuclear imaging, therapy studies with radiolabeled antibodies alone were initiated (Carrasquillo et al. 1984; Montz et al. 1986; Goldenberg 1988). The first clinical milestone in RAIT was the report by DeNardo et al. (1987), showing that radiolabeled antibody therapy alone could elicit responses in patients with B-cell lymphomas. This stimulated the development of other antibodies for the therapy of this radiosensitive neoplasm, ultimately leading to the approval of two radiolabeled anti-CD20 antibodies for the treatment of non-Hodgkin lymphoma (NHL).

External beam radiation delivers high-dose-rate radiation for short bursts divided over several weeks, whereas RAIT is typically given as an intravenous injection, attempting to deliver low-dose radiation to tumor systemically. It can take 1–2 days before a radiolabeled IgG achieves its maximum tumor accretion, with peak uptake usually <0.01 % of the injected dose per gram tumor; however, the radioactivity deposited in the tumor can be detected for several weeks (Sharkey and Goldenberg 2005). The low uptake and slow kinetics of radiolabeled IgG targeting not only means that RAIT delivers a lower dose rate than external beam irradiation, but that it is continually present for a period determined by the physical half-life of the radionuclide and the biological half-life of the antibody in the tumor. This continuous, low-dose-rate radiation exposure can be at least as effective as intermittent, high-dose rate, external irradiation (Buchsbaum and Roberson 1996; Hernandez and Knox 2004).

Since the radiolabeled IgG remains in the blood for an extended time, the highly radiosensitive red bone marrow is continuously exposed to the radiation, causing the dose-limiting myelosuppression typical for RAIT. In the more radiosensitive hematological malignancies, this can elicit significant anti-tumor responses. However, solid tumors, which are more radioresistant, are less affected by such doses. In an attempt to reduce the duration in the blood, the use of smaller forms of the antibodies, such as a F(ab’)₂ or Fab’, and more recently, molecularly engineered antibody sub-fragments, have been explored (Fig. 1). These fragments have more favorable pharmacokinetic properties that improve the tumor/blood ratios because of having a more rapid blood clearance (Kenanova and Wu 2006). The fraction of the injected activity delivered to the tumor is lower with an antibody fragment than an IgG, but because hematological toxicity is reduced, higher doses of radioactivity can be administered. While there have been some reports of improved therapeutic responses using smaller-sized antibodies, the smaller forms, <60,000 daltons, clear from the blood by the kidneys, which could lead to renal toxicity, especially with radiometals (Behr et al. 1999, 2000; Buchegger et al. 1996). Thus, while radiolabeled fragments reduce the risk of hematological toxicity, because the radiation dose required to kill tumors is higher than the limit tolerated to the kidney, radiometal-labeled antibody fragments that are unable to sustain a higher level in the tumor than the kidneys may never prove to be effective therapeutics. In contrast, radioiodinated fragments are not retained in the kidneys, and therefore a favorable therapeutic window potentially can be achieved (Sharkey et al. 1990; Kenanova et al. 2007).

Multi-step pretargeting methods, such as those using bispecific antibodies (discussed in “Radioimmunothera​py of Tumors:​ Pretargeting with Bispecific Antibodies”), represent a promising new method for imaging and therapy (Sharkey et al. 2005). This involves separating the targeting of the tumor with an antibody from the delivery of radiation either for imaging or for RAIT. The bispecific antibody, which is given in the first step, has at least one arm that binds to the tumor antigen, while a second binds to a hapten. The hapten, which is then given later, is typically incorporated in a small peptide that can be radiolabeled. The unlabeled bispecific antibody given first circulates and binds to the tumor cells, and once it has cleared from the blood and non-targeted tissues, the radiolabeled peptide is given. The small-sized radiolabeled peptide enters the extravascular space very quickly, where it binds to the other arm of the bispecific antibody found on the tumor cells. Within minutes, the non-targeted peptide clears from the blood, typically by urinary excretion within a few hours, while the peptide that localized to the bispecific antibody remains bound to the tumor. This method has been shown in xenograft models to enhance tumor/blood ratios by as much 40-fold, while increasing tumor accretion by as much as 10-fold compared to a directly radiolabeled antibody fragment (Sharkey et al. 2005; McBride et al. 2006). We have found that this pretargeting can increase the total radiation delivered to tumors 1.5-fold, and because uptake occurs so quickly, the dose rate is also increased 3-fold, resulting in improved therapeutic responses (Karacay et al. 2005). Also important is that the small radiolabeled peptide does not have as high accretion in the kidney as found with directly radiolabeled antibody fragments; therefore, tumor/kidney ratios are favorable.

Table 3 lists some of the more commonly used radionuclides bound to antibodies for cancer RAIT. Medium-energy beta-emitters, such as ¹³¹I (~0.5 MeV) and ¹⁷⁷Lu (~0.8 MeV), traverse ~0.5 to 1.0 mm; high-energy beta-emitters, such as ⁹⁰Y or ¹⁸⁸Re (~2.1 MeV), penetrate up to 11 mm, thus enabling beta-emitters to kill several hundred cells in their path, which is referred to as the bystander, or crossfire effect (Kassis and Adelstein 2005). This is an important attribute of RAIT as compared to other immunoconjugates, since the radioimmunoconjugates can be therapeutic even with heterogeneity of antigen expression. Thus, radionuclide selection is most related to tumor size and extent. Higher energy beta-emitters can kill across a longer path-length, but the absorbed fraction is higher for the lower energy beta-emitters (i.e., probability of hitting the nuclear DNA), making them more efficient for cell destruction. Alpha-emitters, such as ²¹³Bi and ²¹¹At, have path lengths of only a few cell diameters, but are also more potent than a low-energy beta particle, requiring fewer “hits” to effect cell killing (Kassis and Adelstein 2005). Low-energy electrons, such as Auger emitters (e.g., ¹²⁵I, ⁶⁷Ga, and ¹¹¹In), have to be in close contact, preferably in the cell or near the nucleus, to be cytotoxic (Mattes 2002). Thus, beta emitters are best used where tumors are ≥0.5 cm in diameter; otherwise, a substantial portion of the energy from the radioactive decay will affect surrounding normal tissues. Alpha and low-energy electron emitters are preferred when there is a small disease burden or with single-cell populations (e.g., leukemia, malignant ascites) (Michel et al. 2003; Kotzerke et al. 2005).

Two radioimmunoconjugates are currently approved in the USA for the treatment of indolent and transformed forms of NHL, ⁹⁰Y-ibritumomab tiuxetan (Zevalin, Spectrum) and ¹³¹I-tositumomab (GlaxoSmithKline) (Sharkey et al. 2005). Both have shown higher objective response rates compared to the unlabeled anti-CD20 antibody used alone (Witzig et al. 2002; Davis et al. 2004). Nevertheless, the pivotal trial comparing ⁹⁰Y-ibritumomab tiuxetan to rituximab did not show a statistical improvement in the duration of the response, but further follow-up has shown that the complete responses, which were more prevalent with 90Y-ibritumomab tiuxetan, have also been more durable (Gordon et al. 2004a, b). ¹³¹I-tositumomab has also shown durable responses, and when used as a front-line therapy, it is better tolerated and may improve responses compared with standard chemotherapy (Fisher et al. 2005; Kaminski et al. 2005; Buchegger et al. 2006). Clinical studies are also evaluating RAIT as a front-line treatment, and that these treatments do not preclude the use of cytotoxic therapies, and in fact may be used in concert as part of a conditioning regimen or consolidation therapy (Bethge et al. 2010; Chatal et al. 2008; Devizzi et al. 2008; Jacobs et al. 2008; Krishnan et al. 2008; Morschhauser et al. 2008; Shimoni et al. 2008; Zhang and Gopal 2008; Zinzani et al. 2008a, b, c; Esmaeli et al. 2009; Hainsworth et al. 2009; Morschhauser et al. 2009; Perrotti et al. 2009; Wang et al. 2009; Burdick et al. 2010; Cicone et al. 2010; Goldsmith 2010; Hoffmann et al. 2010; Hohloch et al. 2010; Kang et al. 2010; Leahy and Turner 2010; Zinzani et al. 2010a, b). However, more randomized clinical trials are needed to assess long-term follow-up and also the risk of late toxicities (e.g., myelodysplasia or leukemia) (Connors 2005). Although there has been some concern about the incidence of late toxicities, there is increasing evidence that RAIT alone might not have been the underlying cause, particularly because many of the patients enrolled on these studies have had extensive prior therapies (Magni et al. 2010; Bennett et al. 2005; Czuczman et al. 2007; Focosi et al. 2008; Friedberg 2008; Link et al. 2010; Roboz et al. 2007; Sacchi et al. 2008). It should be emphasized that, because the red marrow is so sensitive to radiation, these agents are restricted to use in patients with minimal bone marrow involvement and good hematologic status. Indeed, a Phase II trial using ⁹⁰Y-ibritumomab tiuxetan in chronic lymphocytic leukemia (CLL) concluded RAIT had significant toxicities, preventing it from being able to provide sufficient benefit in this setting (Jain et al. 2009).

Other radioimmunoconjugates also are being studied in lymphoma and leukemia (DeNardo et al. 1998; Foss et al. 1998; McDevitt et al. 2001; Buchmann et al. 2002; Ma et al. 2002; Zhang et al. 2002, 2006, 2007; Vallera et al. 2003; Glatting et al. 2006) (Table 4). Promising anti-tumor responses have been reported from a Phase I/II trial with a fractionated, weekly dosing of ⁹⁰Y-epratuzumab tetraxetan (anti-CD22 IgG) in both follicular and other NHL (mantle cell and diffuse large B cell) (Morschhauser et al. 2010). Twelve out of 13 patients (92 %) with follicular NHL who received >30 mCi of the ⁹⁰Y-antibody in their treatment had a complete response; one had a partial response. The median progression-free survival of this group was 24.6 months, with half still in remission 2–3 years of follow-up. Equally impressive was the fact that 10 of these follicular NHL patients were refractory to a prior anti-CD20 therapy, and 9 of these had complete responses lasting 21.5 months, which is longer than the 6.8-month progression-free survival in rituximab-resistant follicular lymphoma patients given ⁹⁰Y-ibritumomab tiuxetan (Witzig et al. 2002). The non-follicular group had an overall response of 58 % (11/19), with six being complete or complete/unconfirmed responses, which is also encouraging. Preclinical studies have also suggested a combination of ⁹⁰Y-anti-CD22 with anti-CD20 antibody therapy may further enhance the clinical response (Mattes et al. 2008). For example, the standard radiolabeled anti-CD20 IgG therapy includes substantial amounts of unlabeled anti-CD20 therapy that is pre-infused before the therapeutic dose of the radioimmunoconjugate, which is given at a relatively low protein dose. While clinical studies concluded that unlabeled anti-CD20 antibody aided tumor localization of the radiolabeled anti-CD20 antibody by blocking the large antigen sink in the spleen or in patients with bulky disease, the rationale for using the currently prescribed amount may be related more to early clinical findings indicting the higher antibody dose enhanced the response even before the therapeutic radioimmunoconjugate was given (Sharkey et al. 2009). Preclinical studies have shown anti-CD20 antibody-induced signaling can enhance the cell’s susceptibility to radiation (Kapadia et al. 2008; Skvortsova et al. 2005, 2006), but also anti-CD20 therapy is itself effective. Thus, combining a radiolabeled anti-CD22 antibody therapy with an anti-CD20 antibody therapy would allow both agents to bind optimally to the tumor, with the anti-CD20 therapy given in advance having the ability to reduce some of the normal B cells that could affect the radiolabeled anti-CD22 targeting, and also initiate signaling events in the tumor that might enhance response.