The clinical utility of naked MAb has been limited by both host (number and activity of effector cells, FcR polymorphism, and interference by inhibitory FcR) and tumor factors (antigen heterogeneity and com plement regulatory proteins). While the CMC and ADCC functions of naked MAb (Fig. 14.1) can be improved by altering the Fc protein structure or by modifying Fc-glycosylation, substantial gains in the clinical potentials of MAb can be achieved using immunoconjugates (Cheung 2004) (Fig. 14.1); these include (a) radioimmunoconjugates to deliver band a- emitters (Goldenberg 2003), (b) immunocyto-kines to deliver cytokines to tumor sites while minimizing systemic toxicities (Davis and Gillies 2003), (c) immunotoxins (Pastan 2003), (d) immunolipo-somes to deliver drugs or toxins (Allen et al. 2002), and (e) bispecific MAb (pretargeted to tumor or by ex vivo arming) to direct cells or ligands selectively to tumor (van Spriel et al. 2000).
MAb have the potential to target and ablate tumors in radioimmunotherapy (RIT) (Cheung 2004; Goldenberg 2003). In preclinical models, ablation of established xenografts is possible (Cheung et al. 1986), although radiation damage to the marrow remains dose limiting. Unlike naked antibodies, the bystander effect of RIT from cross-firing of the radioisotopes accounts for most of the toxicities of radioimmuno-conjugates, hence limiting their efficacy. Most clinical applications of RIT utilize b-emitting radioimmunoconjugates. b-particles have a relatively long range (0.8-5 mm) and low linear energy transfer (approximately 0.2keV/pm), resulting in radiation to both antigen-negative tumors as well as innocent bystanders; thus, b-emitters (131I or 90Y) can treat bulky diseases effectively but are not optimal for the killing of single cells or micrometastasis. Because of its g-emission, 131I also permits dosimetry studies, although it also poses a radio-hazard at high treatment doses, necessitating patient isolation. 131I is also dehalogenated in vivo with potential to damage the thyroid gland. 90Y is a pure b-emitter; its lack of g-emissions allows outpatient treatment. However, 90Y, which is a pure b-emitter, requires more extensive chemical modification of the MAb than 131I and is deposited in bone when dissociated from the complex. Alpha particles are helium nuclei. When compared with b-particles, they have a shorter range (50-80 mm) and a higher linear energy transfer (ap proximately 100keV/pm) (McDevitt et al. 1998). As few as one or two a-particles can destroy a target cell. RIT using a-emitters should result in less nonspecific toxicity to normal bystanders as well as more efficient single cell killing, an ideal setting for controlling minimal residual disease. Alpha-particle-emitting isotopes, such as astatine-211 and bismuth-213,have been tested in clinical trials with minimal ex-tramedullary toxicities (Zalutsky and Vaidyanathan 2000; Jurcic et al. 2002).
UJ13A (anti-NCAM) was the first antibody to undergo clinical testing for radioimaging and radio-immunotherapy (Lashford et al. 1987). 131I-3F8 (anti-GD2, 6-28 mCi/kg) achieved responses in both soft tissue masses and bone marrow (Larson et al. 2000). The use of myeloablative 131I-3F8 (20 mCi/kg) to consolidate remission was tested in patients (>1 year of age) newly diagnosed with stage-4 NB (Cheung et al. 2001a). Extramedullary toxicities were limited to hypothyroidism, which occurred despite aggressive thyroid protection using potassium iodide, liothyro-nine (T3), and potassium perchlorate. 131I-MAb was also tested in RIT of leptomeningeal cancers in children by intraventricular administration (Lashford et al. 1988; Kramer et al. 2000). Estimated radiation doses of 14.9-56 cGy/mCi to the cerebrospinal fluid were achieved with 131I-3F8, with less than 2 cGy/mCi to blood and other organs outside the CNS (Kramer et al. 2000).
Multistep Targeting (MST). In order to improve tumor uptake and reduce systemic toxicity, a multistep procedure which pretargets the antibody before the binding of the cytotoxic ligand to the tumor has been successfully employed (Cheung 2004; Goldenberg 2003; Boerman et al. 2003; Goldenberg et al. 2003; Paganelli et al. 2001; Cremonesi et al. 1999; Cheung et al. 2004) (Fig. 14.1). In the first step, an anti-body-streptavidin conjugate or fusion protein is allowed to localize to tumors in vivo, and any excess is cleared from the blood. A small radiolabeled biotiny-lated ligand is then injected intravenously. By virtue of the high-affinity interaction, the ligand penetrates tissues rapidly and is strongly taken up by the antibody conjugate at the tumor site. Because of the short transit time of the toxic ligand (radionuclide or tox in), a substantial improvement in the therapeutic ratio is achievable without sacrificing the percent injected dose per gram in tumor. Neuroblastoma is uniquely suitable for MST because of its abundance of surface ganglioside GD2. Anti-GD2 5F11-single-chain Fv-fragment (scFv)-streptavidin is a homote-tramer with improved avidity and highly favorable tumor-to-nontumor ratios in MST, achieving >50% improvement in radiation dose ratio of tumor to blood. In addition, because biotinylated polypeptides can achieve selective tumor targeting when MST is applied,a large repertoire of agents can potentially be explored for targeting to NB (Cheung et al. 2004).
Cell-mediated cytotoxicity can be highly effective against tumors in vitro and in animal models. Im-munocytokines (Davis and Gillies 2003; Lode and Reisfeld 2000) have shown remarkable success in activating and redirecting effectors to human tumors. The majority of these studies have focused on NK, NKT, T cells (Davis and Gillies 2003), and granulo-cytes (Metelitsa et al. 2002). They are active in ADCC in vitro activating effector cells appropriately through their cytokine receptors. In vivo administration of the ch14.18-IL-2 fusion protein induces long-term anti-tumor immunity (Davis and Gillies 2003; Lode and Reisfeld 2000), and provides greater protection against localized or metastatic murine neuroblas-tomas than does treatment consisting of the identical amounts of ch14.18 antibody and IL-2 given as separate molecules (Lode et al. 1997). Following initial successes with IL-2 and GM-CSF immunocytokines, constructs containing other cytokines have also been tested with encouraging results (Davis and Gillies 2003); these include IL-12, tumor necrosis factor (TNF)-a, and lymphotoxin. Clinical testing of the humanized form of this immunocytokine hu14.18-IL-2 is underway in adults with melanoma and children with NB (King et al. 2002). Immune activation was evidenced by increased serum IL-2 receptor levels, lymphocytosis, and induction of an antibody response against the hu14.18-IL-2. Clinical efficacy is yet to be established.
Ribosome inactivating toxins can be potent cancer drugs. One major limitation is the lack of tumor selectivity (Reiter 2001). Two-chain toxins [e.g., ricin and diphtheria toxin (DT)] utilize their B-chain for cell binding and their A chain for inhibition of protein synthesis, while other toxins [Pseudomonas exotoxin (PE), Pokeweed antiviral protein (PAP), gelonin] have a built-in cell attachment site. When conjugated to MAb, they become immunotoxins. In recombinant toxins (e.g., PE40, PE38, or diphtheria toxin DAB486), the cell-binding domains are replaced by scFv. Anti-GD2 monoclonal MAb have been conjugated to different toxins: ricin toxin A chain (Wargalla and Reisfeld 1989; Manzke et al. 2001), DT (Thomas et al. 2002), PE (Fur et al. 2001a), and gelonin (Mujoo et al. 1991). A common toxicity is the vascular leak syndrome, characterized by fluid overload, dyspnea, and sensory-motor neuropathies. Other natural compound toxins have also been explored as immunocon-jugates including cobra venom factor (Juhl et al. 1997) and staphylococcal enterotoxin A (SEA; Holzer et al. 1995). Anti-GD2 immunoliposomes have also been explored in vitro for delivering adriamycin (Ohta et al. 1993) and fenretinide (Raffaghello et al. 2003).
Cellular Immunoconjugates (Bispecific Antibodies)
Tumor-selective MAb can be rendered cytophilic by conjugation with MAb specific for trigger molecules on T lymphocytes, NK cells, and granulocytes. These molecules include CD3 (Manzke et al. 2001), CD28 (Bauer et al. 1999),Fc receptors (CD64,CD16) (Michon et al. 1995a,b), and FcaRI (CD89) (van Spriel et al. 2000). While one binding site of the bispecific antibody (van Priel et al. 2000; Friedrich et al. 2002; Schef-fold et al. 2002) engages effector cells, the other binding site determines tumor specificity. Since serum IgG competes for FcR, MAb made to recognize the FcR outside its Fc-binding site have been developed to circumvent this concern. Although bispecific MAb have potential in targeting small ligands (e.g., in MST), their clinical application in cellular immuno-conjugates has been complicated by the generalized cytokine release from leukocytes and the inherent limitations of trafficking of effector cells into tumors (Friedrich et al. 2002).
Besides the ability to block receptors from interaction with their natural ligand, MAb can inhibit receptor dimerization or receptor interaction with co-receptors (Agus et al. 2002). While most of the MAb targeting effort has been focused on individual tumor cells, alternative strategies directed at tumor neovasculature (Halin and Neri 2001) or tumor stroma (Hofheinz et al. 2003) are promising approaches. MAb can be made to neutralize the angiogenic factor VEGF (e.g.,beva-cizumab, Avastin) (Presta et al. 1997), or to block the VEGF-R2/KDR (e.g., IMC-1C11, chimeric anti-KDR; see Chap. 16) (Zhu et al. 2003; Posey et al. 2003). Targeting tumor vasculature may have significant advantages over direct tumor targeting, in that endothelial cells, unlike tumor cells, are less likely to acquire resistance. Another angiogenesis target is aVp3 integrin which initiates endothelial proliferation, migration, and matrix remodeling. Based on the preclinical antitumor activity of MAb specific for aVp3 (Gutheil et al. 2000), and the involvement of aV03 in NB (Lode et al. 1999), the chimeric IgG1 (MEDI-522) currently in clinical trial may have potential in treating NB.
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