Target antigens

Neuroblastoma expresses a wide variety of antigens on its surface. Antigens that have been used as targets for mAbs include the gangliosides GD2, GD3 and GM3, and the glycoproteins CD56 (NCAM), L1-CAM, GP58 and GP95 (Ref. Reference Cheung, Sondel, Cohn and Cheung16). GD2 is a disialoganglioside antigen that has been used extensively as a target in mAb therapy and has been the primary target of antibody recognition in neuroblastoma. It is expressed on tumours of neuroectodermal origin that include neuroblastoma, melanoma, osteosarcoma, and small-cell lung cancer (Refs Reference Sondel and Hank9, Reference Mujoo18). In normal tissues, GD2 expression is limited to neurons, skin melanocytes, and peripheral pain fibres (Ref. Reference Svennerholm19), making it well suited for targeted antitumour therapy.

In 1984, a murine mAb (mAB126) was produced against cultured human neuroblastoma cells (LAN1). This antibody was directed to the GD2 antigen on neuroblastoma and melanoma cells. Patients with neuroblastoma were found to have significantly elevated free GD2 levels in serum compared with normal children and children with other tumours (Ref. Reference Schulz20). In addition, unlike other tumour antigens that have been described in the past [e.g. CALLA in acute leukaemia (Ref. Reference Nadler21)], GD2 expression was not lost from the cell surface of neuroblastoma cells, even when bound to antibody (Ref. Reference Kramer22). (This concept is further detailed below in ‘Mechanisms of tumour resistance to antibody therapy’.)

3F8

The first mAb tested in clinical trials was the anti-GD2 mAb 3F8 (Refs Reference Cheung17, Reference Kushner23, Reference Cheung and Modak24, Reference Sondel, Gillies, Morse, Clay and Lyerly25, Reference Yeh26). This murine IgG3 antibody has been shown to have significant antitumour effects on both primary tumours and bone-marrow metastases. Labelled 3F8 has demonstrated selective localisation to tumours expressing GD2, with very little nonspecific uptake in the liver or spleen (Ref. Reference Yeh26). In the initial Phase I and Phase II trials using 3F8 in patients with stage 4 neuroblastoma, there was no significant antitumour effect on bulky disease but some response was seen in bone marrow disease and microscopic bone disease (Refs Reference Cheung, Sondel, Cohn and Cheung16, Reference Cheung27, Reference Cheung28, Reference Cheung29, Reference Chueng30) (Table 1). Side effects included hypertension, hypotension, fever, vomiting, diarrhoea and urticaria. The most common complaint was pain, which can be severe (grade 3–4) and dose limiting. Pain with the use of anti-GD2 antibodies has been attributed to recognition by antibodies of GD2 expressed on peripheral pain fibres (Refs Reference Lammie31, Reference Xiao, Yu and Sorkin32, Reference Yuki33). HAMA (human antimouse antibody) can develop in patients treated with 3F8 and has resulted in termination of therapy (Ref. Reference Kushner, Kramer and Cheung34) (see section ‘Mechanisms of tumour resistance to antibody therapy’).

Table 1

Clinical trials of monoclonal antibodies (mAbs) as single agents

The mechanism of tumour cell lysis using 3F8 antibody is similar to that of other IgG mAb therapies. 3F8 has been shown to activate tumour cell destruction by both CMC and ADCC in vitro (Refs Reference Cheung35, Reference Kushner and Cheung36). As a result of the lack of complement-inhibitory proteins CD55 and CD59 on neuroblastoma cells, this tumour is particularly sensitive to complement-mediated tumour cell lysis in vitro (Refs Reference Cheung35, Reference Chen37).

14.18

14.18 is a separate IgG3 murine mAb targeted to the GD2 antigen (Ref. Reference Mujoo18). In an attempt to enhance ADCC, a class switch variant called 14.G2a has also been prepared (Ref. Reference Mujoo38). The 14.G2a antibody activates complement and mediates ADCC with monocytes, neutrophils, NK cells and lymphokine-activated killer (LAK) cells (Refs Reference Saarinen39, Reference Munn and Cheung40). A chimaeric anti-GD2 antibody, ch14.18, was subsequently created to reduce the immunogenicity associated with the murine antibody (Fig. 1; see also section ‘Mechanisms of tumour resistance to antibody therapy’). The chimaeric antibody is less immunogenic and is more effective than 14.G2a in mediating lysis of neuroblastoma cells with human effector cells such as granulocytes and NK cells (Ref. Reference Barker41). The ch14.18 and 14.G2a antibodies have undergone clinical testing both as single-agent therapies and in combination approaches; in this section, we focus on the former.

Figure 1

Monoclonal antibodies and immunocytokines. (a) A chimaeric monoclonal antibody (mAb) combines the constant region of a human antibody with the variable domain of a murine antibody. The antigen specificity is conferred by the murine variable domain. (b) In the humanised mAb, the murine framework determinants of both the heavy and light chains are replaced with human framework determinants, but the antigen specificity of the original murine mAb is retained. (c, d) Fusion proteins or immunocytokines combine the mAb with covalently linked cytokines, such as molecules of interleukin 2 (IL-2), to the end of each of the heavy chains at the C-terminus. Adapted, with permission, from Ref. 58 (© 1999 Elsevier).

As a single agent, 14.G2a has been used in three separate Phase I clinical trials. In 1992, in Tübingen, Germany, nine patients with stage 4 neuroblastoma were treated with 14.G2a: all patients developed HAMA during or shortly after therapy; six patients were evaluable for response (Ref. Reference Handgretinger42). In a trial conducted at M.D. Anderson (Houston, USA), 18 adults with melanoma or neuroblastoma received the 14.G2a mAb in a dose-escalation trial by continuous intravenous (IV) infusion over 5 days: the maximum tolerated dose (MTD) was 100 mg/m²/day (Ref. Reference Murray43); 16/18 patients developed HAMA. Lastly, at the University of Alabama (Birmingham, USA), 12 adults with melanoma or neuroblastoma received the antibody by 1 h daily infusion on days 1, 3, 5 and 8: all patients developed HAMA (Ref. Reference Saleh44). Responses were variable among all patients but 14.G2a did show promise as a therapeutic agent (Table 1). The toxicities seen in these trials included fever, diarrhoea, nausea, vomiting, transient neuropathy, tachycardia, hypotension, anaphylactoid reactions and pain (Refs Reference Handgretinger42, Reference Murray43, Reference Saleh44).

The chimaeric antibody has also been tested as a single-agent therapy in two separate trials involving patients with stage 4 neuroblastoma. In a Phase I trial in Tübingen, Germany, nine heavily pretreated patients with stage 4 neuroblastoma were treated with 19 courses of chimaeric anti-GD2 antibody at dose levels 30, 40 and 50 mg/m²/day for 5 days per course. The MTD was 50 mg/m²/day. No HAMA responses were seen (Ref. Reference Handgretinger45) (Table 1). Similar results were seen in ten patients with refractory neuroblastoma and one with osteosarcoma. Patients in this study received 20 courses of chimaeric antibody at dose levels of 10, 20, 50, 100 or 200 mg/m². Dose escalation was permitted for a single patient if tolerated. The MTD was not reached in this study. Ten patients were evaluable for response; results are summarised in Table 1 (Ref. Reference Yu46). The chimaeric antibody was well tolerated in both studies with few side effects seen at dosages of 10 mg/m² or less, and encouraging results were seen in bone marrow and microscopic bone disease. Toxicities in each were similar to those seen with the murine antibody, including pain, tachycardia, hypertension, fever, urticaria and transient neuropathy (Refs Reference Handgretinger45, Reference Yu46). Optic nerve atrophy was seen in two patients in the Tübingen study. Both patients had received prior radiotherapy and this was implicated as the cause of this adverse event. The effect was reversible in both patients (Ref. Reference Handgretinger45). Seven out of ten patients in the latter study did develop a HACA (human antichimaeric antibody) response as measured in post-treatment serum samples (Ref. Reference Yu46). Pharmacokinetic and immunological studies in association with these trials have shown that the chimaeric antibody has a longer plasma half-life and is less immunogenic than its murine counterpart, potentially improving its clinical utility (Ref. Reference Uttenreuther-Fischer, Huang and Yu47).

Use of mAbs with cytokines

As the science of mAb-based tumour cell lysis was elucidated, it became clear that the antibody must accomplish three separate tasks to kill a tumour cell: first, the antibody must recognise and bind to the tumour cell; second, it must bind long enough to the tumour cell without being internalised to properly signal immune effector mechanisms; and third, the activated immune effector mechanisms must be able to create a destructive signal (Ref. Reference Sondel and Hank9). Since mAb therapy relies on ADCC and/or CMC to kill tumour cells, strong effector functions are required. However, owing to immune suppression from metastatic cancer and/or chemotherapy, effector-cell function may be compromised in cancer patients (Refs Reference Cheung, Sondel, Cohn and Cheung16, Reference Kushner and Cheung36, Reference Hank48). The addition of cytokines to mAb therapy can augment effector-cell function and improve the overall effect of immune therapy (Ref. Reference Sondel and Hank9).

Although chemotherapy may be strongly immunosuppressive, the effect on granulocytes is more transient (Ref. Reference Barker49). GM-CSF (granulocyte–monocyte colony-stimulating factor) has been found to both increase granulocyte cell populations and increase adhesion molecules on lymphocytes, which enhances ADCC against neuroblastoma cells (Refs Reference Kushner and Cheung36, Reference Furman50, Reference Baldwin51, Reference Metelitsa52). It has been used in clinical trials in combination with the mAbs ch14.18 (Ref. Reference Ozkaynak53), 14.G2a (Ref. Reference Frost54) and 3F8 (Refs Reference Kushner, Kramer and Cheung34, Reference Kushner and Cheung36). The results of all four trials suggest that GM-CSF in combination with anti-GD2 mAb improves the antitumour response, particularly in bone marrow disease (Refs Reference Kushner, Kramer and Cheung34, Reference Kushner and Cheung36, Reference Ozkaynak53, Reference Frost54). However, this has not yet been tested directly in a randomised controlled clinical trial.

Interleukin 2 (IL-2) is a strong pro-inflammatory agent with effects on both the innate and adaptive immune system. IL-2 increases both the number and activation state of NK cells. Activated NK cells bind the Fc portion of the mAb through their FcγRIII and enact ADCC (Refs Reference Sondel55, Reference Mule56). IL-2 also stimulates antigen-specific T cells to kill tumour cells, an example of breaking tolerance to tumour antigens (Refs Reference Mule56, Reference Sondel and Hank57). In a Phase I trial through the Children's Cancer Group (predecessor to the Children's Oncology Group, which conducts multi-institutional clinical trials in paediatric oncology in the USA) involving 33 patients, IL-2 was administered by three 96 h infusions over successive weeks, on days 1, 8 and 15 (Ref. Reference Frost54). The mAb 14.G2a was given as a daily 2 h infusion on days 9 through 13. This was timed to take advantage of the lymphocytosis and maximal NK cell cytotoxic activity seen in several in vitro analyses carried out prior to the study (Ref. Reference Hank, Albertini, Sondel, Pinedo, Longo and Chabner58). One patient had a partial response with a 70% decrease in size of an abdominal tumour over 3 months, facilitating complete resection. Three additional patients had a transient reduction in microscopic bone marrow disease but no reduction in tumour burden in other locations. Serum samples were obtained from these patients and were found to contain sufficient levels of 14.G2a to result in ADCC of GD2-positive tumour cell targets in vitro (Ref. Reference Hank59).

Use of mAbs with β-glucans

3F8 therapy is enhanced when used in combination with the glucose polymer β-glucan, which is homologous with polysaccharides normally found on the cell wall of fungi or yeast (Ref. Reference Xia60). These sugars act as strong signals to the innate immune system, much like lipopolysaccharide (LPS) found on the cell envelope of Gram-negative bacteria. While LPS has been found to be too toxic to use in human patients, β-glucans are well tolerated and have been shown to stimulate ADCC mediated by NK cells, monocytes and neutrophils, as well as increase production of tumour necrosis factor α (TNF-α) (Refs Reference Vetvicka, Thornton and Ross61, Reference Di Renzo, Yefenof and Klein62, Reference Vetvicka, Thornton and Ross63, Reference Czop and Austen64, Reference Thornton65). When mAb binds to a tumour cell, the complement cascade is activated and coats the tumour cell with the complement fragment iC3b. The receptor for iC3b is the CR3 molecule on leukocytes. Soluble β-glucans can be used to prime CR3 on the leukocytes, leading to dual ligation of the CR3 receptor on leukocytes to both iC3b on the tumour and soluble β-glucan; this dual ligation subsequently enhances tumour cytotoxicity (Refs Reference Xia60, Reference Vetvicka, Thornton and Ross61) (Fig. 2).

Figure 2

Monoclonal antibody therapy is enhanced when combined with β-glucan. (a) When anti-GD2 mAb binds to the GD2 antigen on neuroblastoma cells, the complement cascade is activated. While many molecules are involved in the complement cascade, one that becomes displayed on the neuroblastoma surface is the complement fragment iC3b. (b) β-glucan is a glucose polymer normally found on the surface of yeast. Soluble β-glucan binds to the iC3b receptor (CR3) on human leukocytes, priming it for interaction with the iC3b on tumour cells. (c) When the β-glucan-primed CR3 recognises iC3b on the neuroblastoma cell, tumour cell cytotoxicity results.

In vivo, the addition of oral or intraperitoneal β-glucan has been shown to be effective against neuroblastoma. In nude mice bearing human neuroblastoma tumours NMB7, LAN-1, SK-N-ER, SK-N-MM or SK-N-JD, the addition of β-glucan to 3F8 mAb therapy resulted in near-complete or complete tumour resolution. By contrast, either agent alone had significantly less effect. Survival was also increased by 2.6–5.5 times compared with control animals depending on the tumour type. The effect was lost when tested on a GD2-negative tumour (Refs Reference Cheung and Modak24, Reference Hong66). Depletion of NK cells resulted in a depressed antitumour effect in nude mice, but synergy was still observed in severe combined immunodeficient (SCID-beige) mice, suggesting that some of the antitumour effect was mediated in an NK-cell-independent manner (Ref. Reference Cheung67). This synergy in combination with oral β-glucan has been seen in other mAb systems using the antigens GD3, CD20, EpCAM and HER-2 as targets for mAb therapy (Ref. Reference Cheung67). The use of β-glucan in conjunction with 3F8 is currently under Phase I clinical investigation.

mAbs linked to toxins

mAbs have been conjugated to toxins as a means of selective toxin delivery. These agents kill tumour cells via antigen-directed internalisation of the toxin and can work independently of immune effector cells (Ref. Reference Manzke68). Several antibody–toxin pairs have been tested. For example, anti-GD2 mAb has been linked to either Pseudomonas exotoxin A (Ref. Reference Tur69) or diphtheria toxin (Ref. Reference Thomas70). Both agents have shown cytotoxicity against GD2-positive neuroblastoma cell lines but neither has yet undergone clinical testing (Refs Reference Tur69, Reference Thomas70). Noncytotoxic antibody fragments, such as the Fab fragment of 3F8, can be made increasingly cytotoxic through linkage of the Fc fragment to cobra venom factor (CVF), which is a functional homologue of activated human complement. In the presence of human complement, these antibodies have been shown to mediate complement-dependent lysis of neuroblastoma cells, with up to 100% killing in some in vitro assays (Ref. Reference Juhl71). Furthermore, linkage of ch14.18 mAb to the superantigen staphylococcal entertoxin A demonstrated effective T-cell-mediated killing of GD2-positive neuroblastoma cells, independent of major histocompatibility complex (MHC) class II expression (Ref. Reference Holzer72). Ricin A has also proven effective against GD2-positive neuroblastoma cell lines in both in vitro studies and in IMR5-tumour-bearing SCID mice (Ref. Reference Manzke68). Although all of these agents have shown promising in vitro activity, few have moved into preclinical testing.

mAbs for targeted chemotherapy delivery

Antigen-specific drug delivery is another possible mechanism of killing tumour cells selectively. Anti-GD2 or -GM2 antibody fragments have been linked to liposomes containing adriamycin and have been shown to result in significant cytotoxicity when placed in culture with GD2- and GM2-positive cell lines (Ref. Reference Ohta73). In a second model, the synthetic retinoid fenretinide, which is known to induce apoptosis in neuroblastoma cell lines, has also been incorporated into liposomes and bound to anti-GD2 mAb. In vivo, this combination prevented the development of metastatic disease in nude mice bearing human HTLA-230 tumours, and was more effective in survival analyses than either empty liposomes or anti-GD2 mAb alone (Ref. Reference Raffaghello74). Further in vivo work is required to determine the efficacy of antibody-directed chemotherapy, but this method may prove helpful in reducing the side effects of systemic administration, especially in heavily pretreated patients.

Radioimmunoconjugates

Radiolabelled mAbs have been used for both disease detection and targeted treatment.

Immunocytokines

While the addition of cytokines to mAb therapy has shown a synergistic effect, systemic cytokine administration has been limited by toxicity arising from the nonspecific activation of the inflammatory cascade, with no specific activity against tumour cells (Ref. Reference Sondel11). To limit the toxicity from cytokines, a new therapeutic agent was developed that linked the cytokine protein to the Fc end of the mAb (Fig. 1). This ‘immunocytokine’ retained the antigen-binding specificity of the original mAb and delivered the cytokine directly to the tumour microenvironment. As a single agent, both the antibody effector function and the cytokine co-signal are provided (Ref. Reference Lode86). The dose of cytokine required is much lower than that required with systemic administration and its half-life may be significantly increased (Refs Reference Sondel and Hank9, Reference Gillies87, Reference Gan88).

One of the best-characterised immunocytokine molecules is ch14.18–IL2, which was formed by linking the gene sequence of IL-2 to DNA encoding the carboxyl end of the constant region of the human IgG1 molecule that composes the ch14.18 mAb (Refs Reference Gillies87, Reference Gillies89). The advantage of this agent is that it can activate cells without Fc receptors (which includes a subpopulation of cytotoxic NK cells) (Ref. Reference Weil-Hillman90) and in vivo data suggest that activated NK cells have augmented IL-2 receptor expression (Ref. Reference Voss91). Local T cells also may be activated through their IL-2 receptor if the T-cell receptor (TCR) does not recognise the tumour antigen initially (Ref. Reference Gillies89).

Several preclinical studies have been completed using the ch14.18–IL2 molecule (Refs Reference Gillies89, Reference Hank92, Reference Lode93, Reference Lode94). In SCID mice bearing neuroblastoma with bone marrow and liver metastases, intravenous administration of ch14.18–IL2 resulted in a significant reduction in tumour burden. This was not seen in animals that received ch14.18 and IL-2 as separate intravenous infusions (Ref. Reference Lode93). In animals depleted of NK cells, the effect of ch14.18–IL2 was lost, suggesting that the effect was dependent on NK cells. The addition of NK-stimulating agents such as interferon γ (IFN-γ) further increased the antitumour effect. CD8⁺ T-cell depletion, however, did not significantly affect the antitumour response (Ref. Reference Lode94). In a second study, using immunocompetent mice, treatment with ch14.18–IL2 prevented metastatic spread to the liver and bone marrow, whereas giving both agents separately did not prevent metastatic disease. Mice given the ch14.18–IL2 molecule survived twice as long as the other treatment groups; however, the effect on bulky tumours was minimal (Refs Reference Lode93, Reference Lode94).

The ch14.18–IL2 molecule has been refined to a humanised form, known as hu14.18–IL2, in the hope of further reducing its immunogenicity in patients (Fig. 1). This agent has recently completed Phase I trials (Ref. Reference Osenga95). Twenty-seven patients with stage 4 neuroblastoma were given infusions for three consecutive days. The maximum tolerated dose was 12 mg/m²/day. Toxicities included hypotension (requiring use of dopamine in two patients), allergic reaction, blurred vision, neutropaenia, thrombocytopaenia and leukopaenia. All toxicities were reversible. Although there were no complete or partial responses, the patients did respond with lymphocytosis and elevated levels of soluble IL-2 receptor, and three patients had some radiographic and/or bone marrow response. No patient, however, had a reduction in bulky disease in other sites. This agent is now being tested in a Phase II clinical trial through the Children's Oncology Group (study number ANBL0332) in patients with recurrent or refractory stage 4 neuroblastoma.

Immunocytokines have also been developed using the cytokine GM-CSF (Ref. Reference Batova96). This has been linked to ch14.18 to create a molecule that has been shown to be very effective at stimulating ADCC against the neuroblastoma cell line NMB7 (Ref. Reference Batova96). When used in combination with granulocytes or monocytes from patients with neuroblastoma, the immunocytokine was equivalent in cell killing to equal amounts of free GM-CSF and ch14.18 mAb. Although the immunocytokine was not found to be superior in vitro, it is advantageous to be able to provide targeted cytokine delivery to stimulate both granulocyte and monocyte proliferation and migration. The ch14.18–GM-CSF immunocytokine has not yet entered clinical testing.

T-cell engineering

The antitumour response seen with mAbs is not limited to the innate immune system, as T-cell activation and tumour-specific memory responses have also been shown in animal models and in certain clinical settings (Ref. Reference Coughlin122). Even though neuroblastoma may appear to be MHC class I and II negative in some instances, MHC molecules can be upregulated, making these cells recognisable for T-cell cytotoxicity. T-cell cytotoxicity can be enhanced through manipulation of the TCR to redirect its specificity toward tumour antigens (Ref. Reference Rossig and Brenner123). One common target is GD2. T cells can be genetically altered to express chimaeric TCRs that consist of the variable domain of an anti-GD2 antibody linked to a cytoplasmic signalling domain. Engagement of this TCR complex initiates cytotoxic effector function. These cells release a variety of pro-inflammatory cytokines including GM-CSF and IFN-γ upon incubation with GD2-positive tumour cells. These modified T cells are also capable of tumour-specific cytotoxicity with minimal effect on GD2-negative targets (Ref. Reference Rossig124). Furthermore, the feasibility of isolating CD8⁺ T cells and altering the TCR specificity with plasmids encoding engineered antigen receptors has been shown in human patients (Refs Reference Gonzalez125, Reference Park126). Infusions of autologous tumour-specific T cells had half-lifes of 1–42 days with minimal toxicity, and their lifespan was inversely proportional to the patient's disease burden, with particularly long lifespans in patients with minimal disease. Incorporation of DNA encoding the novel antigen receptors has been achieved via a variety of methods, including uptake of naked plasmid DNA via electroporation and retrovirus transfection (Refs Reference Rossig124, Reference Jensen127). Virtually any tumour antigen may be targeted with this method. Although this has been used more extensively for leukaemia and lymphoma, human clinical trials targeting neuroblastoma are also under way (Refs Reference Park126, Reference Serrano128, Reference Cooper129, Reference Savoldo130). Survival and proliferation of engineered T cells can also be enhanced by modification of the intracellular signalling domain. By attaching the additional elements normally provided by costimulatory molecules to the intracellular domain, a single TCR can engage a GD2-positive target and stimulate the intracellular signals necessary for cytotoxicity, T-cell survival and proliferation (Ref. Reference Pule131).

T-cell purification and stimulation can be made more effective when combined with adjunctive immunotherapy. Patients who have undergone stem cell transplantation require several weeks to months to regenerate a fully functional immune system. The infusion of large numbers of tumour-specific effector cells purified ex vivo is an attractive alternative to waiting for an autologous immune response, especially in the critical period of minimal residual disease. Human γδ T cells are a subset of the T-cell population that have been shown to be particularly cytotoxic to tumour cells, including neuroblastoma (Ref. Reference Ferrarini132). Their survival is enhanced when infused with IL-7 (Ref. Reference Laky133). The use of combination therapy of allogeneic γδ T cells with IL-7 and the anti-GD2 mAb hu14.18 has shown a significant survival benefit in a murine model of disseminated neuroblastoma using tail-vein injected NB-1691 cells (Ref. Reference Otto134).

Single-chain variable antibody fragments

One limitation of mAb therapy is poor penetration into solid tumours. Single-chain antibodies consisting of a single variable heavy chain and light chain, also known as single-chain variable fragments (ScFvs), have better tumour penetration and reduced antigenicity (Ref. Reference Whittington, Hancock and Kemshead135). These agents can be easily linked to toxins to improve their therapeutic effect. In addition, antibody binding and toxin administration can be divided into two steps in a method known as multistep targeting. In this technique, tumours are labelled with mAb conjugated to a second molecule. A toxin with affinity for the tumour-bound antibody is then administered. In this way, the toxin is more selective and there is less systemic exposure to the toxic agent. An anti-GD2 mAb bound to streptavidin has shown excellent targeting when coupled with radiolabelled biotin (Ref. Reference Cheung136). Making a dimer out of two ‘single-chain antibodies’ further enhances their affinity and results in slower clearance compared with ScFvs (Ref. Reference Occhino137).

Antibody engineering

Other modifications of immunoglobulin structure and function are being considered in the production of therapeutic reagents. Amino acid substitutions in the antigen-binding portions of the variable region of a humanised antibody can be designed to make the molecule even less immunogenic to the human immune system (Ref. Reference Roque-Navarro138). The desired result is preventing the development of a neutralising antibody against the therapeutic antitumour mAb. Intentional modifications in the Fc end of the immunoglobulin can alter effector functions of the mAb. Changes in glycosylation or amino acid substitution can enhance interactions with Fc receptors on effector cells, thus augmenting ADCC (Ref. Reference Siberil139). Other modifications can alter the interaction with complement, either enhancing or inhibiting complement activation. As complement activation by anti-GD2 antibodies is thought to play an important role in the neuropathic pain associated with anti-GD2 mAb treatment, but complement activation may not be critical in the observed antitumour effects, some effort is now being directed into the testing of a mAb modified to lose its ability to activate complement while retaining its ADCC capability.