B-lymphocytes produce five classes of antibodies, or immunoglobulin (Ig) molecules (IgA, IgD, IgE, IgG, IgM). IgG secreted by memory B cells is the antibody with the highest concentration in circulation. This molecule is composed of two longer (heavy
) chains and two shorter (light
) chains.  The specificity of antigen binding is determined by the amino acid sequence of the variable region
of the IgG molecule. After initial exposure to the cognate antigen, B cells produce IgM, which is followed by class switch and production of IgG of the same specificity.4
Structure of immunoglobin and monoclonal antibody fragments
Monoclonal antibodies against human differentiation antigens
There has been dramatic progress in the clinical development of MoAb-based cancer therapeutics over the past 2 decades. Köhler and Milstein first demonstrated that monoclonal antibodies (MoAb) directed against specific human differentiation antigens could be generated from hybridomas derived from immunized mice,13
which made possible the large-scale production of such reagents for therapeutic use in humans. Subsequently, it was reported that fragments of antibody variable domains (Fv) could be linked together to make recombinant proteins capable of antigen binding.14
Methodologies have since been developed to produce fully human MoAbs and their fragments for clinical use and to generate humanized constructs with reduced immunogenicity. 
Monoclonal antibody-based therapies for cancer
For effective MoAb-targeting, the cognate antigen should be expressed in relatively high levels on the surface of the malignant cells and there should be limited-to-no expression on normal tissues. Ideally, there should also be minimal shedding of antigen from the cell surface, since high levels of free antigen could serve as a decoy, thus diminishing MoAb binding to the target.
MoAbs have the potential to kill cancer cells through direct and indirect effector pathways. Certain antibody-receptor binding interactions directly inhibit cell growth or induce cell death (apoptosis) through effects on intracellular signaling pathways. Indirect killing can occur by antibody-dependent cellular cytotoxicity (ADCC), complement-activation, and/or cell-mediated cytokine production. The mechanism involved, or whether killing occurs at all, varies with the specific MoAb, antigen and cancer. Importantly, immune-mediated cytotoxicity requires functional immune effector mechanisms, which are commonly deficient in patients with cancer.15,16
Monoclonal antibodies for hematologic malignancies
Hematologic malignancies, the most common pediatric cancers, are excellent candidates for MoAb-based therapeutics.17
Malignant blasts from patients with leukemia and lymphoma express lineage-specific human differentiation antigens with otherwise limited tissue distribution, and numerous MoAbs have been developed that effectively target such antigens. In 1997, the U.S. Food and Drug Administration (FDA) approved the first MoAb for the treatment of cancer, rituximab (Genentech, Inc. San Francisco, CA, U.S.A.). This agent is a MoAb directed against the B-lymphoid lineage antigen CD20. Rituximab combined with chemotherapy has been demonstrated to improve disease-free survival in adults with non-Hodgkin's lymphoma (NHL).18
Results of a recent Children's Oncology Group (COG) trial of rituximab with chemotherapy indicate that this agent can be safely administered to pediatric patients with relapsed CD20+ lymphoma and leukemia.19*
The COG and the Berlin-Frankfurt-Muenster cooperative group (BFM) are currently conducting clinical trials of rituximab with chemotherapy for children and adolescents with newly diagnosed CD20+ hematologic malignancies. A COG study of a MoAb that targets the B-lineage antigen CD22, epratuzumab (Immunomedics, Inc., Morris Plains, NJ, U.S.A.), in combination with standard chemotherapy for children with acute lymphoblastic leukemia (ALL) is also in progress. Results in the initial cohort treated with an upfront epratuzumab monotherapy phase were recently published.20*
The clinical activity of epratzumab as a single agent was limited and no complete or partial remissions were observed. In this preliminary analysis, the feasibility of giving epratuzumab in combination with standard chemotherapy was demonstrated and the rates of complete remission and clearance of MRD appeared favorable in comparison to historical results with chemotherapy. It is unlikely that MoAbs will have adequate single agent activity to be effective for pediatric hematologic malignancies when used alone. However, rare cases of complete remissions in adults and children with ALL have been reported with MoAbs targeting CD20, CD33, and CD52.21,22,23
Monoclonal antibodies for solid tumors
Recent success has also been achieved using MoAbs to target antigens expressed on pediatric solid tumors. The ganglioside GD2 is a neuroectodermal-restricted antigen expressed by neuroblastoma. A number of MoAbs targeting GD2 have been studied in children with high-risk neuroblastoma.24,25,26,27,28,29
The anti-GD2 MoAb 3F8 cleared MRD after autologous SCT in children with stage IV neuroblastoma.30
The anti-GD2 MoAb ch14.18, in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) after autologous SCT, enhanced event-free and overall survival in comparison to standard therapy.31**
Importantly, this regimen had limited activity in patients with bulky neuroblastoma, consistent with a model wherein immune-based therapies are more effective when administered in the setting of MRD.
The insulin-like growth factor (IGF) signaling pathway plays a role in cell survival in certain pediatric sarcomas.32,33*
A number of MoAbs against the IGF-1 receptor are undergoing clinical trial in pediatric and adult patients with sarcomas and dramatic responses have been observed suggesting that IGF-1 receptor blockade can interrupt critical cell survival signals.32
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor is expressed on a variety of cancers, and anti-TRAIL receptor MoAbs have shown activity against pediatric sarcomas in pre-clinical models.34
A phase I trial of an anti-TRAIL MoAb for children with solid tumors is in progress at the National Cancer Institute (NCI).
CTLA4 is a receptor on the surface of T cells that diminishes immune responses. Blockade of CTLA4 signaling through anti-CTLA4 MoAbs inhibits this suppressive signal and augments T cell mediated immune reactivity. This agent has activity against melanoma and other tumors in adults,35
and a pediatric Phase I trial is being performed at the NCI.
Conjugated monoclonal antibodies
The cytotoxicity of MoAbs can be dramatically increased by linkage to toxic moieties such as chemotherapeutic agents, bacterial and plant toxins, and radioisotopes. Conjugating highly cytotoxic agents to MoAbs should improve the therapeutic index since the MoAb directs killing to those cells that express the target antigen, thus limiting non-specific damage to normal tissues. Importantly, MoAb-based agents armed with potently cytotoxic compounds do not require active immune response mechanisms for activity. As a result, they can be effective even in profoundly immunocompromised patients.
MoAbs have been conjugated to a number of active chemotherapeutic agents. The first MoAb conjugate to receive FDA approval in the treatment of cancer was gemtuzumab ozogamicin (Pfizer, New York, NY, U.S.A.). This agent targets the myeloid antigen CD33 and is linked to calicheamicin, a potent antitumor antibiotic. Clinical trials of gemtuzumab ozogamicin have been conducted for children with acute myelogenous leukemia (AML).36,37**
Approximately 30% of pediatric patients with relapsed AML respond to gemtuzumab ozogamicin as a single agent. Clinical trials designed to assess the efficacy of gemtuzumab ozogamicin in combination with chemotherapy are being conducted by the COG,38
while the Nordic Society of Paediatric Haematology and Oncology is studying its role as consolidation prior to SCT in children with high-risk AML. This agent has also been used to treat occasional cases of ALL with CD33 expression and anecdotal cases of successful remission induction have been reported.39
Immunotoxins are engineered proteins consisting of a MoAb-based targeting moiety that mediates cell binding and a toxin that induces cell death upon internalization.  A 38 kD truncated derivative of Pseudomonas
exotoxin A (PE38) has been used at the NCI to develop recombinant immunotoxins that target human differentiation antigens.40
A recombinant immunotoxin that targets the B-lineage antigen CD22, CAT-3888 or BL22 (MedImmune LLC, Gaithersburg, MD, U.S.A.), is highly active in adults with B-lymphoid malignancies.41,42
A recently completed pediatric Phase I study demonstrated an acceptable toxicity profile and clinical activity in children with CD22+ ALL and NHL.43
A follow-up pediatric trial of a second-generation agent with higher CD22 binding affinity and increased pre-clinical activity,44
CAT-8015 or HA22, (MedImmune LLC, Gaithersburg, MD, U.S.A.), is in progress.45*
Anti-CD22 immunotoxins appear to have synergistic in vitro
cytotoxicity against childhood ALL blasts when combined with chemotherapy.46
Pseudomonas-based immunotoxins: structure and mechanism of cytotoxicity Inset:
MoAbs that target leukemia-associated antigens have been linked to radioactive isotopes, most commonly β-emitters (e.g., 90
Rhenium) and less frequently α-emitters (e.g., 213
Bismuth). These agents are concentrated in the bone marrow and consequently cause severe myelosuppression. Thus, the application of radioimmunotherapy for hematologic malignancies is limited primarily to myeloablative conditioning prior to SCT,47
and there have been very few trials in the pediatric age group.48,49
Studies are being conducted with a 3F8-131
Iodine conjugate for children with GD2-expressing brain tumors.50
Potential side effects and limitations of monoclonal antibody-based therapeutics
In general, most of the MoAb-based therapeutics currently being advanced for clinical use have been well tolerated with markedly reduced risks of organ toxicity in comparison to chemotherapy and radiation. Nonetheless, there are a variety of specific side effects associated with individual MoAbs and immunotoxins. Acute infusion reactions are relatively common, although these can usually be managed or prevented by treatment with antipyretics, antihistamines, and/or corticosteroids. Depletion of normal blood cells that express the target antigen can be expected. For example, rituximab is associated with B cell depletion, humoral immunosuppression, and risk of certain viral infections. Immunotoxins are associated with a number of unique toxicities, for example, vascular leak syndrome.
Despite the creation of human/murine (“humanized”) antibodies, some components of many MoAbs are derived from foreign species. Similarly, toxins represent foreign proteins. Thus, patients may develop antibodies to foreign protein epitopes (i.e., human anti-mouse and/or human anti-toxin antibodies) that bind and diminish or neutralize therapeutic activity. Notably, there may be pre-existing antibodies due to prior vaccination (e.g., Diphtheria) or infection (e.g., Pseudomonas).