ADCC is mediated by immune effector cells binding to rituximab-coated B cells. This requires rituximab to bind through the Fc region to Fc receptors for IgG (FcγRs) on immune effector cells, such as natural killer (NK) cells, monocytes, macrophages, and granulocytes [3]. ADCC is better mediated through the IgG1 constant region, and polymorphisms in the genes encoding FcγRs may affect the response to rituximab therapy [5].

Rituximab can recruit components of the complement cascade through the Fc region. The first step is the binding of C1q components to the Fc region. C1q activation triggers the classical complement cascade, which leads to the generation of the membrane attack complex that causes direct lysis of the B cells by disrupting the cell membrane [3]. CDC is better mediated through the IgG1 constant region, and it has been demonstrated that CDC by rituximab correlates not only with the CD20 expression level but also with segregation of CD20 into specialized microdomains on the plasma membrane known as lipid rafts [7].

Upon CD20 cross-linking after rituximab treatment, induction of apoptosis involving activation of caspase-9, caspase-3, and poly-adenosine diphosphate ribose polymerase (PARP) cleavage has been described in primary CLL cells, and downregulation of the antiapoptotic proteins XIAP and Mcl-1 has been described as well [8]. In addition, downregulation of the antiapoptotic protein bcl-2 by rituximab treatment has been observed in some B-cell lines [9]. Furthermore, it has been suggested that the transactivation of src family tyrosine kinases and subsequent increase of the intracellular calcium level upon CD20 translocation into lipid rafts are another proposed mechanism of action of rituximab-induced apoptosis [10, 11]. Additive and synergistic effects between rituximab- and chemotherapy-induced apoptosis have been observed suggesting that rituximab-induced apoptosis uses a final pathway similar to chemotherapeutic agents, leading to the use of combined chemoimmunotherapy regimens.

It has been observed that the CD20 expression level correlates with rituximab-mediated CDC. There are some in vitro data showing that CDC is more rapidly and efficiently induced by rituximab in cells with a higher CD20 expression level [12]. Therefore, loss or down modulation of CD20 may impact the efficacy of rituximab. As mentioned previously, CD20 is not shed or internalized following the binding of rituximab in normal individuals. However, it has been demonstrated that shaving/trogocytosis of CD20 is mediated by FcγR on acceptor cells, such as NK cells, monocytes, or macrophages in patients with CLL [13]. It has also been demonstrated that type I (rituximab-like) mAb-induced antigenic modulation occurs as a result of internalization of type I (rituximab-like) mAb-CD20 complexes by the B cells themselves in several cell lines [14] and the internalization is augmented by FcγRIIb [15]. In addition, CLL and mantle cell lymphoma (MCL) samples demonstrate more rapid internalization than follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) [16], suggesting that internalization may explain the differing sensitivity of B-cell malignancies to rituximab.

High levels of sCD20 are present in the plasma of patients with CLL. Plasma sCD20 has been shown to compete with cell surface CD20 for rituximab binding and significantly diminish the binding of rituximab to the surface of CLL cells [16]. It has been reported that response to rituximab is correlated with dose in patients with CLL, suggesting that rituximab dosing may be adjusted according to sCD20 level [17].

The influence of FcγR polymorphisms on rituximab-mediated ADCC has been previously described. There are three classes of FcγR: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). The polymorphism in FcγRIIIa consisting of a valine (V) or a phenylalanine (F) in amino acid position 158 results in a different affinity for IgG1-FcγRIIIa binding. The FcγRIIIa-158V homozygous genotype (V/V) has a greater affinity for human IgG1 than V/F or F/F. Patients with follicular lymphoma with the V/V genotype have a better clinical response to rituximab than patients with V/F or F/F. The polymorphism in FcγRIIa consisting of a histidine (H) or an arginine (R) in amino acid position 131 also results in a different affinity for IgG1-FcγRIIa binding, with better clinical results for FcγRIIa-131H homozygous patients. However, not all results demonstrate that these polymorphisms are clinically relevant [18–20]. It has been described that FcγRIIIa and FcγRIIa polymorphisms do not predict the clinical response to rituximab in patients with CLL [21].

The role of complement regulatory proteins such as CD46, CD55, and CD59 in rituximab-mediated CDC has been previously described. Expression of high levels of CD46 or CD55 prevents the activity of C3 convertase, whereas expression of CD59 prevents the assembly of the active membrane attack complex, resulting in interference of completion of the complement cascade. There are some in vitro data showing that complement regulatory proteins may play an important role in rituximab-mediated CDC [22–24]. Currently, however, there is little in vivo data showing that CD46, CD55, or CD59 expression level correlates with response to rituximab.

The influence of the C1qA-276 polymorphism on rituximab-mediated CDC has also been previously described [20, 25]. However, there are very few data indicating that the polymorphism predicts response to rituximab.

It has been demonstrated that rituximab is widely distributed in body organs. By gamma camera measurements, radioactivity was detected in the heart, liver, lungs, spleen, and kidneys of patients with relapsed NHL who received iodine-131-labeled rituximab. The ratios of tumor to whole-body radioactivity increased over time, whereas those of organ to whole body decreased, indicating evidence of specific antibody binding [27]. It has been reported that rituximab levels in cerebrospinal fluid were detected after infusions with a C _max of 0.55 μg/ml compared with a C _max of 400 μg/ml in peripheral blood [28].

Little is known about the mechanisms of rituximab metabolism. It is speculated that rituximab is degraded by a nonspecific catabolism of proteins in the liver and other organs.

It has been demonstrated that radioactivity was excreted mainly through the kidneys in patients with relapsed NHL who received iodine-131-labeled rituximab. The percentage of radioactivity excreted in the urine was almost 50% at the time of the whole-body half-life [27].

The combined use of rituximab and other agents with different mechanisms of action may be one of the optimal solutions for overcoming rituximab resistance.

The use of other anti-CD20 mAbs, such as ofatumumab, obinutuzumab, tositumomab, ⁹⁰Y-ibritumomab tiuxetan, and ¹³¹I-tositumomab, is another optimal solution for overcoming rituximab resistance.

Infusion reactions are one of the most commonly occurring adverse reactions with rituximab. Infusion reactions are mediated by cytokine release (cytokine release syndrome), not by immunoglobulin E (IgE) (type 1 hypersensitivities). When cytokines, such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), or interleukin-6 (IL-6), are released into the circulation from cells targeted by rituximab as well as immune effector cells recruited to the area, systemic symptoms such as rigor, fever, headache, nausea, sweating, itching, skin rash, cough, fatigue, hypertension, hypotension, tachycardia, angioedema, facial edema, hypoxemia, bronchospasm, pulmonary infiltrates, interstitial pneumonia, acute respiratory distress syndrome, myocardial infarction, ventricular fibrillation, and myocardial shock can occur. Infusion reactions usually occur within 30–120 min, most often with the first infusion. The symptoms are generally mild to moderate in severity and manageable in most patients. A slow initial rate of infusion is recommended to reduce the risk of infusion reactions. Prophylactic administrations of histamine antagonists (diphenhydramine) and antipyretic analgesics (acetaminophen) are also recommended. Steroids (hydrocortisone) should be considered in patients receiving rituximab not already in combination with steroids [41, 42].

LON is a recognized late complication of rituximab-combined chemotherapy, occurring at least 4 weeks after treatment. The incidence of LON has been reported at about 25%, and a high incidence of LON has been reported in patients treated with an intensive primary chemotherapy regimen, such as high-dose therapy followed by stem cell transplantation [43]. LON has been attributed to rituximab, but the mechanism for developing LON remains unclear. It has been suggested that perturbation of stromal-derived factor-1 (SDF-1) during B-cell recovery following rituximab treatment retards neutrophil emergence from the bone marrow. SDF-1 is a chemokine required for early B-cell development and retention of the B-lineage and granulocytic precursors in the bone marrow microenvironment [44]. It has been demonstrated that the FcγRIIIa 158 V/V and V/F polymorphisms are associated with developing LON. This suggests that in patients with a high-affinity genotype, depletion of B cells may be more profound, subsequently stimulating increased bone marrow lymphopoiesis and resulting in less effective granulopoiesis [45]. The clinical course of LON was generally self-limited and not associated with severe infections.

Reactivation of hepatitis B virus (HBV) infection is a well-recognized complication in hepatitis B surface antigen (HBsAg)-positive cancer patients when they undergo cytotoxic chemotherapy. However, HBV reactivation has been reported to occur not only in HBsAg-positive patients but also in patients with a resolved HBV infection who are negative for HBsAg but positive for antibodies against the hepatitis B core antigen (anti-HBc) and/or antibodies against the hepatitis B surface antigen (anti-HBs) undergoing cytotoxic chemotherapy. HBV reactivation has been previously reported to be much less common in patients with resolved HBV, but with the recent increase in the use of rituximab, reports have increased. Since HBV reactivation in patients with resolved HBV is associated with a higher risk of fulminant hepatic failure, patients with resolved HBV receiving rituximab should be closely monitored with HBV DNA for a more prolonged period of time, even after completion of rituximab, so as to promptly start administration of nucleoside/nucleotide analogs to prevent HBV reactivation [46–50].

It has been observed that opportunistic infections, such as pneumocystis jiroveci pneumonia (PCP) or some viral infections, increase in patients receiving rituximab. However, the exact influence of rituximab on incidence of infection is still controversial. It has been suggested that, although rituximab by itself does not increase the incidence or change the spectrum of infections in hematologic patients, a possible influence of concomitant immunosuppressive medication such as chemotherapy and/or significant doses of glucocorticoids on the frequency of infections may be present. Screening and/or prophylaxis should be considered in patients receiving rituximab in combination with other immunosuppressive medications to minimize infectious complications. Sulfamethoxazole-trimethoprim prophylaxis against PCP is generally recommended [51].

Although the mechanism of action of alemtuzumab has yet to be clarified, proposed mechanisms of action of alemtuzumab include ADCC, CDC, and induction of apoptosis. ADCC is mediated by immune effector cells binding to alemtuzumab-coated cells, and this requires alemtuzumab to bind through the Fc region to FcγRs on immune effector cells. CDC is mediated by the recruitment of components of the complement cascade through the Fc region. Both ADCC and CDC are better mediated through the IgG1 constant region [55–57]. Alemtuzumab has been shown to induce not only caspase-dependent apoptosis but also caspase-independent and lipid raft-dependent apoptosis in primary CLL cells [59].