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Although the CD20-targeted monoclonal antibody rituximab (RTX) has revolutionized the therapeutic landscape for B-cell malignancy, relapsed and refractory disease due to RTX resistance continue to constitute major challenges, illustrating the need for better therapies. Here, we apply drug-free macromolecular therapeutics (DFMT) that amplifies CD20 cross-linking to enhance apoptosis in RTX-resistant cells. Bispecific engager (anti-CD20 Fab' conjugated with oligonucleotide1) pretargets CD20 and the deletion of Fc-region minimizes its premature endocytosis in resistant cells that rapidly internalize and consume CD20/RTX complexes. Second-step delivery of multivalent polymeric effector (linear copolymer conjugated with multiple copies of complementary oligonucleotide 2) simultaneously hybridizes multiple CD20-bound engagers and strengthens CD20 ligation. Moreover, the restoration of CD20 expression by the pretreatment of cells with a polymer-gemcitabine conjugate, a CD20 expression enhancer, unleashes the full potential of DFMT in the CD20-deficient resistant cells. Hence, amplification of CD20 cross-linking is achieved by (1) the enhancement of surface CD20 accessibility, (2) the increase in CD20 expression, and (3) multimeric CD20 binding, which ultimately translates into the amplified activation of a wide range of innate apoptotic responses. We demonstrated that the altered molecular signaling pathway that originally results in RTX resistance could be circumvented and compensated by other DFMT-augmented pathways. Of note, our preliminary data provide proof-of-concept that CD20 cross-linking amplification emerges as an important strategy for overcoming RTX resistance.

CD20 is a differentiation related cell surface phosphoprotein that is expressed during early pre-B cell stages until plasma cell differentiation, and is a suitable molecular target for B-cell malignancies by monoclonal antibodies such as rituximab, ofatumumab, obinutuzumab and others. CD20 expression is confirmed in most B-cell malignancies; however, the protein expression level varies in each patient, even in de novo tumors, and down-modulation of CD20 expression after chemoimmunotherapy with rituximab, resulting in rituximab resistance, has been recognized in the clinical setting. Recent reports suggest that genetic and epigenetic mechanisms are correlated with aberrantly low CD20 expression in de novo tumors and relapsed/refractory disease after using rituximab. Furthermore, some targeting drugs, such as lenalidomide, bortezomib and ibrutinib, directly or indirectly affect CD20 protein expression. CD20-negative phenotypically-changed DLBCL after rituximab use tends to show an aggressive clinical course and poor outcome with resistance to not only rituximab, but also conventional salvage chemo-regimens. Understanding of the mechanisms of CD20-negative phenotype may contribute to establish strategies for overcoming chemo-refractory B-cell malignancies. In this manuscript, recent progress of research on molecular and clinical features of CD20 protein and CD20-negative B-cell malignancies was reviewed.

Background: Mantle cell lymphoma (MCL), a B-cell lymphoma, pursues a relatively aggressive course, is resistant to long-term remission, and is associated with a poor prognosis. There is a pressing need for innovative treatment approaches against MCL. One such approach is targeted delivery of cytotoxic drugs to MCL cells., Materials and Methods: In the current investigation, we pursued a strategy to employ retinoid-based or curcumin-based nanoscale delivery particles, called nanodisks (NDs), for targeted drug delivery to MCL cells (Granta), and human follicular lymphoma (HF-1) cells. The cells were incubated with NDs made of CD20 single-chain variable antibody fragment (scFv)/apolipoprotein A-1 fusion protein, and loaded with either all-trans retinoic acid (ATRA) or curcumin, and cell apoptosis was measured using flow cytometry., Results and Conclusion: At 10 μM, curcumin-ND induced cell death more effectively than ATRA-ND. Combination of curcumin-ND and ATRA-ND significantly enhanced the biological activity of these drugs against lymphoma cells compared to individual treatments., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Purpose: Obinutuzumab (GA101), a novel glycoengineered type II anti-CD20 monoclonal antibody, demonstrated responses in single-arm studies of patients with relapsed/refractory non-Hodgkin lymphoma. This is the first prospective, randomized study comparing safety and efficacy of obinutuzumab with rituximab in relapsed indolent lymphoma. The primary end point of this study was the overall response rate (ORR) in patients with follicular lymphoma after induction and safety in patients with indolent lymphoma., Patients and Methods: A total of 175 patients with relapsed CD20(+) indolent lymphoma requiring therapy and with previous response to a rituximab-containing regimen were randomly assigned (1:1) to four once-per-week infusions of either obinutuzumab (1,000 mg) or rituximab (375 mg/m(2)). Patients without evidence of disease progression after induction therapy received obinutuzumab or rituximab maintenance therapy every 2 months for up to 2 years., Results: Among patients with follicular lymphoma (n = 149), ORR seemed higher for obinutuzumab than rituximab (44.6% v 33.3%; P = .08). This observation was also demonstrated by a blinded independent review panel that measured a higher ORR for obinutuzumab (44.6% v 26.7%; P = .01). However, this difference did not translate into an improvement in progression-free survival. No new safety signals were observed for obinutuzumab, and the incidence of adverse events was balanced between arms, with the exception of infusion-related reactions and cough, which were higher in the obinutuzumab arm., Conclusion: Obinutuzumab demonstrated a higher ORR without appreciable differences in safety compared with rituximab. However, the clinical benefit of obinutuzumab in this setting remains unclear and should be evaluated within phase III trials., Competing Interests: Authors' disclosures of potential conflicts of interest are found in the article online at www.jco.org. Author contributions are found at the end of this article., (© 2015 by American Society of Clinical Oncology.)

Activation of the complement system by tumor cells was long believed to only benefit the host. Overexpression of complement inhibitors by many tumor cell types and results obtained in several experimental animal models were all in agreement with this hypothesis. However, recent reports imply that the situation is more complex than initially believed and that under certain circumstances tumor cells may use complement to their own advantage, e.g. by recruitment of suppressor T cells or promoting local angiogenesis. Such a dual role of complement may also be apparent when considering the effect of therapeutic monoclonal antibodies (mAb) used to successfully treat B cell malignancies, such as CD20 mAbs. Some argue that besides direct tumor cell killing by mAbs, two main immune effector mechanisms, complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC), may be competing with each other. Experiments aiming at answering the question whether complement is our friend or foe in mAb therapy ended up with seemingly contradictory conclusions. Herein, we revisit the existing knowledge on this pivotal issue based on rituximab and other anti-CD20 mAb as a model of therapeutic agents., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We have previously shown that rituximab has a significant radiosensitizing effect on Raji cells in vitro. To investigate whether calcium signals participate in rituximab- and radiation-induced cell death in Raji cells, confocal laser scanning microscopy was used to detect kinetic changes in intracellular free calcium concentration ([Ca2+]i). Cell survival, the rates of apoptosis in Raji cells and the kinetics of γ-H2AX foci induction and loss were also evaluated. X-irradiation of Raji cells induced an initial increase of [Ca2+]i in both the presence and absence of extracellular calcium, followed by a decrease in [Ca2+]i over time. Rituximab enhanced both the amplitude and the duration of intracellular calcium signals in the irradiated cells. EGTA significantly inhibited radiation- or radiation/rituximab combination treatment-induced apoptosis. However, the calcium chelators EGTA and BAPTA/AM conferred no survival advantage on the irradiated cells. Furthermore, although no significant difference was seen after 1h, the treatment of cells with a combination of irradiation and rituxiamb caused an increase of γ-H2AX foci when compared with irradiated cells after 8h. Both EGTA and BAPTA/AM suppressed the number of γ-H2AX foci induced by either radiation or radiation combined with rituximab. Our results suggest that rituximab increases the level of [Ca2+]i in irradiated Raji cells. The entry of calcium from the extracellular space plays an essential role in [Ca2+]i-dependent radiation-induced apoptosis in Raji cells. The calcium chelators inhibited the formation of γ-H2AX foci, which are thought to prevent the activation of Ca2+/Mg(2+)-dependent endonucleases and subsequent DNA fragmentation. The calcium chelators most likely modulate only particular features of apoptosis and fail to change the fates of cells that are already committed to die., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Monoclonal antibodies such as rituximab and alemtuzumab show considerable therapeutic efficacy in chronic lymphocytic leukaemia (CLL). Aiming to further improve antineoplastic efficacy, the trifunctional bispecific antibody FBTA05 was developed. FBTA05 is thought to function by simultaneously binding B cells and T cells by its variable regions and by recruiting FcγR-positive accessory immune cells by its intact Fc region. As it was previously shown that this antibody shows considerable cytotoxicity towards a spectrum of B-cell lymphoma cell lines, we here tested its potential efficacy ex vivo against malignant B-CLL cells. Therefore, we assessed the capacity of increasing concentrations of FBTA05 to bind to neoplastic cells, to induce cytotoxicity (comparing it with rituximab and alemtuzumab) and cytokine release. We evaluated the results with respect to the extent of CD20 expression, the effector:target cell ratio as well as with the patients' overall effector cell status. Thus, we show that, although FBTA05-elicited cytotoxicity was comparable with that induced by alemtuzumab, it considerably exceeded the antineoplastic effects of rituximab. Noteworthy, FBTA05 shows effective elimination of malignant B cells even if CD20 surface expression is low. Importantly, a high grade of cytotoxicity was associated with the induction of T-cell proliferation and the concomittant release of interferon-γ and interleukin-6, thus overcoming the detrimental effects of an unfavourable effector:target cell ratio. In conclusion, we here present novel evidence for the therapeutic efficacy of the trifunctional, bispecific antibody FBTA05 in CLL and provide evidence for the importance of immune-mediated mechanisms conveying the cytotoxic effects against malignant B lymphocytes.

Purpose: To report the pharmacokinetics of ¹³¹I-rituximab a patient with a CD20 positive non-Hodgkin Lymphoma who has received ¹³¹I-rituximab as consolidation treatment after remission induction and to evaluate the effect of radioiodination on the biological properties of rituximab., Results: The patient was a 65-year-old male with a relapsed CD20 positive follicular non-Hodgkin Lymphoma. After induction therapy the patient was in partial remission. Following administration of a diagnostic dose of 185 MBq ¹³¹I-rituximab, remaining lesions were identified on the wholebody scans. The patient then received a therapeutic dose of 1000 MBq ¹³¹I-rituximab. The uptake by the tumor in the right axilla was 0.17-0.21% of the injected dose. The calculated biological half-life of ¹³¹I-rituximab was 684 hrs. This biological half-life corresponded well with the half-life of unlabeled rituximab which was approximately 720 hrs., Discussion and Conclusion: Even though radioiodination of rituximab results in a reduced binding capacity, whole body scans demonstrated localization of ¹³¹I-rituximab in the tumor area. This observation supports the specific targeting of ¹³¹I-rituximab. The half-life of ¹³¹I-rituximab corresponded to the half-life of unlabeled rituximab. Hence, the pharmacokinetics of ¹³¹ I-rituximab was not relevantly affected by the radioiodination process.

The aim of adoptive T-cell therapy of cancer is to selectively confer immunity against tumor cells. Autoimmune side effects, however, remain a risk, emphasizing the relevance of a suicide mechanism allowing in vivo elimination of infused T cells. We investigated the use of human CD20 as suicide gene in T-lymphocytes. Potential effects of forced CD20 expression on T-cell function were investigated by comparing CD20- and mock-transduced cytomegalovirus (CMV) specific T cells for cytolysis, cytokine release and proliferation. The use of CD20 as suicide gene was investigated in CMV specific T cells and in T cells genetically modified with an antigen specific T-cell receptor. No effect of CD20 on T-cell function was observed. CD20-transduced T cells with and without co-transferred T-cell receptor were efficiently eliminated by complement dependent cytotoxicity induced by therapeutic anti-CD20 antibody rituximab. The data support the broad value of CD20 as safety switch in adoptive T-cell therapy.

Here we describe two cases of mucosa-associated lymphoid tissue (MALT) lymphoma with monoclonal immunoglobulins (Igs). The first case was a 77-year-old man with primary lymphoma of the lung. Immunoelectrophoresis detected IgM-kappa in serum and kappa light chain excretion into urine. Three months after treatment with single-agent rituximab, a large amount of pleural fluid was found to have accumulated. The fluid contained CD5(-), CD10(-), CD19(+), CD38(+) and CD138(-/+) lymphoma cells with lymphoplasmacytoid appearance. Although a small fraction of the cells were CD20(+), the majority of the lymphoma cells were negative and expressed surface-membrane IgM-kappa at low levels. The cells possessed a karyotype of 46, XY, t(11;18)(q21;q21). The second case was a 55-year-old man who underwent total gastrectomy due to gastric perforation. Surgical specimens demonstrated the histopathological features of MALT lymphoma associated with plasma cell differentiation. The lymphoma cells had a 46, XY, t(11;18)(q21;q21) karyotype. Monoclonal Igs detected were serum IgA (M)-kappa and urinary kappa light chain. The patient was subsequently treated with six cycles of R-CVP (rituximab, cyclophosphamide, vincristine and prednisolone) ; however, serum monoclonal Ig levels were not affected. The lymphoma cells in both cases may have contained two populations, a rituximab-sensitive CD20(+) population and a rituximab-resistant population that had differentiated into the Ig-secreting plasma cell stage.

Purpose: The treatment of primary central nervous system lymphoma (PCNSL) and its subset, primary intraocular lymphoma (PIOL), remains of limited efficiency, and salvage therapies are often used without prior testing in adequate animal models. Most PNCSL/PIOL are aggressive B-cell malignancies. Two animal models that closely mimic the human situation were established to evaluate the efficiency of intravitreal and intracerebral anti-CD20 monoclonal antibody (rituximab) injections., Methods: Human CD20-transfected murine B-lymphoma cells (38C13 CD20(+)) were inoculated in the vitreous through the pars plana or in the caudate nucleus with the use of a stereotaxic frame in immunocompetent syngeneic mice. Animals were monitored clinically and by funduscopic and histologic examination. Rituximab was injected intravitreally or intracerebrally. Occurrences of exophthalmia, neurologic disturbance, and weight loss were monitored over 2 months., Results: Inoculation of 38C13 CD20(+) cells in the eye or the brain resulted in tumor occurrence after a median of 15 days or 22 days, respectively, with histologic characteristics closely resembling those of PIOL and PCNSL. Local rituximab injections eradicated tumor colonization in more than half the graft recipients and inhibited tumor progression significantly in the others compared with progression in mice that underwent grafting with the control 38C13 cell line (no human CD20 expression) and in mice that underwent grafting with 38C13 CD20(+) cells that received local injections of an irrelevant antibody (trastuzumab)., Conclusions: Inoculation of native or human CD20-transfected murine 38C13 cells in the vitreous or the brain of immunocompetent mice provides useful novel models for evaluating the biology and treatment of PIOL and PCNSL. Intravitreal and intracerebral rituximab injections reduced tumor occurrence and growth in each model.

The anti-CD20 monoclonal antibody rituximab has been less successful in treating chronic lymphocytic leukemia (CLL) than lymphoma, possibly due to the lower density of CD20 on B lymphocytes from CLL patients than on those from lymphoma patients. This lowering may result from insufficiency of one of the transcription factors of cd20. Of these, purine-rich box-1 (PU.1) is poorly expressed in CLL. To estimate its weight in CD20 expression, pu.1 cDNA was transfected into CLL B cells and shown to raise the membrane expression of CD20 and to improve the rituximab-induced lysis of transfected cells. Granulocyte macrophage colony-stimulating factor and all-trans-retinoic acids were not involved in the defective expression of PU.1 or the excessive methylation of the pu.1 gene, because 6 of 14 CLL samples tested were normally methylated. This was confirmed by the failure of DNA methyltransferase inhibitors to restore pu.1 transcription in hypermethylated CLL, and, in fact, the expression of PU.1 was down-regulated by excessive expression of the FMS proto-oncogene-like tyrosine kinase 3 (Flt3) receptor. This abnormality is consistent with our finding of elevated levels of Flt3 ligand (FL) in 20 of 23 CLL sera tested. We propose that FL-dependent increased Flt3 signaling prevents the expression of PU.1, which down-regulates that of CD20, and accounts for resistance of leukemic B cells to rituximab-induced lysis.

Immunotherapy with rituximab (chimeric anti-CD20 monoclonal antibody, Rituxan), alone or in conjunction with chemotherapy, has significantly improved the treatment outcome of lymphoma patients. Via an elusive mechanism, a subpopulation of patients becomes unresponsive and/or relapses. To recapitulate various aspects of acquired resistance, rituximab-resistant (RR) clones were established from lymphoma lines and compared with parental cells. Surface CD20 expression was diminished in the clones. The clones neither responded to rituximab-mediated growth reduction or complement-dependent cytotoxicity nor underwent apoptosis in response to cross-linked rituximab. Rituximab failed to chemosensitize the RR clones, which exhibited constitutive hyperactivation of the nuclear factor-kappaB and extracellular signal-regulated kinase 1/2 pathways, leading to overexpression of B-cell lymphoma protein 2 (Bcl-2), Bcl-2-related gene (long alternatively spliced variant of Bcl-x gene), and myeloid cell differentiation 1 and higher drug resistance. Unlike parental cells, rituximab neither inhibited the activity of these pathways nor diminished the expression of resistant factors. Pharmacologic inhibitors of the survival pathways or Bcl-2 family members reduced the activity of these pathways, diminished antiapoptotic protein expression, and chemosensitized the RR clones. These novel in vitro results denote that continuous long-term rituximab exposure culminates in RR clones that do not respond to rituximab-mediated effects, have altered cellular signaling dynamics, and exhibit different genetic and phenotypic properties compared with parental cells. The data also reveal that although RR clones exhibit higher resistance to rituximab and cytotoxic drugs, these clones can be chemosensitized following treatment with pharmacologic inhibitors (e.g., dehydroxymethylepoxyquinomicin, bortezomib, PD098059) that target survival/antiapoptotic pathways. The findings also identify intracellular targets for potential molecular therapeutic intervention to increase treatment efficacy. The significance and potential clinical relevance of the findings are discussed.

Thrombotic thrombocytopenic purpura (TTP) is a haematological syndrome characterised by a dramatic onset requiring an urgent treatment with plasma exchange (PE). However, the prognosis is still dismal for PE related complications, a rate of failure and remarkable frequencies of relapse. TTP post transplantation is largely described as an outstanding, unusual complication of allogenic transplantation, but it is rarely mentioned after autologous transplantation. We describe a 62-year-old Caucasian patient who presented with TTP, accompanied by renal failure, after an autologous transplantation for multiple myeloma. PE together with hemodialysis was rapidly initiated but without any benefit. Since empirical administration of Rituximab, anti CD20 monoclonal antibody,was reported to be effective, we administered four courses of Rituximab inducing a complete remission of TTP and subsequently of the renal failure. This response to Rituximab in TTP post transplantation is suggestive of a possible implication of B-lymphocytes in the pathogenesis of TTP and it paves the way for an investigational approach in this settings.

Rituximab, an anti-CD20 human-mouse chimeric monoclonal antibody has been shown to improve response rates when it is combined with standard salvage chemotherapy in patients with relapsed or refractory intermediate-grade B-cell non-Hodgkin's lymphoma. A vast majority of these patients subsequently undergo high-dose therapy followed by stem cell transplantation. However, the impact of rituximab on stem cell mobilization kinetics is not well characterized. The purpose of this study was to study the effect of high-dose rituximab given with chemotherapy on stem cell mobilization in patients with intermediate-grade B-cell non-Hodgkin's lymphoma. Thirty-six patients received ifosfamide, etoposide, and rituximab followed by filgrastim for stem cell mobilization. The chemotherapy regimen was well tolerated. Thirty-four of 36 patients (94%) were able to mobilize at least 2 x 10(6) CD34+ cells/kg body weight after a median of 2 apheresis procedures. The median CD34+ cell dose collected per kilogram of recipient body weight was 6.5 x 10(6) (range, 4.65-31.15). All patients who subsequently underwent high-dose chemotherapy and stem cell transplantation experienced sustained engraftment. In conclusion, high-dose rituximab given during stem cell mobilization does not negatively affect stem cell mobilization kinetics.

In the present phase II study, we tested the efficacy of a single course of rituximab (375 mg/m2 on days 1, 8, 15 and 22) as treatment for relapsed myeloma. The rationale for this study was the identification of a population of clonotypic CD20+ B cells that are believed to be precursors of malignant plasma cells. In addition, CD20 was expressed on 10% and 50% of bone marrow plasma cells in two of the ten patients enrolled. Following rituximab treatment, none of the patients achieved an objective response. Two patients had stable disease at month 6, the predefined end of the study, while, at that time, two patients were classified as having progressive disease. One patient opted to withdraw from the study at month 3, at which time he had stable disease. The other five patients had to be withdrawn early from the post-treatment observation because of need of salvage therapy for progressive disease. WHO grade

Intravascular lymphoma is a rare aggressive systemic neoplasm with neurological and cutaneous presentation, which commonly eludes the diagnosis ante mortem. We document a case of intravascular lymphoma in a 65-year-old man who presented with altered mental status, fevers and weight loss. The diagnosis was made by random skin biopsies demonstrating an intravascular, CD20 positive cellular infiltrate. Initial treatment consisted of cyclophosphamide, vincristine, doxorubicin, prednisone and rituximab. Because of disease progression, the therapy was switched to weekly rituximab after the first cycle. A complete response was observed with resolution of symptoms and laboratory abnormalities. The patient remains in remission 3 years after completion of therapy. This is the first case report to describe a sustained remission following single-agent rituximab in the patient with neurologic involvement of intravascular lymphoma who failed antracycline-based therapy. Single-agent rituximab has activity in this disease and may be considered as a treatment option.

Currently available monoclonal antibodies against the B cell surface antigen CD20 have been employed to explore whether specific inhibition of B cells may help improve the outcome of idiopathic membranous nephropathy (IMN) and may avoid the side effects of steroids and immunosuppressants. This prospective, observational study evaluated the 1-yr outcome of eight IMN patients with persistent (>6 mo) urinary protein excretion > 3.5 g/24 h given four weekly infusions of the anti-CD20 antibody rituximab (375 mg/m(2)). At 3 and 12 mo, proteinuria significantly decreased from mean (+/- SD) 8.6 +/- 4.2 to 4.3 +/- 3.3 (-51%, P < 0.005) and 3.0 +/- 2.5 (-66%, P < 0.005) g/24 h, albumin fractional clearance from 2.3 +/- 2.1 to 1.2 +/- 1.7 (-47%, P < 0.05) and 0.5 +/- 0.6 (-76%, P < 0.003), and serum albumin concentration increased from 2.7 +/- 0.5 to 3.1 +/- 0.3 (+21%, P < 0.05) and 3.5 +/- 0.4 (+41%, P < 0.05) mg/dl. At 12 mo, proteinuria decreased to < or =0.5 g/24 h or < or =3.5 g/24 h in two and three patients, respectively. Proteinuria decreased in the remaining patients by 74%, 44%, and 41%, respectively. Body weight, diastolic BP, and serum cholesterol progressively decreased in parallel with an improvement of edema in all patients. Renal function stabilized (Delta1/creatinine: +0.002 +/- 0.007). CD20 B lymphocytes fell below normal ranges up to study-end. No patient had major drug-related events or major changes in other laboratory parameters. Rituximab thus promotes sustained disease remission in patients otherwise predicted to progress to ESRD, and it is safe. The long-term risk/benefit profile of this novel, disease-specific approach seems much more favorable to that of commonly employed immunosuppressive drugs.

Waldenström's macroglobulinemia is a low-grade lymphoplasmacytoid lymphoma characterized by CD20 expression on malignant cells. Several studies have indicated that the anti-CD20 monoclonal antibody rituximab has activity against this disease. Thus, we performed a prospective study in which 17 previously untreated patients with symptomatic macroglobulinemia were treated with rituximab 375 mg/m2 intravenously for 4 weeks. Three months after completion of rituximab, patients without evidence of progressive disease received repeat 4-week courses of this agent. Six patients (35%) achieved a partial response after extended treatment with rituximab. Median time to response was 3 months. The median time to progression (TTP) for all patients was 13 months. One of 6 responding patients has progressed at 10 months, while the other 5 patients remain progression free with a follow-up range of > 22-40 months. Eight patients (47%) were rated as stable disease, and their median TTP was 9 months. Treatment with rituximab was well tolerated and was not associated with myelosuppression; one third of the patients experienced infusion-related toxicity, usually fever and chills of mild degree. Our prospective trial of extended rituximab therapy for previously untreated patients with Waldenström's macroglobulinemia indicates that this agent is active, well tolerated, and might be associated with a long period without the need for further treatment. Studies that will combine rituximab with chemotherapy or with other monoclonal antibodies might be of interest.

Background and Objectives: Monoclonal antibody IDEC-C2B8 (rituximab) has been shown to be highly effective in the treatment of non-Hodgkin's lymphomas (NHL). The present study was designed to investigate relationships between the efficacy of IDEC-C2B8 and expression of CD20, presence of complement, and effects of differently acting chemotherapeutic agents used in lymphoma treatment (doxorubicin, mitoxantrone, cladribine, bendamustine)., Design and Methods: DOHH-2, WSU-NHL and Raji lymphoma cell lines and ex vivo cells from patients with chronic lymphocytic leukemia (CLL) (n=17) and leukemic B-cell lymphomas (n=9) were studied. Additionally, the effect of interleukin (IL)-2, IL-4, IL-6, IL-13, granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)alpha on expression of CD20 molecules per cell was determined., Results: We demonstrate that 10 mg/mL rituximab saturated 80-95% of CD20 molecules per cell in all tested lymphoma samples. Although rituximab induced only a minor increase of apoptosis, combinations of rituximab with different cytotoxic drugs significantly decreased the IC(30)- and IC(50) dosages of the chemotherapeutic agents necessary for induction of apoptosis irrespective of addition of complement, demonstrating a chemosensitizing effect of rituximab in combination with cytotoxic drugs in the neoplastic lymphocytes. This effect seemed to be independent of the percentage of saturated CD20 molecules. After addition of caspase inhibitors to the cell lines incubated with rituximab and cytotoxic agents, caspase-7 and -8 were found, by Western blotting, to be the executioner caspases, possibly explaining the rituximab-sensitized apoptosis. Preincubation of lymphoma cells with cytokines did not alter the expression of CD20; IL-2 and IL-4 even decreased the rate of apoptosis., Interpretation and Conclusions: We conclude that rituximab sensitizes lymphoma cells to the effect of differently acting cytotoxic drugs used in lymphoma treatment, that this effect does not require complement, and that caspase-7 and -8 may represent the main executioner caspases in chemosensitization by rituximab.

The use of serotherapy to treat patients with plasma cell dyscrasias (PCDs) has been sought by us and others. Candidate antigens that have been targeted or proposed for targeting in PCDs include the immunoglobulin idiotype, CD19, CD38, CD54, CD126, HM1.24, and Muc-1 core protein. Unfortunately, many of these antigens are not ideal for use in serotherapy since they are not selectively expressed, are either shed or secreted, or have not been fully characterized. Serotherapy with an anti-CD19 monoclonal antibody (B4) conjugated to a blocked ricin toxin had no significant activity in patients with multiple myeloma (MM). Circulating CD20+ clonotypic B cells have been detected in the circulation of most MM and Waldenstrom's macroglobulinemia (WM) patients. Plasma cells from most WM patients express CD20, but most MM patient plasma cells either lack CD20 or express it weakly. In view of recent successes with anti-CD20-directed serotherapy in other B-cell malignancies, we initiated a phase II trial to study the anti-CD20 monoclonal antibody rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, CA, and Genentech, Inc, San Francisco, CA) in patients with MM. We describe two PCD patients (one with WM and one with MM) who responded to therapy. By flow cytometric analysis, CD20+ plasma cells and B cells present in the bone marrow and peripheral blood of a patient with MM disappeared with response to rituximab therapy. However, residual CD20- tumor cells remained in the bone marrow following rituximab therapy, and after 6 months this patient progressed with CD20- myeloma cells. As a potential strategy to overcome this limitation, we demonstrated that interferon-gamma at pharmacologically achievable levels induced CD20 expression on these CD20- plasma cells, consistent with our recent findings that interferon-gamma is a potent inducer of CD20 expression on MM patient plasma cells and B cells. We also characterize a response to rituximab with a decrease in paraprotein and resolution of anemia in a patient with WM whose response to rituximab is ongoing after 19+ months. This preliminary experience supports the potential use of serotherapy targeting CD20 in PCDs. Our studies further suggest that interferon-gamma may enhance CD20 expression on MM plasma cells, thereby increasing their susceptibility to anti-CD20 monoclonal antibody therapies.