Generation and characterization of a monoclonal antibody against human BCL6 for immunohistochemical diagnosis.

BACKGROUND: Human B-cell lymphoma 6 (BCL6) gene, usually coding protein of 706 amino acids, is closely associated with large B cell lymphoma. Researches showed that protein mutation or change of expression levels usually happened in the mounting non-hodgkin lymphoma (NHL). Thus BCL6 is considered to be involved in germinal center (GC)-derived lymphoma. RESULTS: The BCL61-350 gene codons were optimized for prokaryotic system. After expression of BCL61-350 in E. coli, the BCL61-350 protein was purified with Ni column. Then the BCL61-350 protein, mixing with QuickAntibody-Mouse5W adjuvant, was injected into Balb/c mice. After immunization and cell fusion, a stable cell line named 1E6A4, which can secrete anti-BCL6 antibody, was obtained. The isotype of 1E6A4 mAb was determined as IgG2a, and the affinity constant reached 5.12×1010 L/mol. Furthermore, the specificity of the mAb was determined with ELISA, western blot and immunohistochemistry. Results indicated that the 1E6A4 mAb was able to detect BCL6 specifically and sensitively. CONCLUSIONS: BCL61-350 antigen has been successfully generated with an effective and feasible method, and a highly specific antibody named 1E6A4 against BCL6 has been screened and characterized in this study, which was valuable in clinical diagnosis.

Safety and tolerability of conditioning chemotherapy followed by CD19-targeted CAR T cells for relapsed/refractory CLL.

BACKGROUND: Subgroups of patients with relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) exhibit suboptimal outcomes after standard therapies, including oral kinase inhibitors. We and others have previously reported on safety and efficacy of autologous CD19-targeted CAR T-cells for these patients; here we report safety and long-term follow-up of CAR T-cell therapy with or without conditioning chemotherapy for patients with R/R CLL and indolent B-cell non-Hodgkin lymphoma (B-NHL). METHODS: We conducted a phase 1 clinical trial investigating CD19-targeted CAR T-cells incorporating a CD28 costimulatory domain (19-28z). Seventeen of 20 patients received conditioning chemotherapy prior to CAR T-cell infusion. Five patients with CLL received ibrutinib at the time of autologous T-cell collection and/or CAR T-cell administration. RESULTS: This analysis included 16 patients with R/R CLL and 4 patients with R/R indolent B-NHL. Cytokine release syndrome (CRS) was observed in all 20 patients but grades 3 and 4 CRS and neurological events were uncommon (10% for each). Ex vivo expansion of T-cells and proportions of CD4+/CD8+ CAR T-cells with CD62L+CD127+ immunophenotype were significantly greater in patients on ibrutinib at leukapheresis. Three of 12 evaluable CLL patients receiving conditioning chemotherapy achieved CR (two had minimal residual disease-negative CR). All patients achieving CR remained progression-free at median follow-up of 53 months. CONCLUSION: Conditioning chemotherapy and 19-28z CAR T-cells were acceptably tolerated across investigated dose levels in heavily pretreated patients with R/R CLL and indolent B-NHL, and a subgroup of patients achieved durable CR. Ibrutinib therapy may modulate autologous T-cell phenotype. TRIAL REGISTRATION: ClinicalTrials.gov NCT00466531. FUNDING: Juno Therapeutics.

YY1 binds to the E3' enhancer and inhibits the expression of the immunoglobulin κ gene via epigenetic modifications.

The rearrangement and expression of immunoglobulin genes are regulated by enhancers and their binding transcriptional factors that activate or suppress the activities of the enhancers. The immunoglobulin κ (Igκ) gene locus has three important enhancers: the intrinsic enhancer (Ei), 3' enhancer (E3'), and distal enhancer (Ed). Ei and E3' are both required for Igκ gene rearrangement during early stages of B-cell development, whereas optimal expression of the rearranged Igκ gene relies on both E3' and Ed. The transcription factor YY1 affects the expression of many genes involved in B-cell development, probably by mediating interactions between their enhancers and promoters. Herein, we found that YY1 binds to the E3' enhancer and suppresses Igκ expression in B lymphoma cells by epigenetically modifying the enhancer. Knocking down YY1 enhanced Igκ expression, which was associated with increased levels of E2A (encoded by the TCF3 gene) and its binding to the E3' enhancer. Moreover, in germinal centre B cells and plasma cells, YY1 expression was reversely associated with Igκ levels, implying that YY1 might facilitate antibody affinity maturation in germinal centre B cells through the transient attenuation of Igκ expression.

Unleashing the clinical power of T cells: CD19/CD3 bi-specific T cell engager (BiTE®) antibody construct blinatumomab as a potential therapy.

Multi-agent chemotherapy is the standard treatment for most B cell malignancies. Since chemotherapy can be associated with significant toxicity and since relapses resistant to chemotherapy often develop, new therapies are needed. Blinatumomab (AMG 103 or MT103) is a late-stage candidate in clinical development, which belongs to a novel class of antibody constructs termed bi-specific T cell engager antibodies. This antibody construct has dual specificity for CD19 and CD3 and can re-direct polyclonal cytotoxic T lymphocytes toward the tumor. This review focuses on the pre-clinical and clinical development of blinatumomab as a powerful new tool in the treatment of B cell malignancies.

Immunosuppressive CD14+HLA-DR(low)/- monocytes in B-cell non-Hodgkin lymphoma.

Immunosuppression is a known risk factor for B-cell non-Hodgkin lymphoma (NHL), yet mechanisms of tumor-associated immunosuppression remain to be fully characterized. We examined the immunophenotype of 40 NHL patients and 27 age-matched healthy volunteers to better understand systemic immune suppression. NHL peripheral blood mononuclear cells had significantly decreased interferon-γ production and proliferation. This suppression was not the result of regulatory T cells, interleukin-6 or interleukin-10, as these factors were not different between NHL and healthy volunteers (controls). We were able to restore T-cell proliferation by removing NHL monocytes, suggesting that these monocytes are suppressive. This suppression was mediated in part through arginine metabolism as exogenous arginine supplementation partially overcame monocytes' suppression of T-cell proliferation in vitro and NHL patients had elevated arginase I in their plasma. NHL monocytes had impaired STAT1 phosphorylation and interferon-α production to CpG stimulation and a dendritic cell differentiation deficiency. Further studies demonstrated that monocytes from NHL patients had decreased HLA-DR and Tumor necrosis factor-α receptor II (CD120b) expression compared with controls (CD14(+)HLA-DR(low/-)CD120b(low)). Patients with increased ratios of CD14(+)HLA-DR(low/-) monocytes had more aggressive disease and suppressed immune functions. In summary, we report that CD14(+)HLA-DR(low/-) monocytes are a major and multifactorial contributor to systemic immunosuppression in NHL.

Adenovirus-mediated LIGHT gene modification in murine B-cell lymphoma elicits a potent antitumor effect.

Here, we investigated the antitumor effect of adenovirus-mediated gene transfer of LIGHT, the tumor-necrosis factor (TNF) superfamily member also known as TNFSF14, in the murine A20 B-cell lymphoma. LIGHT gene modification resulted in upregulated expression of Fas and the accessory molecule--intercellular adhesion molecule-1 (ICAM-1) on A20 cells and led to enhanced A20 cell apoptosis. LIGHT-modified A20 cells effectively stimulated the proliferation of T lymphocytes and interferon (IFN)-gamma production in vitro. Immunization of BALB/c mice with a LIGHT-modified A20 cell vaccine efficiently elicited protective immunity against challenge with the parental tumor cell line. Adenovirus-mediated gene transfer of LIGHT by intratumoral injection exerted a very potent antitumor effect against pre-existing A20 cell lymphoma in BALB/c mice. This adenovirus-mediated LIGHT therapy induced substantial splenic natural killer (NK) and cytotoxic T lymphocyte (CTL) activity, enhanced tumor infiltration by inflammatory cells and increased chemokine expression of CC chemokine ligand 21 (CCL21), IFN-inducible protein-10 (IP-10) and monokine induced by IFN-gamma (Mig) from tumor tissues. Thus, adenovirus-mediated LIGHT therapy might have potential utility for the prevention and treatment of B-cell lymphoma.

Novel anti-CD20 antibody TGLA with enhanced antibody-dependent cell-mediated cytotoxicity mediates potent anti-lymphoma activity.

Rituximab is the first anti-cancer antibody approved by the FDA for the treatment of B-cell lymphoma. However, its efficacy remains variable and often modest. Some patients are initially unresponsive to rituximab or later develop resistance to it, and require alternative therapies. Rituximab activity has been thought to involve antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and apoptosis. Present studies suggest that the patients unresponsive to rituximab may be helped with other CD20 antibodies with enhanced activities. In this study, we characterized a novel anti-CD20 chimeric antibody, TGLA, which binds to various B-cell lines specially and shares an epitope with rituximab. TGLA shows equal activities with rituximab, such as CDC, cell growth arrest and so on. Interestingly, TGLA also shows significant ADCC activity. Immunotherapeutic studies further show that TGLA is far more effective in delaying tumor growth than rituximab. These findings suggest that the ADCC-enhanced anti-CD20 antibody TGLA might be an alternative therapeutic agent for B-cell lymphoma.

TRU-016, a humanized anti-CD37 IgG fusion protein for the potential treatment of B-cell malignancies.

TRU-016, under development by Trubion Pharmaceuticals Inc and Facet Biotech Corp, is an intravenously administered anti-CD37 IgG fusion protein for the potential treatment of B-cell malignancies, including chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL), as well as for autoimmune and inflammatory diseases. TRU-016 was created by humanizing SMIP-016, a mouse/human chimeric protein that demonstrated antitumor activity against lymphoid malignancies in preclinical studies, including in human B-cell tumor mouse xenograft models. In addition, TRU-016 demonstrated synergistic or additive activity in NHL cells in combination with rituximab, rapamycin, doxorubicin and bendamustine. In a phase I/II clinical trial in refractory or relapsed patients with CLL or small lymphocytic lymphoma, TRU-016 was well tolerated, with clinical benefit and a reduced absolute lymphocyte count observed in all cohorts dosed at > 0.1 mg/kg. TRU-016 is a promising therapeutic agent for patients with B-cell lymphoid malignancies, especially patients refractory to standard treatment.

Combination treatment of malignant B cells using the anti-CD20 antibody rituximab and the anti-malarial artesunate.

Chemotherapy of non-Hodgkin's lymphoma is frequently hampered by drug resistance. The monoclonal antibody rituximab specifically targets the CD20 antigen and sensitizes B-cell lymphoma cells to standard anticancer drugs. In the present investigation, we analyzed, whether a combination of rituximab and artesunate may act in a complementary manner and eventually synergize in tumor cell killing. Artesunate is an anti-malarial drug, which also exerts profound activity towards cancer cells. While rituximab alone was minimally cytotoxic, rituximab increased cytotoxicity to artesunate in Ramos cells. Artesunate induced apoptosis, induced Fas/CD95 expression and the formation of reactive oxygen species (ROS) and resulted in a breakdown of mitochondrial membrane potential. This argues for the involvement of both receptor-driven extrinsic and mitochondrial intrinsic routes of apoptosis. Rituximab increased Fas/CD95 expression and ROS formation and decreased mitochondrial membrane potential ultimately leading to increased apoptosis induced by artesunate. The transcription factors YY1 and Sp1 are upstream regulators of apoptosis by controlling the expression of apoptosis-regulating genes. YY1 and Sp1 were down-regulated and Fas/CD95 was up-regulated by rituximab and artesunate indicating that artesunate activated the Fas/CD95 pathway and that rituximab increased the susceptibility of tumor cells to artesunate-induced apoptosis. Furthermore, rituximab affected the expression of antioxidant genes. The antibody decreased artesunate-induced up-regulation of catalase expression and increased artesunate-induced down-regulation of glutathione S-transferase-phi expression. Manganese-dependent superoxide dismutase expression was not changed by artesunate. Antioxidant proteins may help to detoxify artesunate-induced ROS. Rituximab reversed the artesunate-induced expression changes of antioxidant genes and, hence, reduced the detoxification capacity of Ramos cells. The effects of rituximab on antioxidant genes represent a novel mechanism of rituximab for chemosensitization.

GA-101, a third-generation, humanized and glyco-engineered anti-CD20 mAb for the treatment of B-cell lymphoid malignancies.

Glycart Biotechnology AG, Genentech Inc, F Hoffmann-La Roche Ltd, Biogen Idec Inc and Chugai Pharmaceutical Co Ltd are developing GA-101, a third-generation, humanized and glyco-engineered anti-CD20 IgG1 mAb, for the potential treatment of B-cell malignancies. Compared with classic type I CD20 antibodies (eg, rituximab), GA-101 binds with high affinity to the CD20 type II epitope, resulting in the induction of antibody-dependent cytotoxicity that is 5- to 100-fold greater than observed upon treatment with rituximab. GA-101 also exhibits superior direct cell killing properties than rituximab. In preclinical studies, GA-101 was significantly more potent and effective in depleting B-cells than rituximab, and induced dose-dependent antitumor activity, complete tumor regression and improved long-term survival in xenograft mouse models of B-cell malignancy. In a phase I/II clinical trial, GA-101 had a similar safety profile to rituximab, and exhibited promising efficacy in patients with relapsed/refractory CD20-positive lymphoid malignancies. At the time of publication, phase I/II trials for GA-101 were ongoing in B-cell malignancies. Based on preclinical and preliminary clinical data, GA-101 appears to be a promising therapeutic agent for CD20-positive B-cell lymphoid malignances, including non-Hodgkin lymphomas and chronic lymphocytic leukemia.

[Human/mouse chimeric anti-CD20 monoclonal antibody enhances antigen presentation in dendritic cells and induces anti-lymphoma CTL effects].

In order to investigate the cellular immunoresponses mediated by chimeric anti-CD20 monoclonal antibody (anti-CD20 McAb) through dendritic cells (DCs), mononuclear cells were isolated from human peripheral blood (PBMNC) and DCs from PBMNCs in vitro were generated with normal methods. Human CD20 positive lymphoma cell line-Raji cells were treated with different methods including treatment with anti-CD20 McAb (group R), treatment with heat-induced apoptosis (group A) and treatment with anti-CD20 McAb+heat-induced apoptosis (group R+A), then Raji cells treated with above-mentioned methods as tumor antigen were loaded on DCs. The phagocytosis of DCs to tumor antigen was observed by transmission electron microscope (TEM), the auto-mixed lymphocyte reaction was performed with antigen-primed DCs, the release of INF-gammafrom effector cells was detected by ELISPOT, the killing of effector cells on Raji cells was assayed by MTT. The results showed that under TEM, no pronounced phagocytosis of DCs was seen in group R, while the phagocytosis of apoptotic bodies could be easily seen in group A and the more cell fraqments were observed in group R+A. The FCM indicated that the expressions of CD80, CD86 and HLA-DR on DCs in 3 experimental groups were higher than those in group control (p<0.05), while expression positive rate in group R+A was higher than those in group R and A (p<0.05). The detection of lymphocyte surface antigen revealed that proportions of CD8+ cells in all experimental groups were higher than those in group control (p<0.05), count of CD56+ cells in group R and R+A increased, as compared with group A and control, difference was significant (p<0.05). ELISPOT assay indicated that amount of cells releasing IFN-gamma in all experimental groups was higher than that in group control, and also number of spots in group R+A significantly higher than that in other groups at effector-targetor ratio=1:10 (p<0.05). The results of killing assay demonstrated that killing rate on Raji cells in all experimental groups increased as compared with group control (p<0.05), while killing rate in group R+A was higher than that in group R and A. It is concluded that anti-CD20 McAb can mediate DC to induce cellular immunoresponse against lymphoma, that is, to stimulate and amplify specific CTLs and NK cells. Anti-CD20 McAb combined with DCs primed by heat-stressed tumor cells as antigen can further enhance cellular immunoresponse against lymphoma.

Rituximab-induced inhibition of antiapoptotic cell survival pathways: implications in chemo/immunoresistance, rituximab unresponsiveness, prognostic and novel therapeutic interventions.

Rituximab (chimeric anti-CD20 monoclonal antibody) is the first Food and Drug Administration approved antitumor antibody and is used in the treatment of B-non-Hodgkin's lymphoma (B-NHL). It is used as single monotherapy or in combination with chemotherapy and has improved the treatment outcome of patients with B-NHL. The in vivo mechanisms of rituximab-mediated antitumor effects include antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cell cytotoxicity (CDC), growth-inhibition and apoptosis. A subset of patients does not initially respond to rituximab and several responsive patients develop resistance to further rituximab treatment. The mechanism of rituximab unresponsiveness is not known. Besides the above-postulated mechanisms, rituximab has been shown to trigger the cells via CD-20. Studies performed with B-NHL cell lines as model systems revealed several novel mechanisms of rituximab-mediated effects that are involved in chemo/immunosensitization and the development of resistance to rituximab. Rituximab has been shown to inhibit the p38 mitogen-activated protein kinase, nuclear factor-kappaB (NF-kappaB), extracellular signal-regulated kinase 1/2 (ERK 1/2) and AKT antiapoptotic survival pathways, all of which result in upregulation of phosphatase and tensin homolog deleted on chromosome ten and Raf kinase inhibitor protein and in the downregulation of antiapoptotic gene products (particularly Bcl-2, Bcl-(xL) and Mcl-1), and resulting in chemo/immunosensitization. Further, rituximab treatment inhibits the overexpressed transcription repressor Yin Yang 1 (YY1), which negatively regulates Fas and DR5 expression and its inhibition leads to sensitization to Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Rituximab-resistant clones were generated as model to examine the mechanism of in vivo rituximab unresponsiveness. These clones showed reduced expression of CD20 and hyperactivation of the above antiapoptotic signaling pathways and failure of rituximab to trigger the cells leading to inhibition of ADCC, CDC and chemo/immunosensitization. Interference with the hyperactivated pathways with various pharmacological and proteasome inhibitors reversed resistance. Furthermore, the above findings have identified several gene products that can serve as new prognostic/diagnostic biomarkers as well as targets for therapeutic intervention in B-NHL.

Regression of gastric large B-Cell lymphoma accompanied by a florid lymphoma-like T-cell reaction: immunomodulatory effect of Ganoderma lucidum (Lingzhi)?

Complete regression of high-grade lymphoma is extremely rare. We report 1 such case that might have been conceivably mediated by Ganoderma lucidum (Lingzhi), an immunomodulatory herbal medicine. A 47-year-old man presented with epigastric pain. Endoscopy revealed a large gastric ulcer, which on biopsy was diagnostic of large B-cell lymphoma. At gastrectomy 11 days later, no evidence was found of large B-cell lymphoma despite thorough sampling. Instead, there was a dense and permeative infiltrate of CD3(+) CD8(+) cytotoxic small T lymphocytes spanning the whole thickness of the gastric wall. In situ reverse transcription polymerase chain reaction for T-cell receptor beta-chain family did not detect a monoclonal T-cell population. We postulate that the cytotoxic T cells may represent an active host-immune response against the large B-cell lymphoma that resulted in a complete regression. On questioning, the patient had taken megadoses of Ganoderma lucidum, which might have triggered the successful immune reaction.

[Radioimmunotherapy for non-Hodgkin lymphoma: historical development and current status]. / Radioinmunoterapia en los linfomas no Hodgkin: desarrollo histórico y estado actual.

Radioimmunotherapy treatment for lymphoma is a novel targeted therapeutic approach. Several years of development of radioimmunotherapeutic compounds came to fruition in February of 2002 when 90Y-ibritumomab tiuxetan (Zevalin, Y2B8) was approved in the USA and later in Europe, for the treatment of relapsed or refractory, low grade or transformed B-cell lymphoma in the USA. 90Y-ibritumomab tiuxetan utilizes a monoclonal anti-CD20 antibody to deliver beta-emitting yttrium-90 to the malignant B-cells. Clinical trials have demonstrated its efficacy, with observed clinical responses in the 80 % range. This product has become available in Europe, with simplified administration, for the treatment of relapsed follicular lymphoma. A similar anti-CD20 radiotherapeutic compound, 131I-tositumomab, was subsequently approved in the USA. Promising studies exploring expanded applications of radioimmunotherapy as consolidation, as part of transplant, or in other histologic types have been recently completed or are under way. Radioimmunotherapy has been shown to be an effective and clinically relevant complementary therapeutic approach for patients with lymphoma, bringing the Nuclear Medicine into lymphoma therapeutics.

Enhanced expression of CD20 in human tumor B cells is controlled through ERK-dependent mechanisms.

Rituximab, a chimeric Ab directed against CD20, induces apoptosis in targeted cells. Although the majority of B cell malignancies express the CD20 Ag, only approximately 50% of patients will respond to single-agent rituximab. The available data suggest that a decreased CD20 expression could account for the lack of response observed in some patients treated with rituximab. Despite the potential critical role of CD20 in the biology of B cell malignancies, the mechanisms controlling its expression are poorly understood. We evaluated the effect of the immune modulator agent bryostatin-1 on the expression of CD20 in non-Hodgkin's lymphoma cells. Using the B cell lines, DB and RAMOS, as well as tumor cells derived from a chronic lymphocytic leukemia patient, we demonstrated that bryostatin-1 enhanced the expression of both CD20 mRNA and protein. The enhanced expression of CD20 was associated with increased transcriptional activity of the CD20 gene, whereas the stability of CD20 mRNA was not affected. The effect of bryostatin-1 on CD20 expression in non-Hodgkin's lymphoma cells was mediated through the MAPK kinase/ERK signal transduction pathway and involved protein kinase C, but was independent of p38 MAPK and was insensitive to dexamethasone. Cells pretreated with bryostatin-1 were more susceptible to the proapoptotic effect of anti-CD20 Ab. Overall, these data demonstrate for the first time that ERK phosphorylation is required for the up-regulated expression of CD20 on B cell malignancies. The findings also suggest that bryostatin-1 and rituximab could be a valuable combined therapy for B cell malignancies.

Recombinant, tumour-derived idiotype vaccination for indolent B cell non-Hodgkin's lymphomas: a focus on FavId.

FavId (Favrille, Inc., San Diego, CA, USA) is a personalized therapeutic vaccine product for B cell non-Hodgkin's lymphoma, custom-manufactured from individual patient's tumour cells. This investigational agent consists of recombinant tumour-specific immunoglobulin (idiotype [Id]) chemically conjugated to the highly immunogenic carrier protein keyhole limpet haemocyanin (Id-KLH). The vaccine product is administered by subcutaneous co-injection with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor (GM-CSF) with the goal of stimulating tumour-specific T cell and humoral immunity. Therapeutic Id vaccines have shown promising results in early phase clinical trials in follicular lymphoma, and several Phase III trials are ongoing. FavId's advantages over other Id vaccine formulations include its rapid and efficient manufacturing technology utilising recombinant baculovirus, with a production time of only 8-12 weeks. In Phase II studies, FavId Id-KLH plus GM-CSF vaccines have been found to be safe, immunogenic and clinically active in follicular lymphoma. At present, FavId is being tested in a randomised, placebo-controlled Phase III trial in follicular lymphoma, aimed at improving the time to disease progression when administered following cytoreduction with rituximab. If found to be efficacious in this pivotal trial, FavId would represent a tumour-selective immunotherapy for lymphoma with little toxicity and a novel mechanism of action.

Modulation of clinical drug resistance in a B cell lymphoma patient by the protein kinase inhibitor 7-hydroxystaurosporine: presentation of a novel therapeutic paradigm.

Emerging evidence suggests that apoptosis is an important mechanism of tumor cell death from antineoplastic therapy. 7-hydroxystaurosporine (UCN-01) is a novel protein kinase inhibitor that increases chemotherapy-induced apoptosis in vitro and is in early phases of clinical development. In this report, we present a 68-year-old patient with chemotherapy-resistant lymphoma treated with UCN-01 and chemotherapy. He had a stage IV plasmacytoid lymphoma that failed to enter remission with high-dose EPOCH II (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin) chemotherapy. Due to disease progression and transformation to large cell lymphoma in the liver and bone marrow, he received UCN-01. Four weeks later, he received "standard-dose" EPOCH because of progression, developed severe neutropenia for 9 days, and expired from Candida sepsis on day 23. At autopsy, there was no histological evidence of residual lymphoma, although PCR for immunoglobulin gene rearrangement analysis revealed a faint clonal band in two of six nodes but none in the liver. Significantly, no B cells were detected by immunohistochemistry in lymph nodes, and a polyclonal ladder pattern associated with the presence of normal B cells was not seen in the immunoglobulin gene rearrangement PCR assay. Profound peripheral lymphopenia (50 cells/microliter) was also observed. Pharmacokinetics showed UCN-01 salivary concentrations, a surrogate for free drug concentrations, to be within an effective range in vitro (45 nmol/L) as a modulator of DNA-damaging agent cytotoxicity. In vitro, UCN-01 is synergistic with multiple cytotoxic agents and increases fludarabine-induced apoptosis in a human breast cell line. These results suggest that UCN-01 sensitized the lymphoma to the cytotoxic effects of EPOCH, possibly by modulating the "threshold" for apoptosis, and may illustrate a new paradigm for reversal of drug resistance.

Radiolabeled antibody therapy of B-cell lymphomas.

Patients with relapsed B-cell lymphomas are currently incurable with conventional doses of chemotherapy or radiotherapy. In recent years, new treatment options have become available for these patients, including the use of chimeric mouse-human anti-CD20 antibodies and radiolabeled anti-CD20 antibodies. The nonradioactive rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, CA, and Genentech, Inc, San Francisco, CA) antibody induces remissions in 60% of patients with relapsed follicular lymphomas, including 5% to 10% complete remissions. Anti-CD20 antibodies radiolabeled with iodine 131 and yttrium 90 given at nonmyeloablative doses yield remissions in 75% to 80% of cases, including 35% to 40% complete remissions. High-dose (131)I-anti-B1 antibody with stem cell transplantation generates objective responses in 85% to 90% of cases, including 75% to 80% complete remissions. Although more patients need to be evaluated with a longer follow-up period, radioimmunotherapy appears to be an effective and well-tolerated addition to the oncologists' armamentarium for relapsed lymphomas.

Preclinical and phase I and II trials of rituximab.

The CD20 antigen is an attractive target for antibody-directed therapy due to its stable, high-level surface expression on normal and malignant B cells. Rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, and Genentech, Inc, San Francisco, CA) is an anti-CD20 chimeric monoclonal antibody that has shown single-agent activity in phase I and II clinical trials in patients with B-cell non-Hodgkin's lymphoma. This antibody has a long serum half-life and low immunogenicity. Infusional symptoms are common with the initial infusion but rare with subsequent treatments. Temporary B-cell depletion occurs but has not been associated with immunodeficiency. The mechanism of action likely includes both immune-mediated effects and direct effects. The generally mild toxicity profile and excellent single-agent activity provide the rationale for use with or following chemotherapy. Additional studies evaluating these and other combinations are under way.