Atomic force microscopy study of the antigen‐antibody binding force on patient cancer cells based on ROR1 fluorescence recognition

Knowledge of drug–target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single‐molecule techniques, the information of individual interactions between ligand‐receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand‐receptor forces under near‐physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single‐molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug–target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti‐CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B‐cell lymphomas but not on normal cells. First, B‐cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20‐rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20‐rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab‐conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20‐Rituximab binding force was measured by single‐molecule force spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

抗CD20单克隆抗体治疗B 细胞淋巴瘤的效应机制

B细胞淋巴瘤是一种主要的非霍奇金淋巴瘤(non-Hodgkin’s lymphoma,NHL),95%以上的B细胞淋巴瘤均表达B细胞分化抗原CD20。尽管目前CD20在B细胞分化发育过程中的具体功能尚未阐明,但其特有的表达方式和在细胞膜上的分布特点决定了其成为B细胞淋巴瘤靶向治疗的主要靶点。近十年来,随着抗CD20单克隆抗体的不断发展和进步,联合传统的CHOP化疗方案,在NHL的治疗中显示出良好的效果。虽然,近年来抗CD20单抗逐渐被用于B细胞相关的自身免疫病的治疗,但相关作用机制尚不明确。在针对NHL的临床治疗中,抗CD20单抗的疗效被认为依赖于效应器机制,主要有ADCC、CDC和细胞凋亡。虽然抗CD20在淋巴瘤的治疗中具有显著的免疫疗效,但部分瘤荷较大的病人出现了耐药和复发,循环中大量B细胞由于单抗的结合而被机体的单核巨噬细胞吞噬或NK细胞杀伤,机体出现成熟B细胞空缺期,同时单核巨噬细胞、NK细胞和补体大量消耗,造成机体免疫效应功能饱和和效应器耗竭。本文就抗CD20单克隆抗体在治疗淋巴瘤中的具体作用机制及可能造成的机体效应器耗竭问题做一简要的概述。     

Expression of the CD31 antigen in normal B-cells and non Hodgkin's lymphomas

The role cell adhesion molecules play in the biological and clinical behaviour of non Hodgkin's lymphomas (NHL) has been reported in several studies. This study reports the findings on B-cells taken from various healthy control tissues and compared them to B-cells from 83 malignant B-lymphomas, that had been classified according to the WHO classification. Flow cytometry was used to investigate the surface expression of CD31, an adhesion molecule involved in B-cell development and vascular adhesion mechanisms. Quantification of the fluorescence signals showed specific patterns of CD31 expression on normal B-cell subpopulations and different NHL groups. Our results demonstrate that CD31 expression is modulated during the differentiation process in normal B-cells, high in pre-B-I cells, low in pre-B-II precursors, intermediate in the mature B-cell subpopulations or, depending on the functional state absent in activated follicular centre cells, present in pre- and post- germinal centre cells. When the CD31 expression is evaluated as fluorescence intensity in NHL, it reveals a heterogeneous pattern related to histogenetic derivation (high in small lymphocytic lymphoma, low in follicular lymphoma, intermediate in marginal zone and large cell lymphomas). These observations suggest that CD31 might well play a critical role in the ontogeny and physiology of B-lymphocytes. Therefore, on the basis of these observations we propose the CD31 molecule as an interesting additional useful parameter to be used for the differential diagnosis of NHL and hypothese that it has a pathophysiologic role in NHL evolution.     

Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations

T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19⁺ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2^nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR⁺ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1⁺ tumors. Moreover, such cells could eliminate ROR1⁺ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR⁺ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.     

Soluble and Membrane-Bound TGF-β-Mediated Regulation of Intratumoral T Cell Differentiation and Function in B-Cell Non-Hodgkin Lymphoma

While the effect of TGF-β on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-β in tumor immunity in B-cell NHL are limited. We found that soluble TGF-β, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-β promoted regulatory T (T_reg) cells by enhancing expression of Foxp3 in CD4⁺ T cells and suppressed effector helper T (T_H) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-β on T cell differentiation. Furthermore, we found that membrane-bound TGF-β is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-β was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-β receptor. We showed that pretreatment of lymphoma B cells with TGF-β significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-β are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.     

Effects of blocking CD24 and CD47 ‘don't eat me’ signals in combination with rituximab in mantle-cell lymphoma and chronic lymphocytic leukaemia

Mantle-cell lymphoma (MCL) is a B-cell non-Hodgkin Lymphoma (NHL) with a poor prognosis, at high risk of relapse after conventional treatment. MCL-associated tumour microenvironment (TME) is characterized by M2-like tumour-associated macrophages (TAMs), able to interact with cancer cells, providing tumour survival and resistance to immuno-chemotherapy. Likewise, monocyte-derived nurse-like cells (NLCs) present M2-like profile and provide proliferation signals to chronic lymphocytic leukaemia (CLL), a B-cell malignancy sharing with MCL some biological and phenotypic features. Antibodies against TAMs targeted CD47, a ‘don't eat me’ signal (DEMs) able to quench phagocytosis by TAMs within TME, with clinical effectiveness when combined with Rituximab in pretreated NHL. Recently, CD24 was found as valid DEMs in solid cancer. Since CD24 is expressed during B-cell differentiation, we investigated and identified consistent CD24 in MCL, CLL and primary human samples. Phagocytosis increased when M2-like macrophages were co-cultured with cancer cells, particularly in the case of paired DEMs blockade (i.e. anti-CD24 + anti-CD47) combined with Rituximab. Similarly, unstimulated CLL patients-derived NLCs provided increased phagocytosis when DEMs blockade occurred. Since high levels of CD24 were associated with worse survival in both MCL and CLL, anti-CD24-induced phagocytosis could be considered for future clinical use, particularly in association with other agents such as Rituximab.     

Binding to CD20 by Anti-B1 Antibody or F(ab')2 is sufficient for induction of apoptosis in B-cell lines

CD20 is a B-cell-specific cell surface protein expressed on mature B lymphocytes and is a target for monoclonal antibody therapy for non-Hodgkin's lymphoma (NHL). Though clear clinical efficacy has been demonstrated with several anti-CD20 antibodies, the mechanisms by which the antibodies activate CD20 and kill cells remain unclear. Proposed mechanisms of action include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and induction of apoptosis. In this report we compared the activity of two anti-CD20 antibodies, Anti-B1 Antibody (tositumomab) and rituximab (C2B8), in a variety of cellular assays using a panel of B-cell lines. Anti-B1 Antibody showed a low level of activity in a CDC assay against complement-sensitive B-cell lines, Ramos and Daudi. We found that there is an inverse correlation between the expression of CD55 and CD59 and CDC mediated by either Anti-B1 Antibody or rituximab. Rituximab was more potent at inducing CDC when compared to Anti-B1 Antibody. Using Raji cells as target cells and human peripheral blood leukocytes as effector cells, Anti-B1 Antibody was a potent inducer of ADCC. The activities of Anti-B1 Antibody and rituximab were nearly identical in the ADCC assay. In addition, Anti-B1 Antibody showed direct induction of apoptosis in all B-cell lines tested. In general, crosslinking Anti-B1 Antibody with a goat anti-mouse Ig did not further enhance the percentage of cells undergoing apoptosis. Importantly, a F(ab')(2) fragment of Anti-B1 Antibody induced apoptosis, while the Fab fragment did not, indicating that the Fc region was not required and dimerization of CD20 may be sufficient for induction of apoptosis. In contrast, rituximab, which binds to an overlapping epitope on CD20 with a three-fold lower affinity than Anti-B1 Antibody, did not efficiently induce apoptosis in the cell lines tested in the absence of crosslinking. In conclusion, these two anti-CD20 antibodies have overlapping, but distinct mechanisms of action on B-cell lines.     

AFM‐ and NSOM‐based force spectroscopy and distribution analysis of CD69 molecules on human CD4+ T cell membrane

Although CD69 is well known as an early T cell‐activation marker, the possibility that CD69 are distributed as nano‐structures on membrane for immune regulation during T cell activation has not been tested. In this study, nanoscale features of CD69 expression on activated T cells were determined using the atomic force microscopy (AFM) topographic and force‐binding nanotechnology as well as near‐field scanning optical microscopy (NSOM)‐/fluorescence quantum dot (QD)‐based nanosacle imaging. Unstimulated CD4⁺ T cells showed neglectable numbers of membrane CD69 spots binding to the CD69 Ab‐functinalized AFM tip, and no detectable QD‐bound CD69 as examined by NSOM/QD‐based imaging. In contrast, Phytohemagglutinin (PHA)‐activated CD4⁺ T cells expressed CD69, and displayed many force‐binding spots binding to the CD69 Ab‐functionalized AFM tip on about 45% of cell membrane, with mean binding‐rupture forces 276 ± 71 pN. Most CD69 molecules appeared to be expressed as 100–200 nm nanoclusters on the membrane of PHA‐activated CD4⁺ T cells. Meanwhile, NSOM/QD‐based nanoscale imaging showed that CD69 were non‐uniformly distributed as 80–200 nm nanoclusters on cell‐membrane of PHA‐activated CD4⁺ T cells. This study represents the first demonstration of the nano‐biology of CD69 expression during T cell activation. Copyright © 2009 John Wiley & Sons, Ltd.     

Renal Involvement in Non-Hodgkin Lymphoma: Proven by Renal Biopsy

Aims

To determine the spectrum of renal lesions in patients with kidney involvement in non-Hodgkin''s lymphoma (NHL) by renal biopsy.

Methods

The clinical features and histological findings at the time of the renal biopsy were assessed for each patient.

Results

We identified 20 patients with NHL and renal involvement, and the diagnosis of NHL was established following the kidney biopsy in 18 (90%) patients. The types of NHL include the following: chronic lymphocytic leukemia/small lymphocytic lymphoma (n = 8), diffuse large B-cell lymphoma (n = 4), T/NK cell lymphoma (n = 3), lymphoplasmacytic lymphoma (n = 2), cutaneous T-cell lymphoma (n = 1), mucosa-associated lymphoid tissue lymphoma (n = 1) and mantle cell lymphoma (n = 1). All presented with proteinuria, and 15 patients had impaired renal function. The pathological findings included (1) membranoproliferative glomerulonephritis-like pattern in seven patients; (2) crescent glomerulonephritis in four; (3) minimal-change disease in three, and glomeruli without specific pathological abnormalities in three; (4) intraglomerular large B-cell lymphoma in one; (5) intracapillary monoclonal IgM deposits in one; (6) primary diffuse large B-cell lymphoma of the kidneys in one; and (7) lymphoma infiltration of the kidney in eight patients.

Conclusion

A wide spectrum of renal lesions can be observed in patients with NHL, and NHL may be first proven by renal biopsies for evaluation of kidney injury or proteinuria. Renal biopsy is necessary to establish the underlying cause of renal involvement in NHL.     

CD22-Binding Peptides Derived from Anti-CD22 Ligand Blocking Antibodies Retain the Targeting and Cell Killing Properties of the Parent Antibodies and May Serve as a Drug Delivery Vehicle

CD22 is a B-cell specific membrane glycoprotein that mediates homotypic and heterotypic cell adhesion; it also regulates B-cell receptor (BCR)-mediated signals. Monoclonal antibodies (mAb) directed at the ligand binding domain of CD22 initiate CD22-mediated signal transduction and apoptosis in B-cell lymphomas (NHL). Amino acid analysis of the complimentary determining regions (CDRs) of six different anti-CD22 ligand blocking mAb revealed a high level of sequence conservation. The heavy chain CDRs 1, 2, and 3 are 85, 40, and 38% conserved, respectively; light chain CDRs 1, 2, and 3, are 95, 90 and 90% conserved, respectively. Based on these conserved sequences, five peptides were designed and synthesized. Only the sequence derived from heavy chain CDR2 (Peptide 5) demonstrated significant B-cell binding. Peptide 5 bound to both malignant and primary B-cells with very little T-cell binding. The affinity had a Km of 5 × 10⁻⁶ M. Peptide 5 mediated killing of several NHL cell lines to a degree similar to that of the parent mAb (HB22.7). Peptide 5’s loop structure was shown to be crucial for B-cell binding and ligand blocking. Mutational analysis revealed that most Peptide 5 amino acids were critical for B cell binding. Using a CD22 transfected COS cell line, we demonstrated CD22-specific binding and CD22 ligand blocking to a degree similar to HB22.7. Finally Peptide 5 was used as a vehicle to deliver a pro-apoptotic peptide into NHL cells. Peptide 5 was fused to a BH3 death domain-containing peptide which demonstrated more effective NHL cell killing than the parent peptide.     

Cytomorphological and immunocytochemical study of non-Hodgkin's lymphoma in pleural effusion and ascitic fluid

INTRODUCTION: Non-Hodgkin's lymphoma (NHL) is often complicated by pleural effusion and ascites. The present study is an attempt to categorize the lymphomatous effusions according to the WHO classification, using archival material. METHODS: May-Grünwald-Giemsa and Papanicolaou-stained smears of 31 lymphomatous effusion specimens were reviewed. Of these, detailed cytological assessment was done on 12 pleural effusions and ten ascitic fluid specimens from 22 patients using the WHO lymphoma classification system. Immunocytochemical studies were performed in 21 specimens. RESULTS: Based on cytomorphological features, the 22 lymphomatous effusion specimens were categorized into lymphoplasmacytoid lymphoma (1), follicle centre cell (FCC) grade-1 (centrocytic) lymphoma (3), FCC grade-2 (centrocytic-centroblastic) lymphoma (3), FCC grade-3 (centroblastic) lymphoma (4), large cell immunoblastic lymphoma (4), lymphoblastic lymphoma (2), anaplastic large cell lymphoma (3) and miscellaneous types (2). Immunocytochemically, the lymphoma cells were T-cell (positive for CD3) and B-cell type (CD20 positive) in five and six cases respectively. CONCLUSION: Cytological examination of pleural effusion and ascitic fluid samples, supported by immunocytochemical studies, may be useful for the classification of lymphomas under the WHO system.     

Authentication of primordial characteristics of the CLBL-1 cell line prove the integrity of a canine B-cell lymphoma in a murine in vivo model

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2(-/-)γ(c) (-/-) mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45(+), MHCII(+), CD11a(+) and CD79αcy(+). PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.     

Long non-coding RNA as a novel biomarker and therapeutic target in aggressive B-cell non-Hodgkin lymphoma: A systematic review

Cancer initiation and progression have been associated with dysregulated long non-coding RNA (lncRNA) expression. However, the lncRNA expression profile in aggressive B-cell non-Hodgkin lymphoma (NHL) has not been comprehensively characterized. This systematic review aims to evaluate the role of lncRNAs as a biomarker to investigate their future potential in the diagnosis, real-time measurement of response to therapy and prognosis in aggressive B-cell NHL. We searched PubMed, Web of Science, Embase and Scopus databases using the keywords “long non-coding RNA”, “Diffuse large B-cell lymphoma”, “Burkitt's lymphoma” and “Mantle cell lymphoma”. We included studies on human subjects that measured the level of lncRNAs in samples from patients with aggressive B-cell NHL. We screened 608 papers, and 51 papers were included. The most studied aggressive B-cell NHL was diffuse large B-cell lymphoma (DLBCL). At least 79 lncRNAs were involved in the pathogenesis of aggressive B-cell NHL. Targeting lncRNAs could affect cell proliferation, viability, apoptosis, migration and invasion in aggressive B-cell NHL cell lines. Dysregulation of lncRNAs had prognostic (e.g. overall survival) and diagnostic values in patients with DLBCL, Burkitt's lymphoma (BL), or mantle cell lymphoma (MCL). Furthermore, dysregulation of lncRNAs was associated with response to treatments, such as CHOP-like chemotherapy regimens, in these patients. LncRNAs could be promising biomarkers for the diagnosis, prognosis and response to therapy in patients with aggressive B-cell NHL. Additionally, lncRNAs could be potential therapeutic targets for patients with aggressive B-cell NHL like DLBCL, MCL or BL.     

Malignant B cells induce the conversion of CD4+CD25- T cells to regulatory T cells in B-cell non-Hodgkin lymphoma

Recent evidence has demonstrated that regulatory T cells (Treg) were enriched in the tumor sites of patients with B-cell non-Hodgkin lymphoma (NHL). However, the causes of enrichment and suppressive mechanisms need to be further elucidated. Here we demonstrated that CD4(+)CD25(+)FoxP3(+)CD127(lo) Treg were markedly increased and their phenotypes were different in peripheral blood (PB) as well as bone marrow (BM) from newly diagnosed patients with B-cell NHL compared with those from healthy volunteers (HVs). Involved lymphatic tissues also showed higher frequencies of Treg than benign lymph nodes. Moreover, the frequencies of Treg were significantly higher in involved lymphatic tissues than those from PB as well as BM in the same patients. Suppression mediated by CD4(+)CD25(+) Treg co-cultured with allogeneic CFSE-labeled CD4(+)CD25(-) responder cells was also higher in involved lymphatic tissues from B-cell NHL than that mediated by Treg from HVs. In addition, we found that malignant B cells significantly induced FoxP3 expression and regulatory function in CD4(+)CD25(-) T cells in vitro. In contrast, normal B cells could not induce the conversion of CD4(+)CD25(-) T cells to Treg. We also showed that the PD-1/B7-H1 pathway might play an important role in Treg induction. Taken together, our results suggest that malignant B cells induce the conversion of CD4(+)CD25(-) T cells to Treg, which may play a role in the pathogenesis of B-cell NHL and represent a promising therapeutic target.     

Use of a Conformational Switching Aptamer for Rapid and Specific Ex Vivo Identification of Central Nervous System Lymphoma in a Xenograft Model

Improved tools for providing specific intraoperative diagnoses could improve patient care. In neurosurgery, intraoperatively differentiating non-operative lesions such as CNS B-cell lymphoma from operative lesions can be challenging, often necessitating immunohistochemical (IHC) procedures which require up to 24-48 hours. Here, we evaluate the feasibility of generating rapid ex vivo specific labeling using a novel lymphoma-specific fluorescent switchable aptamer. Our B-cell lymphoma-specific switchable aptamer produced only low-level fluorescence in its unbound conformation and generated an 8-fold increase in fluorescence once bound to its target on CD20-positive lymphoma cells. The aptamer demonstrated strong binding to B-cell lymphoma cells within 15 minutes of incubation as observed by flow cytometry. We applied the switchable aptamer to ex vivo xenograft tissue harboring B-cell lymphoma and astrocytoma, and within one hour specific visual identification of lymphoma was routinely possible. In this proof-of-concept study in human cell culture and orthotopic xenografts, we conclude that a fluorescent switchable aptamer can provide rapid and specific labeling of B-cell lymphoma, and that developing aptamer-based labeling approaches could simplify tissue staining and drastically reduce time to histopathological diagnoses compared with IHC-based methods. We propose that switchable aptamers could enhance expeditious, accurate intraoperative decision-making.     

Treatment of non-Hodgkin’s lymphoma xenografts with the HB22.7 anti-CD22 monoclonal antibody and phosphatase inhibitors improves efficacy

Purpose  To examine the role of phosphatase inhibition on anti-CD22, HB22.7-mediated lymphomacidal effects. Experimental design  CD22 is a cell-surface molecule expressed on most B cell lymphomas (NHL). HB22.7 is an anti-CD22 monoclonal antibody that binds a unique CD22-epitope, blocks ligand binding, initiates signaling, and has demonstrated lymphomacidal activity. The SHP-1 tyrosine phosphatase is associated with the cytoplasmic domain of CD22. Sodium orthovanadate (NaV) is a phosphatase inhibitor. The SHP-1-CD22 interaction presents an opportunity to manipulate CD22-mediated signaling effects. In vitro cell culture assays and in vivo human NHL xenograft studies were used to assess the effects of phosphatase inhibition. Results  NaV caused dose dependent killing of NHL cells in vitro; when HB22.7 was given with NaV, antibody-mediated cell death was augmented. Flow cytometry showed that NaV-pretreatment resulted in less CD22 internalization after ligation with HB22.7 than did control cells. Studies in mice bearing Raji NHL xenografts showed that the combination of NaV and HB22.7 shrank NHL tumors more rapidly, had a higher complete response rate (80%), and produced the best survival compared to controls; no toxicity was detected. Studies using Raji cells stably transfected with SHP-1DN confirmed that these observations were due to SHP-1 inhibition. Conclusion  The relatively specific association of SHP-1 with CD22 suggests that CD22-specific signal augmentation by phosphatase inhibitors can improve the clinical outcome of anti-CD22 based immunotherapy.     

Common germinal-center B-cell origin of the malignant cells in two composite lymphomas, involving classical Hodgkin's disease and either follicular lymphoma or B-CLL

BACKGROUND: Classical Hodgkin's disease (HD) and B-cell non-Hodgkin lymphoma (NHL) occasionally occur in the same patient. Such composite lymphomas represent interesting models to study the pathogenesis of B-cell lymphomas and the relationship between HD and B-cell NHL. MATERIALS AND METHODS: We analyzed two composite lymphomas (a combination of classical HD with follicular lymphoma [FL] and a combination of classical HD with B-cell chronic lymphocytic leukemia [B-CLL]) by micromanipulation of single cells from tissue sections and amplification of immunoglobulin V region genes for the clonal relationship of the tumor cells. RESULTS: In both cases, clonally related variable (V) genes with both shared as well as distinct somatic mutations were obtained from the two lymphomas, showing that in each of the cases the distinct tumor cells were members of a common germinal center (GC) B-cell clone. FL cells from two different lymph nodes of patient 1 showed a similar mutation pattern, suggesting that infiltration of these lymph nodes by tumor cells was not restricted to a particular FL cell or subclone. In the FL, a single cell was identified with a mutation signature indicating that premalignant cells can persist in the tissue. CONCLUSIONS: The cases presented here further underline the close relationship between HD and B-cell NHL and the role of the GC in lymphomagenesis. Whereas the latter was already suggested for FL and HD, the present study indicates that also in the B-CLL subset characterized by mutated Ig genes, important steps in malignant transformation happen in the GC, and that HRS cells can derive from CD5-positive B cells.     

Cancer/testis antigens expression and autologous serological response in a set of Brazilian non-Hodgkin’s lymphoma patients

Background

Based on their tumor-associated expression pattern, cancer/testis antigens (CTAs) are considered potential targets for cancer immunotherapy. We aim to evaluate the expression of CTAs in non-Hodgkin??s lymphoma (NHL) samples and the ability of these patients to elicit spontaneous humoral immune response against CTAs.

Methods

Expression of MAGE-A family, CT7/MAGE-C1, CT10/MAGE-C2, GAGE and NY-ESO-1 was analyzed by immunohistochemistry in a tissue microarray generated from 106 NHL archival cases. The humoral response against 19 CTAs was tested in 97 untreated NHL serum samples using ELISA technique.

Results

11.3?% of NHL tumor samples expressed at least 1 CTA. MAGE-A family (6.6?%), GAGE (5.7?%) and NY-ESO-1(4.7?%) were the most frequently expressed antigens. We found no statistically significant correlation between CTA positivity and clinical parameters such as NHL histological subtype, Ann Arbor stage, international prognostic index score, response to treatment and overall survival. Humoral response against at least 1 CTA was observed in 16.5?% of NHL serum samples. However, overall seroreactivity was low, and strong titers (>1:1000) were observed in only two diffuse large B-cell lymphomas patients against CT45.

Conclusion

Our findings are in agreement with most of published studies in this field to date and suggest an overall low expression of CTAs in NHL patients. However, as many new CTAs have been described recently and some of them are found to be highly expressed in NHL cell lines and tumor samples, further studies exploring the expression of different panels of CTAs are needed to evaluate their role as candidates for immunotherapy in NHL patients.     

A fully human CD19/CD3 bi-specific antibody triggers potent and specific cytotoxicity by unstimulated T lymphocytes against non-Hodgkin’s lymphoma

The use of a bi-specific antibody (BsAb) is an attractive and specific approach to cancer therapy. We have constructed a fully human recombinant single chain Fv BsAb against CD19 and CD3 that was an effective treatment in an animal model of non-Hodgkin's lymphoma (NHL). The CD19/CD3 BsAb was expressed in CHO cells and purified by Ni-column chromatography. Flow cytometry revealed that the CD19/CD3 BsAb specifically bound to both CD19 and CD3-positive cells. In vitro, the CD19/CD3 BsAb could stimulate T cell proliferation and induce the lysis of cultured Raji cells in the presence of unstimulated T lymphocytes. In vivo, the CD19/CD3 BsAb efficiently inhibited tumour growth in SCID mice of NHL, and the survival time of the mice was significantly prolonged. Therefore, our CD19/CD3 BsAb is a useful tool that could be a suitable candidate for treatment of NHL.     

Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab

Although rituximab has revolutionized the treatment of hematological malignancies, the acquired resistance is one of the prime obstacles for cancer treatment, and development of novel CD20-targeting antibodies with potent anti-tumor activities and specificities is urgently needed. Emerging evidence has indicated that lysosomes can be considered as an “Achilles heel” for cancer cells, and might serve as an effective way to kill resistant cancer cells. HLA-DR antibody L243 has been recently reported to elicit potent lysosome-mediated cell death in lymphoma and leukemia cells, suggesting that HLA-DR could be used as a potential target against lymphoma. In this study, we generated a bispecific immunoglobulin G-like antibody targeting both CD20 and HLA-DR (CD20–243 CrossMab) through CrossMab technology. We found that the CrossMab could induce remarkably high levels of complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and anti-proliferative activity. Notably, although HLA-DR is expressed on normal and malignant cells, the CrossMab exhibited highly anti-tumor specificity, showing efficient eradication of hematological malignancies both in vitro and in vivo. Our data indicated that combined targeting of CD20 and HLA-DR could be an effective approach against malignancies, suggesting that CD20–243 CrossMab would be a promising therapeutic agent against lymphoma.