CD3/Bars: A Novel Bispecific Format for the Treatment of B-Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3516-3516
Author(s):  
Moritz Bewarder ◽  
Klaus-Dieter Preuss ◽  
Natalie Fadle ◽  
Evi Regitz ◽  
Lorenz Thurner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphoma Cell ◽  
Bispecific Antibodies ◽  
B Cell Lymphomas ◽  
Lymphoma Cells

Abstract Background Autoantigens are suspected to play an important role in the pathogenesis of different types of B cell neoplasia. Suggestive of this hypothesis is the restricted usage of a stereotyped IgHv repertoire in CLL, MCL and DLBCL. Further evidence supporting this notion is the identification of specific autoantigens as the target of the B-cell receptor from malignant lymphomas and myelomas, such as paratarg-7 as antigenic target for 15% of paraproteins of MGUS and MM patients. ARS2 was previously identified as the autoantigenic target for the B-cell receptor of approximately 25% of DLBCLs of the ABC type, here termed ARS2 reactive lymphomas. We had recently shown that the B-cell receptor antigens can be used to target B-cell lymphoma cells in vitro and in vivo in an approach designated as BARs (B-cell receptor antigens for reverse targeting), the first therapeutic strategy in oncology with absolute and exclusive specificity for the malignant clone. To test whether BARs can substitute the B-cell binding antibody (e.g. CD19) in T-cell engaging bispecific antibodies, we designed a bispecific CD3/BAR product consisting of a recombinant single chain fragment (scFv) against CD3 linked to ARS2 (CD3-ARS2). One arm of this construct should engage the T cell co-receptor CD3 of human T cells, and the other site should bind to the B cell receptor of ARS2 reactive lymphomas thus specifically redirecting and activating T cells against lymphoma cells. Material and methods VL and VH genes of the CD3 - OKT3 hybridoma and the DNA sequence of the 33 amino acids containing the B-cell receptor binding epitope of ARS2 were cloned into a pcDNA 3.1 vector by standard cloning techniques. VH and VL were linked by a glycine-serine linker, as was VL to the ARS2 epitope resulting in VH-(Gly₄Ser₁)ⁿ-VL-(Gly₄Ser₁)ⁿ-ARS2 peptide chain. The final cloning product was transfected in HEK cells for production of the bispecific construct. Binding capacity to lymphoma cell lines (OCI-Ly3, U2932, HBL-1) and PBMCs was assessed by flow cytometry. Western blot analysis was used for detection of CD3-ARS2 after incubation with the monoclonal anti-His Tag antibody. Cytotoxicity was evaluated by LDH release assay. Results The CD3 - ARS2 bispecific construct bound to CD3 on T cells and the B-cell receptor of ARS2 reactive lymphoma cells. CD3/ARS2 induced rapid cytotoxicity exclusively in ARS2 reactive lymphoma cell lines at concentrations as low as 250 ng/ml with an effector - target ratio of 10:1. Specific T-cell mediated cytotoxicity reached 40% after 4 hours. Lymphoma cell lines with BCRs of a specificity other than ARS2 were not affected. Conclusion The CD3/BAR construct is a novel therapeutic principle for the treatment of B-cell lymphomas, suggesting that BARs might also be useful as part of CAR-T cells. Compared to CD3/CD19 bispecific antibodies the CD3/BARs are exclusively cytotoxic against the malignant clone and spare normal B-cells. This should considerably reduce the acute toxicity of T-cell engaging bispecific constructs and circumvent long-term B cell depletion. Experiments comparing the cytotoxic capacity of CD3/BARs with CD3/CD19 bispecific antibodies are underway, as are analyses evaluating possible synergisms of these constructs. Disclosures No relevant conflicts of interest to declare.

scholarly journals BAR-Bodies: B-Cell Receptor Antigens As the Targeting Moiety of Antibodies in Substitution for the Variable Region of Heavy and Light Chains

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2940-2940
Author(s):  
Moritz Bewarder ◽  
Lorenz Thurner ◽  
Frank Neumann ◽  
Natalie Fadle ◽  
Evi Regitz ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphoma Cell ◽  
Lymphoma Cells ◽  
Advisory Committees

Abstract Background Chronic antigenic stimulation of the B-cell receptor (BCR) seems to play a critical role in the pathogenesis of B-cell lymphomas. We recently identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type and 45% of mantle cell lymphomas (MCLs), respectively. These BCR antigens can be used to target lymphoma cells in an approach we designated as BAR (B-cell receptor antigens for reverse targeting). The optimal therapeutic format BARs can be integrated in has yet to be found. Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed and tested an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains. Material and methods To create the ARS2 BAR-body, we exchanged the heavy and light chain variable region sequences of an IgG1 antibody with a sequence of similar length (approximately 120 amino acids) of the ARS2 protein (aa 343 - 466) containing the DLBCL reactive epitope (aa 343 - 375). The construct was assembled in a pCR2.1 vector, then transferred to a pSfi FLAG Tag vector for fusion with the FLAG tag and transfected into HEK293 cells for production. Purification of the BAR-body was performed via anti-FLAG antibody affinity chromatography. The BAR-body was detected by western blot analysis and binding capacity to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and the not ARS2-reactive control DLBCL cell line TMD8 was assessed by flow cytometry. ARS2 BAR-body induced cytotoxicity of lymphoma cells with an ARS2 reactive BCR was measured by LDH release assays with human PBMCs as effector cells at an E:T ratio of 10:1. Results We cloned, expressed and characterized an ARS2 containing BAR-body incorporating 4 molecules of the lymphoma-reactive epitope of ARS2 resulting in an antibody like construct using a BAR (ARS2) as binding moiety instead of normal variable regions. The ARS2 BAR-body could successfully be cloned and expressed as confirmed by western blot analysis, which showed the construct at approximately 150 kD as was to be expected. The BAR-body bound specifically to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and did not bind to the DLBCL cell line TMD8, which has a B-cell receptor of different specificity or to lymphoma cell lines of different entities. In LDH release assays with 5 x 104 PBMCs and 5 x 103 lymphoma cells (E:T ratio of 10:1) the ARS2 BAR-body induced PBMC mediated specific lysis of the ARS2 reactive lymphoma cell lines U2932 and OCI-Ly3 but not the control DLBCL cell line TMD8 starting at a concentration of 0,1µg/ml. Cytotoxic effects were dose dependent, reached a maximum of 50% specific lysis at a concentration of 1µg/ml and did not increase at concentrations of 10µg/ml. Conclusion Here, we show that BARs can substitute for the variable domains as binding moiety in antibody like constructs to target the BCR of B-cell lymphomas. Because approaches using their specific cognate antigen for targeting the malignant B cells have an exclusive specificity for the BCR of the malignant clone, they can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells, because they leave normal B-lymphocytes unaffected. By incorporating BARs into the well-known format of an antibody we hope to capitalize on years of experience with this therapeutic format from conducting and interpreting in vivo experiments to the translation of the BAR approach into the clinic. Disclosures Stilgenbauer: Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

TGF-β Is Selectively Expressed on Lymphoma B Cells and Regulates the Differentiation of Intratumoral T Cells in B-Cell Non-Hodgkin Lymphoma (NHL)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1586-1586
Author(s):  
Zhi-Zhang Yang ◽  
Deanna Grote ◽  
Steven C. Ziesmer ◽  
Thomas E. Witzig ◽  
Anne J. Novak ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Tumor Microenvironment ◽  
B Cell ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Lymphoma Cell ◽  
Lymphoma Cells

Abstract Abstract 1586 Transformation growth factor (TGF-β) is a highly pleiotropic cytokine critical to a variety of cellular events such as cell differentiation and apoptosis. TGF-β is synthesized as a prepro-TGF-β precursor and secreted after being processed in Golgi apparatus as a latent form that non-covalently combines both TGF-β and latency-associated protein (LAP). Our previous work in B-cell NHL has shown that the intratumoral T cell composition results in the establishment of a profoundly inhibitory tumor microenvironment. However, the underlying mechanism is only partially understood. In this study, using patient specimens and lymphoma cell lines, we evaluated the role of TGF-β in the tumor microenvironment and determined the effect of TGF-β on the generation of intratumoral TH1, TH17 and Treg cells in B-cell NHL. First, we determined expression of TGF-β and found that a latent form of TGF-β was specifically expressed on the surface of CD19+ B cells, but not on other types of cells from B-cell lymphoma biopsy specimens. By screening cell lines, we found that latent TGF-β was also expressed on the surface of lymphoma cell lines, confirming the finding. Second, we tested whether surface expression by lymphoma cells led to the secretion of TGF-β in culture medium. Using an ELISA assay, we detected variable levels of latent TGF-β in the culture medium of primary malignant B cells (median 100 pg/ml per million cells, range: undetectable −229 pg/ml, n=7). Similarly, lymphoma cell lines secreted variable amounts of TGF-β from undetectable to 200 pg/ml per million cells. Next, we determined the effect of TGF-b on intratumoral T cell proliferation and differentiation. As expected, exogenous addition of TGF-β inhibited the proliferation of T cells. Notably, the proliferation of intratumoral T cells was significantly reduced when co-cultured with lymphoma cells bearing an active form of TGF-β compared to that with lymphoma cells without TGF-β. Using flow cytometry, we showed that the addition of exogenous TGF-β enhanced Foxp3 expression in activated CD4+, CD4+CD45RA+ or CD4+CD45RO+ intratumoral T cells, suggesting that TGF-β promotes the generation of Treg cells in tumor microenvironment. In contrast, TGF-β suppressed the expression of IFN-γ in activated CD4+ T cells and inhibited the up-regulation of IL-12 and IL-23-induced IFN-γ expression in CD4+ cells, indicating that TGF-β suppresses the generation of TH1 cells. TGF-β alone slightly inhibited IL-17 expression in CD4+ T cells; however, TGF-β, in the presence of IL-6 and IL-23, up-regulated IL-17 expression in CD4+ T cells, suggesting proinflammatory cytokines are able to reverse the suppression induced by TGF-β. These results suggest that TGF-β controls the generation of TH1, TH17 and Treg cells contributing to the imbalance of effector TH cells and inhibitory Treg cells in the tumor microenvironment of B-cell NHL. Since malignant B-cells produce TGF-β, these results further support the important role of malignant B cells in the regulation of intratumoral T cell differentiation and the host immune response. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Novel Approach for Treatment of T-Cell Malignancies: Targeting T-Cell Receptor Vβ Families

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Jie Wang ◽  
Katarzyna Urbanska ◽  
Prannda Sharma ◽  
Mathilde Poussin ◽  
Reza Nejati ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Receptor ◽  
Brentuximab Vedotin ◽  
T Cell Lymphomas ◽  
T Cell Malignancies

Background: Peripheral T-cell lymphomas (PTCL) encompass a highly heterogeneous group of T-cell malignancies and are generally associated with a poor prognosis. Combination chemotherapy results in consistently poorer outcomes for T-cell lymphomas compared with B-cell lymphomas.1 There is an urgent clinical need to develop novel approaches to treatment of PTCL. While CD19- and CD20-directed immunotherapies have been successful in the treatment of B-cell malignancies, T-cell malignancies lack suitable immunotherapeutic targets. Brentuximab Vedotin, a CD30 antibody-drug conjugate, is not applicable to PTCL subtypes which do not express CD30.2 Broadly targeting pan-T cell markers is predicted to result in extensive T-cell depletion and clinically significant immune deficiency; therefore, a more tumor-specific antigen that primarily targets the malignant T-cell clone is needed. We reasoned that since malignant T cells are clonal and express the same T-cell receptor (TCR) in a given patient, and since the TCR β chain in human α/β TCRs can be grouped into 24 functional Vβ families targetable by monoclonal antibodies, immunotherapeutic targeting of TCR Vβ families would be an attractive strategy for the treatment of T-cell malignancies. Methods: We developed a flexible approach for targeting TCR Vβ families by engineering T cells to express a CD64 chimeric immune receptor (CD64-CIR), comprising a CD3ζ T cell signaling endodomain, CD28 costimulatory domain, and the high-affinity Fc gamma receptor I, CD64. T cells expressing CD64-CIR are predicted to be directed to tumor cells by Vβ-specific monoclonal antibodies that target tumor cell TCR, leading to T cell activation and induction of tumor cell death by T cell-mediated cytotoxicity. Results: This concept was first evaluated in vitro using cell lines. SupT1 T-cell lymphoblasts, which do not express a native functioning TCR, were stably transduced to express a Vβ12+ MART-1 specific TCR, resulting in a Vβ12 TCR expressing target T cell line.3 Vβ family specific cytolysis was confirmed by chromium release assays using co-culture of CD64 CIR transduced T cells with the engineered SupT1-Vβ12 cell line in the presence of Vβ12 monoclonal antibody. Percent specific lysis was calculated as (experimental - spontaneous lysis / maximal - spontaneous lysis) x 100. Controls using no antibody, Vβ8 antibody, and untransduced T cells did not show significant cytolysis (figure A). Next, the Jurkat T cell leukemic cell line, which expresses a native Vβ8 TCR, was used as targets in co-culture. Again, Vβ family target specific cytolysis was achieved in the presence of CD64 CIR T cells and Vβ8, but not Vβ12 control antibody. Having demonstrated Vβ family specific cytolysis in vitro using target T cell lines, we next evaluated TCR Vβ family targeting in vivo. Immunodeficient mice were injected with SupT1-Vβ12 or Jurkat T cells with the appropriate targeting Vβ antibody, and either CD64 CIR T cells or control untransduced T cells. The cell lines were transfected with firefly luciferase and tumor growth was measured by bioluminescence. The CD64 CIR T cells, but not untransduced T cells, in conjunction with the appropriate Vβ antibody, successfully controlled tumor growth (figure B). Our results provide proof-of-concept that TCR Vβ family specific T cell-mediated cytolysis is feasible, and informs the development of novel immunotherapies that target TCR Vβ families in T-cell malignancies. Unlike approaches that target pan-T cell antigens, this approach is not expected to cause substantial immune deficiency and could lead to a significant advance in the treatment of T-cell malignancies including PTCL. References 1. Coiffier B, Brousse N, Peuchmaur M, et al. Peripheral T-cell lymphomas have a worse prognosis than B-cell lymphomas: a prospective study of 361 immunophenotyped patients treated with the LNH-84 regimen. The GELA (Groupe d'Etude des Lymphomes Agressives). Ann Oncol Off J Eur Soc Med Oncol. 1990;1(1):45-50. 2. Horwitz SM, Advani RH, Bartlett NL, et al. Objective responses in relapsed T-cell lymphomas with single agent brentuximab vedotin. Blood. 2014;123(20):3095-3100. 3. Hughes MS, Yu YYL, Dudley ME, et al. Transfer of a TCR Gene Derived from a Patient with a Marked Antitumor Response Conveys Highly Active T-Cell Effector Functions. Hum Gene Ther. 2005;16(4):457-472. Figure Disclosures Schuster: Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria.

scholarly journals T Cells Can Induce Somatic Mutation in B Cell Receptor-Engaged BL2 Burkitt’s Lymphoma Cells Independently of CD40-CD40 Ligand Interactions

2000 ◽  
Vol 164 (3) ◽  
pp. 1306-1313 ◽  
Author(s):  
Stéphane Denépoux ◽  
Nathalie Fournier ◽  
Catherine Péronne ◽  
Jacques Banchereau ◽  
Serge Lebecque
Keyword(s):  
T Cells ◽  
B Cell ◽  
Somatic Mutation ◽  
Cell Receptor ◽  
Cd40 Ligand ◽  
Burkitt’S Lymphoma ◽  
B Cell Receptor ◽  
Lymphoma Cells ◽  
Ligand Interactions ◽  
Burkitt’S Lymphoma Cells

Murine Epidermal Vgamma5/Vdelta1-T-Cell Receptor+ T Cells Respond to B-Cell Lines and Lipopolysaccharides.

1995 ◽  
Vol 105 (s1) ◽  
pp. 58S-61S ◽  
Author(s):  
Christopher L. Reardon ◽  
Kent Heyborne ◽  
Moriya Tsuji ◽  
Fidel Zavala ◽  
Robert E. Tigelaar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Receptor ◽  
Cell Lines ◽  
Cell Receptor

Anti-CD45 Mediated Cytolysis of Human T-Cell Lymphoma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4637-4637
Author(s):  
Gerald G. Wulf ◽  
Anita Boehnke ◽  
Bertram Glass ◽  
Lorenz Truemper
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cytolytic Activity ◽  
T Cell Lymphoma ◽  
Lymphoma Cell ◽  
Lymphoma Cells ◽  
Human T Cell

Abstract Anti-CD45 mediated cytoreduction is an effective means for T-cell depletion in rodents and humans. In man, the CD45-specific rat monoclonal antibodies YTH24 and YTH54 are IgG2b subclass, exert a predominantly complement-dependent cytolytic activity against normal T-lymphocytes, and have been safely given to patients as part of conditioning therapies for allogeneic stem cell transplantation. The efficacy of such antibodies against human lymphoma is unknown. Therefore, we evaluated the cytolytic activity of YTH24 and YTH54 by complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), as well as by direct apoptotic and antiproliferative effects, against a panel of Hodgkin disease (HD) and non-Hodgkin lymphoma (NHL) cell lines, and against primary specimens. Significant CDC activity (>50% cytolysis) of the antibodies YTH54 and YTH24 was observed against three of five T-cell lymphoma lines, but against only one of nine B-cell lymphoma lines and none of four HD cell lines. The combination of YTH54 and YTH24 induced ADCC in all T-cell lymphoma cell lines and three primary leukemic T-cell lymphoma specimens, but were ineffective in B-cell lymphoma and HD cell lines.There were only minor effects of either antibody or the combination on lymphoma cell apoptosis or cell cycle arrest. In summary, anti-CD45 mediated CDC and ADCC via the antibodies YTH24 and YTH54 are primarily effective against lymphoma cells with T-cell phenotype, and may be an immunotherapeutic tool for the treatment of human T-cell lymphoma.

B-Cell Receptor Signaling Suppresses TAp63-Induced Apoptosis Via PI3-K/mTOR Pathway and Upregulation of MiR-21 in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 562-562
Author(s):  
Leigh Ann Humphries ◽  
Darcy Bates ◽  
Claire Godbersen ◽  
Prabhjot Kaur ◽  
Alexey V. Danilov
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
Mtor Pathway ◽  
B Cell Receptor ◽  
Lymphoma Cell ◽  
Raji Cells ◽  
Transcript Levels

Abstract Abstract 562 p63, an ancestral homolog of p53, encodes two major variants that have variable expression and context-specific functions in malignant tissues. We and others have shown that N-terminally truncated ΔNp63 promotes tumor growth in carcinomas. Meanwhile, the full-length TAp63 variant, which predominates in lymphoid malignancies, is anti-oncogenic in solid tumor models, where it mediates Ras-induced cellular senescence, suppresses anchorage-independent growth, and induces apoptosis. In hepatoma cells, TAp63 activates both extrinsic and intrinsic apoptosis pathways and enhances chemosensitivity. CLL clonal B cells have a low proliferative potential and disrupted apoptotic mechanism as a result of intrinsic defects and interaction with the microenvironment. At the crossroads of those pathways, the B-cell receptor (BCR) serves as a key survival molecule in CLL. Little is known about whether p63 regulates B-cell survival in CLL. Here we sought to investigate the role of TAp63 in regulation of apoptosis in CLL B cells and lymphoma cell lines and determined whether B-cell receptor signaling modulates p63. Forty-eight patients with B-CLL were enrolled in this study. CLL B cells were isolated from peripheral blood using standard Ficoll-Hypaque technique and co-cultured with M210B4 murine stroma cell line layers in RPMI supplemented with 15% fetal bovine serum (FBS). B-cell lymphoma cell lines Daudi, DOHH, Raji, OCI-LY3, OCI-LY19, SU DHL-4 and SUDHL-10 were maintained in RPMI with 10% FBS. CLL B cells and Raji cells were transfected with TAp63α expression vector or with siRNAs targeting p63 or miR-21 using Lonza Nucleofector with B-cell nucleofection solution (CLL B cells) and Solution V (Raji cells). Apoptosis was quantified by means of Annexin V/7-AAD staining and flow cytometry. B-cell receptor was engaged with 5 mg/mL (Raji cells) or 10 mg/mL IgM (CLL B cells). Expression of p63 and miR-21 was evaluated by immunoblotting and real-time RT-PCR. Median age of patients was 63 years. Median follow up was 3 years. Most patients presented in Rai stage 0–1 (80%). TAp63α was the predominantly expressed p63 variant in CLL cells and 7 lymphoma cell lines. Compared with normal B cells, TAp63 mRNA transcript levels were low in 28 CLL patient samples (using an arbitrary cutoff of <10% normal; 58.3%) and normal/elevated in 20 samples (41.7%). Patients with high expression of p63 were more likely to exhibit unmutated IGHV (U-CLL; p=0.016). siRNA-mediated knockdown of p63 in CLL cells resulted in protection from spontaneous apoptosis of CLL cells cultured on M210B4 murine bone marrow stroma (p<0.01) and was accompanied by a reduction in Noxa, Puma and Bax transcript levels. By contrast, enforced expression of TAp63α enhanced apoptosis. p63 knockdown in the Raji lymphoma cells resulted in increased proliferation and metabolic activity (p<0.05). B-cell receptor engagement suppressed p63 expression in CLL cells and Raji lymphoma cells. Pre-incubation of Raji cells with Syk inhibitor R406 and inhibitors of the PI-3K/mTOR pathway (LY294002, rapamycin, and CAL-101) resulted in the reversal of this phenomenon. Meanwhile, chemical inhibition of MEK, Erk, JNK, and p38 and siRNA-mediated knockdown of the transcription factor FOXO (a downstream targets of PI-3K) had no effect on p63 expression. Since TAp63α is a known target of miR-21, a microRNA that has been implicated in the pathogenesis of CLL, we examined their relationship in CLL and lymphoma. We found that TAp63 transcript levels inversely correlated with the expression of miR-21 in CLL B cells, but not in lymphoma cell lines. BCR stimulation led to increased miR-21 levels in CLL B cells, whereas knockdown of miR-21 resulted in upregulation of TAp63 in 3 out of 5 tested samples. TAp63α is the predominantly expressed p63 variant in the peripheral blood CLL cells and B-cell lymphoma cell lines, where it modulates the apoptosis program. BCR signaling repressed TAp63α via PI-3K/mTOR pathway and via upregulation of miR-21. This may be particularly relevant in U-CLL, where baseline p63 levels were higher. These data provide additional insights and rationale for targeting the BCR pathway and miR-21 in CLL and lymphoma. Disclosures: No relevant conflicts of interest to declare.

Massive Parallel Sequencing of Full-Length B-Cell Receptor Sequences Reveals HLA-Dependent Shaping of the B-Cell Immune Repertoire

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4143-4143
Author(s):  
Marvyn T. Koning ◽  
Sander A.J. van der Zeeuw ◽  
Marcelo Navarrete ◽  
Cornelis A.M. van Bergen ◽  
Valeri Nteleah ◽  
...  
Keyword(s):  
T Cell ◽  
Follicular Lymphoma ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Full Length ◽  
Lymphoma Cells ◽  
Hla Alleles ◽  
Hla Binding

Abstract Peptides of the B-cell receptor (BCR) may be presented in HLA molecules and therefore be recognized as epitopes by T cells. Bioinformatic evidence indicates that follicular lymphoma cells are selected against expression of a clonal BCR with a high cumulative predicted binding of BCR-derived peptides to the respective patient's HLA complex (Strothmeyer, Blood 2010). This observation suggests T-cell-mediated immunosurveillance against outgrowth of follicular lymphoma cells according to BCR HLA binding strength. Here, we investigate whether this phenomenon pertains to peripheral B cells in 6 healthy donors: 2 donors homozygous for HLA A01*01 / B08*01, 2 homozygous for HLA A02*01 / B7*02, and 2 donors heterozygous for these alleles. Unbiased representation of full-length V(D)J sequences was considered essential for correct data interpretation. PCR primers annealing to conserved motifs of BCR variable regions (e.g. BIOMED-2 protocol) fail to amplify a fraction of BCR, particularly those modified by somatic hypermutation. Therefore, we developed an improved anchored PCR strategy: cDNA was synthesized from poly(A)-RNA from peripheral blood with primers that anneal to specific Ig constant regions. In the same reaction, the 3' cDNA end is extended by switching to an oligonucleotide template containing an anchor sequence (SMART technology; Clontech). Anchor-tagged cDNA was amplified with a primer annealing to the anchor in combination with a nested constant region-specific reverse primer. Dumbbell adapters were added to the termini of 250 ng of purified PCR products. Circular consensus sequencing of single molecules was performed on the PacBio platform (Pacific Biosciences). Using one SMRT PacBio cell per amplicon, separate sequence libraries were created for μ, γ, κ, and λ BCR transcripts. Sequences covered by at least five reads were selected with SMRT Portal software to obtain >95% of sequences without sequence errors as demonstrated on multiple B-cell lines. Selected sequences were analysed by HighV-QUEST software (Alamyar, Immunome Research 2012). After exclusion of non-BCR sequences and duplicate BCR transcripts, a median of 5318 (range: 670-8752) individual BCR sequences was obtained per library. Binding affinity of nonamers in in-silico-translated BCR were calculated for the 4 HLA alleles by the NetMHC 3.4 algorithm. The fractions of BCR lacking any weak HLA binding peptide (NetMHC IC50 <500nM) within a library were compared between donors positive or negative for any HLA molecule. μ VDJ transcripts without HLA binding peptides were significantly more frequent for all HLA alleles in donors that actually express that particular allele (Table). With the exception of HLA A01*01, similar results were observed for γ transcripts. While the fraction of κ VJ transcripts without an HLA binder was overall higher in HLA A01*01 and B08*01, HLA-positive individuals had higher proportions of non-HLA binding sequences. λ transcripts were less likely to contain HLA binders with respect to HLA B07*02 and B08*01 but not to the HLA A alleles. Analogous analyses were performed for CDR3 regions as annotated by HighV-QUEST plus six amino acids on either flank. In 10 of 16 analyses, CDR3 sequences were less likely to contain an HLA binder in HLA-positive individuals; in three analyses an opposite effect was seen (Table). These results indicate that the peripheral BCR repertoire is shaped by HLA alleles in healthy individuals, most likely by T-cell mediated recognition of BCR peptides. Ongoing studies expand this fundamental finding with respect to the IC50 threshold, the number of nonamers, and additional HLA alleles. Our results warrant investigation of the potential role of HLA-dependent shaping of the BCR repertoire for the immune defense and the development of autoimmune disease and B-cell lymphoma. Table 1V(D)J without HLA binding peptideCDR3 without HLA binding peptideHLADonorμγκλμγΚλ A01*01Positive21%41%61%37%87%90%98%70%Negative16%42%59%38%92%92%96%65%P<0.001n.s.<0.01n.s.<0.001n.s.<0.01<0.001 A02*01Positive6%4%3%32%77%77%77%70%Negative4%1%2%32%75%69%78%78%P<0.001<0.001<0.01n.s.<0.01<0.001n.s.<0.001 B07*02Positive31%13%3%13%79%73%91%96%Negative27%8%2%6%79%69%90%98%P<0.001<0.01<0.01<0.001n.s.<0.05<0.05<0.001 B08*01Positive30%35%64%64%89%87%92%96%Negative14%28%62%61%88%82%90%93%P<0.001<0.001<0.01<0.001<0.01<0.001<0.01<0.001 Disclosures No relevant conflicts of interest to declare.

Pre-Clinical Characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b/CD3 As a Potential Therapy for B Cell Malignancies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4507-4507 ◽  
Author(s):  
L. Laura Sun ◽  
Xiaocheng Chen ◽  
Yvonne Chen ◽  
Mark S. Dennis ◽  
Diego Ellerman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Antibody Fragments ◽  
Bispecific Antibodies ◽  
Cell Targeting ◽  
B Cell Malignancies

Abstract T-cell recruiting bispecific antibodies and antibody fragments have been used to harness the cytotoxic potential of T cells for cancer treatment. As an example, encouraging clinical responses have been reported with the B cell targeting Blinatumomab, a 55-kDa fusion protein composed of two single-chain antibody fragments (scFvs). However, the therapeutic promise of many reported bispecific antibodies and fragments is often limited by unfavorable pharmacokinetics and administration schedule, immunogenicity, and a propensity towards aggregation. We have adopted a knobs-into-holes (KIH) antibody format and produced T-cell dependent bispecific antibodies (TDB), which allow one arm to target various B cell antigens while the other arm recruits T cells by binding to the CD3e subunit of the T-cell receptor. These B cell targeting TDBs are full length, humanized IgG1 antibodies with natural antibody architecture. Single dose pharmacokinetic/pharmacodynamic studies in cynomolgus monkeys show the KIH format TDBs are well tolerated in life, result in potent B cell depletion in peripheral and lymphoid tissue, and demonstrate pharmacokinetic properties resembling conventional antibody therapy. One B cell antigen targeted is CD79b, a component of the B cell receptor complex. CD79b is restricted to B cells, is highly prevalent on B cell leukemia and lymphomas, and has been clinically validated by an anti-CD79b antibody-drug conjugate as a safe and effective therapeutic target for B cell malignancies (ASCO 2014 abstract#8519). In our present work, we show that anti-CD79b/CD3 TDB can be produced and purified from E.coli, free of homodimer and aggregates. Anti-CD79b/CD3 TDB is a conditional agonist, activating CD3+T cells only in the presence of CD79b expressing B cells. In vitro, it induces potent B cell killing in a T-cell dependent manner, and is broadly active against lymphoma cell lines with a wide range of CD79b antigen levels. Compared to bispecific antibodies targeting some other B cell antigens, anti-CD79b/CD3 TDB appears to be more potent in autologous B cell killing assays with human PBMCs isolated from healthy donors. Taking advantage of antibodies with a range of binding affinities, we show that the B cell cytotoxic potency of anti-CD79b/CD3 TDB can be enhanced with increased binding affinity of either the anti-CD79b arm or the anti-CD3 arm in vitro. To assess the therapeutic potential of anti-CD79b/CD3 TDB, we further demonstrate that it is active in killing B lymphoma cells isolated from leukemia and lymphoma patients. Collectively, these preclinical data suggest anti-CD79b/CD3 TDB may be a promising agent for clinical development in B cell malignancies. Disclosures Sun: Genentech: Employment. Chen:Genentech: Employment. Chen:Genentech: Employment. Dennis:Genentech: Employment. Ellerman:Genentech: Employment. Johnson:Genentech: Employment. Mathieu:Genentech: Employment. Oldendorp:Genentech: Employment. Polson:Genentech: Employment. Reyes:Genentech: Employment. Stefanich:Genentech: Employment. Wang:Genentech: Employment. Wang:Genentech: Employment. Zheng:Genentech: Employment. Ebens:Genentech: Employment.

Targeting DKK1 for the Immunotherapy of B-Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 465-465
Author(s):  
Jianfei Qian ◽  
Sungyoul Hong ◽  
Liang Zhang ◽  
Yuhuan Zheng ◽  
Haiyan Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
Specific Ctls

Abstract Abstract 465 Immunotherapy may complement the current treatments for lymphomas. The lack of suitable shared lymphoma-associated antigens limits its applicability. Therefore, identification and utilization of novel and more potent tumor-associated antigens, particularly those shared among patients, are urgently needed to improve the efficacy of immunotherapy in the diseases. Recent studies have shown that Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by myeloma and other tumor cells, and is absent from normal tissues and organs except placenta and prostate. In the present study we demonstrated that DKK1 is also overexpressed in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Using DKK1 peptide-pulsed dendritic cells (DCs), we successfully generated HLA-A*0201+ DKK1-specific CTL lines and clones in vitro. These CTLs effectively lysed DKK1+/HLA-A*0201+ lymphoma cell lines Jeko-1 and Granta 519 cells, but not DKK1-/HLA-A*0201+ BJAB, RL and Mino cells nor DKK1+/HLA-A*020- CA46 and Daudi cells. Furthermore, the T-cell clones efficiently killed DKK1+/HLA-A*0201+ primary B-cell lymphoma cells from patients but not lymphoma cells from DKK1–/HLA-A*0201+ patients. HLA-ABC or HLA-A*0201 blocking mAbs significantly inhibited T cell-mediated cytotoxicity against peptide-pulsed T2 cells (P < .01, compared with medium control). No inhibitory effect was observed with mAb against HLA-DR and isotype control IgG. The results indicate that the cytotoxicity was attributed to MHC class I and more specifically, HLA-A*0201-restricted CD8+ CTLs. The CTLs did not kill DKK1–/HLA-A*0201+ DCs, B cells, or PBMCs, These results suggest that the CTLs recognized DKK1 peptides that are naturally processed and presented in the context of HLA-A*0201 molecules on lymphoma cells. To determine the in vivo antitumor activity, NOD-SCID and SCID-hu mice were used for lymphoma cell lines and primary lymphoma cells, respectively. Mice were treated with DKK1-specific CTLs after tumor established in NOD-SCID and SCID-hu mice. Control mice were treated with naïve CD8+ T cells or PBS alone. Tumor burden was measured according to levels of circulating human B2M, and survival rates were determined. Low levels (< 50 ng/ml) of circulating human B2M were detected in group treated DKK1-specific CTLs, while high levels (≥ 150 ng/ml) of circulating human B2M were detected in control mice. In SCID-hu model, X-ray examination showed that established tumors were eradicated in 60% mice treated with DKK1-specific CTLs, while large tumor burdens were found in all control mice. In NOD-SCID model, 40% of mice survived with the treatment of DKK1-specific CTLs. TUNEL assay further confirmed that tumor cells were lysed by DKK1-specific CTLs not naïve CD8+ T cells. These results indicate that DKK1-specific CTLs are able to eradicate established, patient-derived primary B- cell lymphoma in the hosts and adoptive transfer of DKK1-specific CTLs may be used for B-cell lymphoma therapy. Disclosures: No relevant conflicts of interest to declare.

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