Targeted Depletion of B-Cell Subsets by Anti-CD22 and Anti-CD79bAntibody Drug Conjugates: Illumination of the Mechanism of Action through Pharmacodynamic Biomarkers

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3923-3923
Author(s):  
Franklin Fuh ◽  
Caroline Looney ◽  
Dongwei Li ◽  
Kirsten Achilles Poon ◽  
Randall Dere ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Mechanism Of Action ◽  
Peripheral Blood ◽  
Employment Equity ◽  
Ki 67 ◽  
Cynomolgus Monkeys ◽  
Drug Conjugates ◽  
Equity Ownership

Abstract Abstract 3923 CD22 and CD79b are cell surface receptors whose expression is limited to B-cells. Both CD22 and CD79b are expressed on NHL and CLL patient B-cells, as well as on relapsed NHL B-cells (Dornan et al., 2009; Polson et al., 2010). In order to develop a target specific therapy for the treatment of CLL and NHL, we generated anti-CD22 and anti-CD79b antibody drug conjugates (ADCs) linked to an auristatin, a potent anti-mitotic drug that disrupts cellular mitosis through inhibition of tubulin polymerization. Preliminary efficacy data have shown that these ADCs have significant activity in preclinical xenograft models of NHL, while minimal activity was observed with the unconjugated antibody (AG Polson et al, 2010). To evaluate the cellular effects and characterize the mechanism of action (MOA) of these ADCs, we have examined the pharmacokinetics and the pharmacodynamic effects in non-human primates. Substantial B cell depletion was observed after administration of either anti-CD22-ADC or anti-CD79b-ADC to cynomolgus monkeys. By comparison, the extent and duration of B cell depletion was less substantial in animals dosed with unconjugated anti-CD22 or anti-CD79b antibodies. We evaluated several potential mechanisms for the depletion, including antibody opsonization, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and internalization of drug conjugates which leads to subsequent intracellular release of cytotoxic drug and cell death. Our data from in vitro anti-CD22 studies showed minimal to no ADCC or CDC activity, suggesting that these mechanisms play little to no role in vivo. Consistent with the expected MOA of ADCs, we observed depletion of proliferating splenic follicular germinal center B cells in cynomolgus monkeys following dosing with anti-CD22 ADC. Furthermore, in studies with either anti-CD22-ADC or anti-CD79b-ADC, preferential depletion of proliferating Ki67+ B lymphocytes (compared to Ki67- B lymphocytes) was observed in peripheral blood of ADC-dosed animals, and not in animals dosed with the unconjugated antibody or with vehicle control. This preferential depletion was observed on Days 8 and 15, but was not seen on Day 21 or at subsequent time points, which correlated with the expected serum clearance of the ADC. In contrast, we observed that administration of either ADC resulted in a dose-dependent decrease in circulating non-proliferating B cells one day after dosing. Taken together, these observations agree with the proposed mechanism of action of initial depletion of both proliferating and non-proliferating B cells via antibody-mediated opsonization at early time points after dosing (1-2 days) with subsequent preferential depletion of proliferating B lymphocytes mediated by the auristatin component of the anti-CD22 and anti-CD79b ADCs. These data support CD22 and CD79b ADCs as promising candidate therapeutics for the treatment of NHL and CLL, as both anti-CD22 and anti-CD79b drug conjugates are capable of targeting Ki-67+ (proliferating) B cells in lymphoid tissues and peripheral blood. In CLL, Ki-67+ cells in bone marrow have been hypothesized to represent pathogenic ‘stem’ cells, and our preliminary data indicate increased percentages of Ki-67+ B cells in the peripheral blood of CLL patients compared to normal healthy adults. As both anti-CD22 and anti-CD79b ADCs specifically deplete proliferating B cells, the development of these ADCs represents an effective way to target proliferating pathogenic B cells in NHL and CLL, and offers a more favorable risk-benefit profile than traditional chemotherapeutic agents. Disclosures: Fuh: Genentech/Hoffmann-La Roche Inc.: Employment, Equity Ownership. Looney:Genentech/Hoffmann-La Roche Inc.: Employment, Equity Ownership. Li:Genentech/Hoffmann-La Roche Inc.: Employment, Equity Ownership. Poon:Genentech/Hoffmann-La Roche Inc.: Employment, Equity Ownership. Dere:Genentech/Hoffmann-La Roche Inc.: Employment, Equity Ownership. Ramakrishnan:Genentech/Hoffmann-La Roche Inc.: Employment, Equity Ownership. Polson:Genentech/Hoffmann-La Roche Inc.: Employment, Equity Ownership. Prabhu:Genentech/Hoffmann-La Roche Inc.: Employment, Equity Ownership. Williams:Genentech/Hoffmann-La Roche Inc.: Employment, Equity Ownership.

CC-122 Degrades the Lymphoid Transcription Factor Aiolos (IKZF3) By Modulating Cereblon and Shows Clinical Activity in a Phase Ib Study of Subjects with Relapsed or Refractory Non-Hodgkin’s Lymphoma and Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3500-3500 ◽  
Author(s):  
Vincent Ribrag ◽  
Silvia Damien ◽  
Mecide Gharibo ◽  
Mercede Gironella ◽  
Armando Santoro ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Single Dose ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Memory T Cells ◽  
Cell Lymphoma ◽  
Employment Equity ◽  
Equity Ownership

Abstract Background: CC-122 is a novel non-phthalimide analog of the IMiDs® immunomodulatory drugs (lenalidomide and pomalidomide) and a first in class PPMTM (Pleiotropic Pathway Modifier) compound with multiple biological activities including potent anti-proliferative activity against B-lineage cells (10-fold greater than lenalidomide), anti-angiogenic activity (100-fold greater than lenalidomide) and immunomodulatory effects (10-fold greater than lenalidomide). The molecular target of CC-122 is cereblon (CRBN), a substrate receptor of the Cullin ring E3 ubiquitin ligase complex (CRL4CRBN). CC-122 promotes ubiquitination of lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) in a CRBN-dependent manner, leading to their subsequent degradation. Following establishment of 3mg once daily (QD) as the maximum tolerated dose (Blood 122:2905 2013), patients with advanced aggressive non-Hodgkin lymphoma (NHL), multiple myeloma (MM), and select solid tumors were enrolled in parallel expansion cohorts of up to 20 evaluable patients. CC-122 was dosed at 3 mg QD in 28-day cycles until disease progression. Results: As of May 1, 2014, 93 total patients were enrolled in the expansion phase of the study. The NHL cohort included 21 patients with diffuse large B-cell lymphoma (DLBCL) and 1 patient with mantle cell lymphoma, and twenty-four patients were enrolled in the MM cohort. Results in solid tumor cohorts will be reported separately. All patients were ECOG performance status 0-2, the median number of prior systemic therapies was 4 (NHL) and 6 (MM). The most common (> 20%) adverse events (AEs) (grades 1-4) included neutropenia (69.6%), anemia (52%), asthenia (50%), pyrexia (35%), diarrhea (30%), cough (30%), thrombocytopenia (28%), and constipation (22%). Grade 3/4 AEs occurring in more than one patient were neutropenia (52%), anemia (26%), febrile neutropenia (13%), and thrombocytopenia (7%). CC-122 dose reduction was required in 36.4% of patients with NHL and 63% of patients with MM, the majority of which was due to neutropenia and occurred during cycle 1 or 2. CC-122 systemic exposure in NHL and MM patients was generally comparable after administration of single and multiple doses. Peak concentrations were observed between 30 minutes and 2 hours (median Tmax concentration = 1.5 h). Four treated patients with DLBCL had objective responses; one patient with complete response (CR) and 3 with partial responses (PR). Responses were observed in patients with germinal center B cell (GCB), non-GCB and Myc/Bcl2 over-expressing DLBCL. Four treated patients with MM had PR, and two of these responders were progression free beyond 10 cycles. A single dose of CC-122 3mg resulted in decreased Aiolos protein expression at 1.5 and 5 hours compared with baseline in peripheral B cells (median 38% and 53%) and T cells (median 31% and 54%) in the combined NHL (n = 16) and MM (n = 19) cohorts. Decrease in expression of Aiolos protein from baseline was also observed in lymph node biopsies of patients with DLBCL. Furthermore, CC-122 treatment decreased CD19+ B cells (median = 57% of baseline), expanded CD4-/CD8+/CD45RA-/CD45RO+ cytotoxic memory T cells (median = 320% of baseline), and expanded CD4+/CD8-/CD45RA-/CD45RO+ helper memory T cells (median = 154% of baseline) in peripheral blood samples from patients with MM (n = 9) and NHL (n = 3-12) subjects. Additionally, ex vivo activation of T cells after a single dose of CC-122 compared with baseline, as measured by IL-2 production, increased by a median of 776% (NHL n = 3 and MM n = 7). Conclusions: CC-122 shows promising initial clinical and pharmacodynamic activity in heavily pretreated relapse/refractory NHL and MM patients. Biomarker analysis indicates that the 3 mg QD dose of CC-122 results in rapid CRBN target engagement and Aiolos degradation in the peripheral blood lymphocytes of patients with NHL and MM patients and in NHL tumor tissue. Exploration of an intermittent dosing to mitigate neutropenia-related dose reductions and interruptions is ongoing and clinical studies exploring drug combinations with CC-122 are underway. Disclosures Ribrag: Celgene Corp: Consultancy. Rasco:Celgene Corp: Membership on an entity's Board of Directors or advisory committees. Wei:Celgene Corp: Employment, Equity Ownership. James:Celgene Corp: Employment. Hagner:Celgene Corp: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership. DiMartino:Celgene Corp: Employment, Equity Ownership. Pourdehnad:Celgene Corp: Employment, Equity Ownership. Stoppa:Celgene Jansen: Honoraria.

scholarly journals A Novel CD20xCD3 Bispecific Fully Human Antibody Induces Potent Anti-Tumor Effects Against B Cell Lymphoma in Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4501-4501 ◽  
Author(s):  
Bindu Varghese ◽  
Jayanthi Menon ◽  
Luis Rodriguez ◽  
Lauric Haber ◽  
Kara Olson ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Tumor Cells ◽  
Bispecific Antibody ◽  
Tumor Regression ◽  
Employment Equity ◽  
Cynomolgus Monkeys ◽  
Nsg Mice ◽  
Equity Ownership

Abstract Bispecific antibodies that redirect effector T cells to kill tumor cells have shown considerable promise in both pre-clinical and clinical studies. However, these bispecific formats can have short half-lives necessitating constant infusion of the molecules into patients. We report here on a novel full-length human IgG CD20xCD3 bispecific antibody (REGN1979) that targets CD20 expressed on normal and malignant B cells and CD3 expressed on T cells in humans and cynomolgus monkeys. Our results demonstrate CD20-target cell-dependent activation and cytokine release by T cells, and efficient redirected T cell lysis of target tumor cells. Raji B cell lymphomas grown as tumors in NOD SCID IL2R gamma deficient (NSG) mice and co-implanted with human peripheral blood mononuclear (PBMC) cells were completely inhibited when treated at the time of implantation with a low dose (0.004 mg/kg; 2x/week) of REGN1979. As expected, T cells were required for this tumor inhibition, since treatment in the absence of human T cells was not effective. REGN1979 bispecific antibody also demonstrated potent activity against other tumor cells expressing CD20, as it significantly delayed CD20-transduced B16F10.9 tumor growth in immune-competent mice. Most importantly, REGN1979 induced dramatic tumor regression in large advanced (500-900 mm3) Raji tumors, associated with long-lasting tumor control. The tumor-infiltrating lymphocytes (TILs) in B cell lymphomas in these untreated NSG mice were found to express the inhibitory receptors Tim-3 and PD-1 and were the predominant fraction of T cells in the tumors and in the circulation. T cells in mice treated with REGN1979 showed decreased Tim-3 and PD-1 expression in the circulation accompanied by complete tumor regression. In further studies, REGN1979 (dosed at 0.4 mg/kg; 2x/week) was superior to rituximab therapy (dosed at 8 mg/kg; 5x/week) and comparable to the CD19xCD3 BiTE (dosed at 0.5 mg/kg; 5x/week) in suppressing established Raji tumors (200-400mm3). Pre-clinical studies in cynomolgus monkeys to assess activity of the bispecific antibody for depleting B cells in circulation and various lymphoid organs showed that a single injection of REGN1979 (0.1 mg/kg) was more potent at depleting CD20+ B cells in the mesenteric lymph nodes than a high dose of rituximab (30 mg/kg). In separate studies, REGN1979 was also found to have a long half-life (>14 days) in the circulation of monkeys following depletion of B cells. These studies show potent activity of a new class of fully human bispecific antibodies for treating tumors, and support clinical testing of REGN1979 in patients with CD20+ cancers. Figure 1 Figure 1. Disclosures Varghese: Regeneron Pharmaceuticals: Employment, Equity Ownership. Menon:Regeneron Pharmaceuticals: Employment, Equity Ownership. Rodriguez:Regeneron Pharmaceuticals: Employment, Equity Ownership. Haber:Regeneron Pharmaceuticals: Employment, Equity Ownership. Olson:Regeneron Pharmaceuticals: Employment, Equity Ownership. Duramad:Regeneron Pharmaceuticals: Employment, Equity Ownership. Oyejide:Regeneron Pharmaceuticals: Employment, Equity Ownership. Smith:Regeneron Pharmaceuticals: Employment, Equity Ownership. Thurston:Regeneron Pharmaceuticals: Employment, Equity Ownership. Kirshner:Regeneron Pharmaceuticals: Employment, Equity Ownership.

scholarly journals Immunotherapy with Long-Lived Anti-CD20 × Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human B Cell Lines and of Circulating and Lymphoid B Cells in Monkeys: A Potential Therapy for B Cell Lymphomas and Leukemias

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3111-3111 ◽  
Author(s):  
Seung Y. Chu ◽  
Sung-Hyung Lee ◽  
Rumana Rashid ◽  
Hsing Chen ◽  
Emily W. Chan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Half Life ◽  
Bispecific Antibodies ◽  
Employment Equity ◽  
Cynomolgus Monkeys ◽  
Equity Ownership ◽  
Anti Cd20

Abstract CD20 is highly expressed on normal and malignant B cells, and is a well-established target of antibody therapeutics for B cell leukemias and lymphomas. However, a limitation of approved anti-CD20 antibodies such as rituximab, ofatumumab, and obinutuzumab is that they are unable to stimulate T cell-mediated killing of CD20+ B cells. To exploit the potent activity intrinsic to T cell immunotherapy while maintaining the favorable dosing regimen of a therapeutic antibody, we have designed novel humanized bispecific antibodies that bind to both CD20+ B cells and CD3+ T cells. Such antibodies act via a mechanism known as "redirected T cell-cytotoxicity" (RTCC), because they stimulate targeted T cell-mediated killing regardless of T cell receptor antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life). We first generated a series of affinity-optimized anti-CD20 × anti-CD3 bispecific antibodies and screened these using RTCC assays in which bispecifics stimulated killing of the CD20+ Ramos B cell line by purified human T cells. From this cell-based screen, we selected two candidates for further study in animal models. The bispecific antibodies XmAb13676 and XmAb13677 have identical T cell-engaging domains with 8 nM affinity for human CD3. XmAb13676 stimulated T cell killing of Ramos cells with an EC50 of ~53 ng/ml (~420 pM), while XmAb13677 (with higher affinity for CD20) had an EC50 of ~2 ng/ml (~16 pM). To assess in vivo half-life, we next dosed mice with 2 mg/kg of XmAb13676 or XmAb13677. In marked contrast to non-Fc domain-containing bispecific antibody formats, XmAb13676 and XmAb13677 had an extended serum half-life in mice of 6.7 and 6.6 days, respectively. Because these bispecifics were optimized for binding to human CD20 and CD3 targets and do not crossreact with mouse antigens, we evaluated efficacy in cynomolgus monkeys. We treated 3 monkeys per group with a single dose of XmAb13676 or XmAb13677 at 0.03, 0.3, or 3 mg/kg. Within 4 hr after dosing, T cells were strongly activated and stimulated depletion of over 97% of circulating CD40+ B cells, with the two 3 mg/kg groups showing greatest depletion. B cells continued to decrease for 24 to 48 hr after dosing, with the high-dose groups remaining at baseline levels for the duration of the study (29 days). CD4+ and CD8+ T cells in the circulation were activated immediately after treatment with XmAb13676 and XmAb13677, and this state was sustained for over 48 hr, as measured by greatly increased levels of the activation markers CD25 and CD69. Bispecific antibodies also induced rapid margination of CD4+ and CD8+ T cells from the circulation, with blood T cell populations returning to baseline from 2 to 7 days after dosing. Notably, CD40+ cells in lymph nodes and in bone marrow were depleted by over 90% at all doses, and at the higher dose levels, these B cell populations had not recovered by 29 days after treatment. Our results demonstrate that bispecific antibodies can recruit and activate T cells to efficiently kill CD20+ B cells not only in the circulation but also in the more resistant reservoir of lymphoid organs. These preclinical data in cynomolgus monkeys provide a rationale for clinical assessment of anti-CD20 × anti-CD3 bispecific antibodies in patients with CD20+ B cell leukemias and lymphomas. Disclosures Chu: Xencor: Employment, Equity Ownership. Lee:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

scholarly journals Sequence Level Analysis of Hodgkin Lymphoma Clonotypes Detected in Peripheral Blood Using a Next-Generation Sequencing Approach

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1610-1610
Author(s):  
Yasuhiro Oki ◽  
Sattva S. Neelapu ◽  
Michelle Fanale ◽  
Larry W. Kwak ◽  
Luis Fayad ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Expression Level ◽  
Employment Equity ◽  
Tumor Biopsy ◽  
Cellular Compartment ◽  
Equity Ownership ◽  
Level Analysis

Abstract Background: Classical Hodgkin lymphoma (CHL) has been established as B-cell malignancy characterized by a clonal expansion of pathognomonic Reed Sternberg cells (Marafioti et al. Blood 2000). A previous report suggests that clonotypic B-cells may be present in the blood of patients with CHL; however, the relationship between these circulating clonotypic B-cells and CHL is unclear. We utilized the LymphoSIGHT™ method, a next-generation sequencing approach, to detect lymphoma-specific clonotypes in peripheral blood in patients with CHL at diagnosis or disease recurrence as well as in follow-up blood samples. This method has the sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood, and has been applied to minimal residual disease (MRD) detection in multiple B-cell malignancies. We evaluated the extent of somatic hypermutation in the lymphoma clonotypes, and performed sequence and expression level analyses of the lymphoma clonotype repertoire. Methods: Frozen primary tumor biopsy samples were first analyzed for clonality at the immunoglobulin heavy chain (IGH) and kappa chain (IGK) loci using the LymphoSIGHT method. Rearranged immunoglobulin gene segments (IGH-VDJ, IGH-DJ and IGK) in the genomic DNA and/or RNA were amplified with locus-specific primer sets, sequenced, and analyzed using standardized algorithms for clonotype determination. Clonotypes with a frequency >5% in the B-cell repertoire of the tumor biopsy were considered to represent tumor clonotypes. IGH-VDJ clonotypes with a frequency >2% in DNA were deemed to be cancer-specific if the clonotype was not present in the RNA. Such clonotypes were then quantitated in serum and peripheral blood mononuclear cells (PBMC), and DNA sequence and/or RNA expression level analysis was performed. Results: A total of 17 CHL patients were analyzed. A high-frequency clonal rearrangement was identified for at least one receptor (IGH-VDJ, IGH-DJ and IGK) in 12 of 17 cases (71%). Lymphoma-specific clonotypes were detected in blood samples from 8 of 11 patients (73%). Notably, a lymphoma-specific clonotype was detected in the serum compartment in 8 of 9 cases (89%) tested (Figure 1A), while it was detected in PBMC only in 3 of 9 cases (33%) tested (Figure 1B). Follow-up samples obtained from three patients in remission were negative for the tumor-specific clonotype in both the serum and cellular compartments. We conducted sequence and expression level analysis of each IGH-VDJ clonal rearrangement. We calculated the number of somatic mutations in each lymphoma-specific clonotype compared to the germline sequence in the interrogated region. In the ten patients with IGH-VDJ clonal rearrangements, we observed a median of 14 somatic mutations (range 0 to 27). This confirms that HRS cells correspond in their developmental stage to germinal or post-germinal center B-cells. While IGH-VDJ clonotypes were observed frequently in DNA obtained at diagnosis, IGH-VDJ clonotypes were not detected in the RNA from the same sample. We evaluated the relationship between the presence of lymphoma-specific clonotypes in the cellular compartment at diagnosis and eventual progression. All three untreated patients that were positive at baseline in the cellular compartment experienced relapse or progression (at 3, 11 and 17 months). In contrast, zero of 5 patients without detectable lymphoma-specific clonotypes in their cellular compartment at baseline experienced relapse (follow up duration 23-45 months, log-rank test p=0.004). Conclusions: This is the first clinical assay that can be used to detect and monitor MRD in CHL. Lymphoma-specific sequences can be identified in serum in 80% of cases. Our preliminary analysis suggests that the presence of lymphoma-specific clonotypes in PBMCs may indicate high risk for recurrence. This study demonstrates proof-of-principle and underscores the promise of a new methodology to measure disease burden and provide prognostic information from a blood test in patients with CHL. Figure 1. Lymphoma clonotype levels in A) cell-free plasma and B) PBMC samples are shown for the different patients. Figure 1. Lymphoma clonotype levels in A) cell-free plasma and B) PBMC samples are shown for the different patients. Figure 2 Figure 2. Disclosures Klinger: Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Kong:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Cell Enrichment for FISH: A Novel Diagnostic Tool in Challenging Cases of Low Bone Marrow Involvement By Mature B-Cell Neoplasms

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2992-2992
Author(s):  
Farzad Nooraie ◽  
J. Dianne Keen-Kim ◽  
Derek Denton Lyle ◽  
Shannon Dingivan ◽  
Austin Mauch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Detection Rate ◽  
Cell Detection ◽  
Employment Equity ◽  
Cell Enrichment ◽  
Equity Ownership ◽  
Blood Specimens

Abstract Cytogenetic studies are useful tools which can provide diagnostic, prognostic and management information for mature B-cell neoplasms. Mature B-cells do not grow well in cytogenetic cultures. Therefore, detection, characterization and differentiation of scant mature B-cell neoplasms or minimal residual disease can be difficult in bone marrow or peripheral blood specimens. FISH provides more sensitive information than G-band chromosome analysis. However, in cases with low-level involvement of the marrow, abnormal FISH results may be missed or not reported when abnormalities do not exceed experimentally determined cut-off values. To increase the sensitivity of abnormal mature B-cell detection, we developed an enrichment method utilizing pan B-cell antibodies. This method separates mature B-cells from the remaining bone marrow and/or peripheral blood cells. We selected 59 bone marrow and peripheral blood specimens from patients referred for mature B-cell neoplasms (except for myeloma) for use in the validation. These specimens had 1% to 10% monoclonal mature B-cells detected by flow cytometry and were enriched using cell-separating technologies. Each specimen was divided into four equal portions, with three of four portions undergoing enrichment using different pan B-cell antibodies. The fourth portion was reserved for standard non-enriched testing and was used for comparison to results obtained in the enriched portions. A variety of corresponding FISH analyses were performed in each of the four portions, based upon the disease state. FISH results were obtained by two independent scoring technologists. Enrichment with B-cell antibodies improved detection of FISH abnormalities that may not have otherwise been observed in the patient specimens. 42% (25/59) of samples had abnormalities detected within the enriched portion that were not detected in the standard non-enriched portion. Of these, 64% (16/25) had a FISH abnormality that was a critical finding for the final diagnosis, prognosis and/or management of the patient. Enrichment also increased the sensitivity of FISH abnormality detection. 29% (17/59) of samples had abnormalities that were detected in both the enriched and non-enriched portions. However, detection was on average 15-fold more sensitive. The average detection rate of FISH abnormalities in the non-enriched portion was 3%, which is at or near the experimentally determined cut-off value for most FISH probes. In contrast, the average detection rate of FISH abnormalities in the enriched portion was 56%. In 5% (3/59) of cases, detection of FISH aberrations in enriched specimens helped to distinguish two separate neoplastic processes in the bone marrow. These results demonstrate the increased opportunity for detecting FISH aberrations in enriched versus non-enriched specimens. Mature B-cell enrichment and subsequent FISH testing in cases of scant mature B-cell neoplastic involvement of the bone marrow and/or peripheral blood is a novel and powerful cytogenetic technique. This technique enriches bone marrow and/or peripheral blood specimens for targeted abnormal cells and increases the number of those cells analyzed by FISH testing, thus allowing for a higher detection rate of genetic abnormalities. Disclosures Nooraie: Genoptix Inc., A Novartis Company: Employment, Equity Ownership. Keen-Kim:Genoptix Inc., A Novartis Company: Employment, Equity Ownership. Lyle:Genoptix Inc., A Novartis Company: Employment, Equity Ownership. Dingivan:Genoptix Inc., A Novartis Company: Employment, Equity Ownership. Mauch:Genoptix Inc., A Novartis Company: Employment, Equity Ownership. Lynes:Genoptix Inc., A Novartis Company: Employment. Castillo:Genoptix Inc., A Novartis Company: Employment. Kolker:Genoptix Inc., A Novartis Company: Employment. Cancino:Genoptix Inc., A Novartis Company: Employment, Equity Ownership.

scholarly journals Interleukin-13 in Combination With CD40 Ligand Potently Inhibits Apoptosis in Human B Lymphocytes: Upregulation of Bcl-xL and Mcl-1

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4415-4424 ◽  
Author(s):  
Jon Lømo ◽  
Heidi Kiil Blomhoff ◽  
Sten Eirik Jacobsen ◽  
Stanislaw Krajewski ◽  
John C. Reed ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Cell Apoptosis ◽  
Cell Types ◽  
Cd40 Ligand ◽  
Interleukin 13

Abstract Interleukin-13 (IL-13) is a novel T-cell–derived cytokine with IL-4–like effects on many cell types. In human B lymphocytes, IL-13 induces activation, stimulates proliferation in combination with anti-IgM or anti-CD40 antibodies, and directs Ig isotype switching towards IgE and IgG4 isotypes. We show here that IL-13 also regulates human B-cell apoptosis. IL-13 reduced spontaneous apoptosis of peripheral blood B cells in vitro, as shown by measurement of DNA fragmentation using the TUNEL and Nicoletti assays. The inhibition of cell death by IL-13 alone was significant but modest, but was potently enhanced in combination with CD40 ligand (CD40L), a survival stimulus for B cells by itself. Interestingly, IL-13 increased the expression of CD40 on peripheral blood B cells, providing a possible mechanism for the observed synergy. IL-13 alone was a less potent inhibitor of apoptosis than IL-4. Moreover, there was no additive effect of combining IL-4 and IL-13 at supraoptimal concentrations, which is consistent with the notion that the IL-4 and IL-13 binding sites share a common signaling subunit. The combination of IL-13 with CD40L augmented the expression of the Bcl-2 homologues Bcl-xL and Mcl-1, suggesting this as a possible intracellular mechanism of induced survival. By contrast, levels of Bcl-2, and two other Bcl-2 family members, Bax and Bak, remained unaltered. Given the importance of the CD40-CD40L interaction in B-cell responses, these results suggest a significant role of IL-13 in the regulation of B-cell apoptosis.

Detection of Diffuse Large B-Cell Lymphoma in Peripheral Blood Using High-Throughput Sequencing Assay.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2666-2666
Author(s):  
Yasuhiro Oki ◽  
Malek Faham ◽  
Victoria Carlton ◽  
Sattva S. Neelapu ◽  
Anas Younes
Keyword(s):  
B Cell ◽  
Peripheral Blood ◽  
High Throughput Sequencing ◽  
B Cell Lymphoma ◽  
Lymph Node Biopsy ◽  
Employment Equity ◽  
Tumor Biopsy ◽  
Blood Samples ◽  
Large B Cell Lymphoma ◽  
Equity Ownership

Abstract Abstract 2666 Background: In patients with diffuse large B-cell lymphoma (DLBCL), circulating lymphoma cells in the bloodstream are rarely detected by conventional morphology or flow cytometry evaluation. We developed a high-throughput sequencing based platform, LymphoSIGHT, to detect evidence of lymphoid malignancies in peripheral blood samples, as this could potentially be used for detection of minimal residual disease after treatment. This sequencing method has a sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood. We herein report the results of our pilot study assessing the ability of this method to detect the lymphoma clone in peripheral blood samples from 5 DLBCL patients at the time of diagnosis. Methods: This study has been approved by IRB and consent has been obtained from patients. Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and peripheral blood samples (plasma and peripheral blood mononuclear cell (PBMC) compartments) collected at initial diagnosis. Amplified products were sequenced to obtain >1 million reads (>10× sequencing coverage per IgH molecule), and were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the lymph node biopsy sample. The presence of the tumor-specific clonotype was then quantitated in cell-free and PBMC compartments from the diagnostic blood sample. A quantitative and standardized measure of clone level among all leukocytes in the diagnostic sample was determined using internal reference DNA. Results: We detected a high-frequency IgH clonal rearrangement in all 5 lymph node biopsy samples. The lymphoma clonotype that was identified in the tumor biopsy was also detected in the plasma and/or PBMC compartment in all 5 patients at diagnosis. Specifically, the lymphoma clonotype was detected in the plasma compartment in 4 patients, while 3 patients demonstrated the presence of the lymphoma clonotype in the PBMC compartment (Table 1). We hypothesize that the positive lymphoma clone in the plasma is due to rapid proliferation and necrosis of the primary tumor, releasing the degraded component of lymphoma into the blood stream. However, in this small sample size, we did not observe an obvious correlation between the level of detection (PBMC or plasma) and clinical parameters (LDH, stage, size of tumor, tumor Ki67, cell-of-origin). All patients achieved complete response after initial treatment and four are being followed. We plan to analyze blood specimens while they are in remission. Conclusions: IgH clonal rearrangements were detected by sequencing in all tumor biopsy samples. Importantly, all peripheral blood samples showed signs of circulating lymphoma material in either the plasma or PBMC compartment at diagnosis. Analysis of diagnostic and post-therapy samples from additional DLBCL patients is ongoing. These data will determine whether the sequencing assay is a strong indicator for response to therapy and relapse monitoring. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Carlton:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Steady-state generation of mucosal IgA+ plasmablasts is not abrogated by B-cell depletion therapy with rituximab

Blood ◽  
2010 ◽  
Vol 116 (24) ◽  
pp. 5181-5190 ◽  
Author(s):  
Henrik E. Mei ◽  
Daniela Frölich ◽  
Claudia Giesecke ◽  
Christoph Loddenkemper ◽  
Karin Reiter ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Steady State ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Ki 67

AbstractThe anti-CD20 antibody rituximab depletes human B cells from peripheral blood, but it remains controversial to what extent tissue-resident B cells are affected. In representative patients with rheumatoid arthritis, we here demonstrate that recently activated presumably short-lived plasmablasts expressing HLA-DRhigh and Ki-67 continuously circulate in peripheral blood after B-cell depletion by rituximab at 26%-119% of their initial numbers. They circulate independent of splenectomy, express immunoglobulin A (IgA), β7 integrin, and C-C motif receptor 10 (CCR10) and migrate along CCL28 gradients in vitro, suggesting their mucosal origin. These plasmablasts express somatically hypermutated VH gene rearrangements and spontaneously secrete IgA, exhibiting binding to microbial antigens. Notably, IgA+ plasmablasts and plasma cells were identified in the lamina propria of patients treated with rituximab during peripheral B-cell depletion. Although a relation of these “steady state”–like plasmablasts with rheumatoid arthritis activity could not be found, their persistence during B-cell depletion indicates that their precursors, that is, B cells resident in the mucosa are not deleted by this treatment. These data suggest that a population of mucosal B cells is self-sufficient in adult humans and not replenished by CD20+ B cells immigrating from blood, lymphoid tissue, or bone marrow, that is, B cells depleted by rituximab.

scholarly journals First-in-Human, Phase 1/2 Trial to Assess the Safety and Clinical Activity of Subcutaneous GEN3013 (DuoBody®-CD3×CD20) in B-Cell Non-Hodgkin Lymphomas

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 758-758 ◽  
Author(s):  
Pieternella Lugtenburg ◽  
Rogier Mous ◽  
Michael Roost Clausen ◽  
Martine E.D. Chamuleau ◽  
Peter Johnson ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Dose Escalation ◽  
Research Funding ◽  
Clinical Activity ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Introduction: CD20-specific monoclonal antibodies (mAbs) have demonstrated efficacy in the treatment of B-cell non-Hodgkin lymphomas (B-NHL); however, a significant proportion of patients (pts) present with refractory disease or will experience relapse. GEN3013 (DuoBody®-CD3×CD20) is the first subcutaneously administered IgG1 bispecific antibody (bsAb) that targets the T-cell surface antigen CD3 and the B-cell surface antigen CD20, triggering T-cell-mediated killing of B cells. In vitro, GEN3013 efficiently activates and induces cytotoxic activity of CD4+ and CD8+ T cells in the presence of B cells (Hiemstra et al. Blood 2018), and results in long-lasting depletion of B cells in cynomolgus monkeys. Subcutaneous (SC) GEN3013 in cynomolgus monkeys resulted in lower plasma cytokine levels, and similar bioavailability and B-cell depletion, compared with intravenous administration. GEN3013 has higher potency in vitro than most other CD3×CD20 bsAbs in clinical development (Hiemstra et al. HemaSphere 2019). SC GEN3013 in pts with B-NHL is being evaluated in a first-in-human, Phase 1/2 trial (NCT03625037), which comprises a dose-escalation part and a dose-expansion part. Here we report preliminary dose-escalation data. Methods: Pts with CD20+ B-NHL with relapsed, progressive, or refractory disease following anti-CD20 mAb treatment, and ECOG PS 0-2 were included. During dose escalation, pts received SC GEN3013 flat dose in 28-day cycles (q1w: cycle 1-2; q2w: cycle 3-6; q4w thereafter) until disease progression or unacceptable toxicity. Risk of cytokine release syndrome (CRS) was mitigated with the use of a priming dose and premedication with corticosteroids, antihistamines, and antipyretics. Primary endpoints were adverse events (AEs) and dose-limiting toxicities (DLTs). Secondary endpoints included pharmacokinetics (PK), immunogenicity (anti-drug antibodies [ADA]), pharmacodynamics (PD) (cytokine measures; laboratory parameters), and anti-tumor activity (tumor size reduction; objective and best response). Results: At data cut-off (June 28, 2019), 18 pts were enrolled into the dose-escalation part of the trial, with safety data available for pts receiving doses starting at 4 µg. Most pts had diffuse large B-cell lymphoma (DLBCL; n=14) and were heavily pre-treated; 10 pts had received ≥3 prior lines of therapy (overall median [range]: 3 [1-11]). The median age was 58.5 years (range: 21-80), and 13 pts were male. At a median follow-up of 1.9 months, pts received a median of 5 doses (range: 1-14); treatment is ongoing in 6 pts. Twelve pts discontinued treatment due to progressive disease. Six pts died (2 during treatment, 4 during survival follow-up), all due to disease progression and unrelated to treatment. The most common (n≥5) treatment-emergent AEs were pyrexia (n=8), local injection-site reactions (n=7), diarrhea (n=5), fatigue (n=5), and increased aspartate aminotransferase (n=5). The most common Grade (G) 3/4 AEs were anemia (n=3) and neutropenia (n=3). Despite increasing GEN3013 doses, all CRS events were non-severe (initial observation: 3/8 pts, G1: n=1, G2: n=2; following modification of premedication plan [corticosteroids for 3 days]: 6/10 pts, G1: n=4, G2: n=2). Increases in peripheral cytokine (IL6, IL8, IL10, IFNγ, TNFα) concentrations after GEN3013 dosing correlated with clinical symptoms of CRS in most pts. No pts had tumor lysis syndrome or neurological symptoms. No DLTs were observed. GEN3013 PK profiles reflect SC dosing; Cmax occurred 2-4 days after dosing. No ADAs were detected. PD effects following GEN3013 dosing were observed at dose levels as low as 40 µg and included rapid, complete depletion of circulating B cells (if present after prior anti-CD20 therapy) and peripheral T-cell activation and expansion. The first evidence of clinical activity was observed at a dose level of 120 µg, with complete metabolic response observed in a pt with DLBCL. Conclusions: Subcutaneously administered GEN3013, a potent CD3×CD20 bsAb, shows good tolerability and early evidence of clinical activity at low dose levels in heavily pretreated pts with relapsed or refractory B-NHL. All CRS events were non-severe and did not lead to discontinuation. No DLTs were observed. Dose escalation is ongoing; updated data will be presented. Dose expansion will begin upon determining the recommended Phase 2 dose (RP2D) (NCT03625037). Disclosures Lugtenburg: Janssen Cilag: Honoraria; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria; Servier: Consultancy, Honoraria, Research Funding; Genmab: Consultancy, Honoraria; BMS: Consultancy; Takeda: Consultancy, Honoraria, Research Funding. Mous:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Sandoz: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Takeda: Honoraria, Research Funding; Janssen Cilag: Consultancy, Honoraria; MSD: Honoraria; Gilead: Consultancy, Honoraria, Research Funding. Clausen:Abbvie: Other: Travel grant to attend ASH 2019. Johnson:Boehringer Ingelheim: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Epizyme: Honoraria, Research Funding; Incyte: Honoraria; Takeda: Honoraria; Genmab: Honoraria; Bristol-Myers Squibb: Honoraria; Kite: Honoraria; Novartis: Honoraria. Rule:Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Astra-Zeneca: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Napp: Consultancy; Kite: Consultancy. Oliveri:Genmab: Employment, Equity Ownership. DeMarco:Genmab: Employment, Equity Ownership. Hiemstra:Genmab: Employment, Equity Ownership, Other: Warrants. Chen:Genmab: Employment. Azaryan:Genmab: Employment. Gupta:Genmab: Employment, Equity Ownership. Ahmadi:Genmab Inc: Employment, Other: stock and/or warrants. Hutchings:Incyte: Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Genmab: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Research Funding; Pfizer: Research Funding.

scholarly journals Immunoglobulin secretory function of B cells from untreated patients with chronic lymphocytic leukemia and hypogammaglobulinemia: role of T cells

Blood ◽  
1983 ◽  
Vol 62 (4) ◽  
pp. 767-774 ◽  
Author(s):  
LA Fernandez ◽  
JM MacSween ◽  
GR Langley
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Cell Function ◽  
Lymphocytic Leukemia ◽  
Suppressor Activity ◽  
Normal Peripheral Blood

Abstract The mechanism of the hypogammaglobulinemia in patients with chronic lymphocytic leukemia (CLL) was studied by determining the generation of specific immunoglobulin-secreting cells in response to mitogen and antigen stimulation in culture. Normal peripheral blood B lymphocytes from 18 normal subjects cocultured with equal numbers of autologous T cells generated cells secreting 2,542 +/- 695 IgG, 2,153 +/- 615 IgA, and 2,918 +/- 945 IgM. Normal B lymphocytes cocultured with normal allogeneic T cells generated similar numbers. However, B lymphocytes from patients with chronic lymphocytic leukemia cocultured with T cells from the same patient generated only 0.5% as many cells secreting IgG and 11% and 23% as many secreting IgA and IgM, respectively. The reason for this markedly defective generation of immunoglobulin-secreting cells was investigated by evaluating T-helper, T-suppressor, and B-cell function using B cells from tonsil and T and B cells from peripheral blood of normal and leukemic individuals. T cells from patients with chronic lymphocytic leukemia provided somewhat greater help than did normal T cells to normal peripheral blood B cells and normal help to tonsil B cells, whether stimulated with mitogen or antigen. T cells from patients with chronic lymphocytic leukemia did not demonstrate increased suppressor function compared to normals with B cells from normal peripheral blood. The hypogammaglobulinemia in these patients therefore was associated with a markedly defective generation of immunoglobulin secreting cells, and as there was normal or increased T- cell helper activity without excessive suppressor activity, it seems likely that this was due to an intrinsic B-cell defect.

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