Annotation Of Pseudogenous Gene Segments By Massively Parallel Sequencing Of Rearranged Lymphocyte Receptor Loci

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3480-3480
Author(s):  
Ryan O Emerson ◽  
Anna Sherwood ◽  
Harlan Robins ◽  
Christopher S Carlson ◽  
Mark Rieder
Keyword(s):  
Immune System ◽  
T Cell ◽  
B Cells ◽  
Gene Segment ◽  
Antigen Receptor ◽  
Cell Receptor ◽  
T And B Cells ◽  
Employment Equity ◽  
Immune Receptor ◽  
Equity Ownership

Abstract The adaptive immune system generates a remarkable breadth of antigen-specific T cell receptors (TCRs) and B cell receptor (BCRs) by combinatoric shuffling of gene segments, enabling the immune system to recognize a diverse and unpredictable set of antigens. To generate this enormous diversity, lymphocytes undergo somatic recombination of noncontiguous variable (V), diversity (D), and joining (J) region gene segments, which collectively encode the CDR3 region along with non-templated deletion or insertion of nucleotides at the V-D, and D-J junctions. Many of the V, D, and J gene segments at immune receptor loci are annotated as non-functional due to defects in primary sequence, motifs necessary for rearrangement, or chromosome position. However, full annotation of functional, pseudogene, or ORF, has proven elusive due to the random and unpredictable nature of non-templated deletions and insertions, the huge space of potential receptors, and the large V and J gene families. We amplified genomic DNA using a highly multiplexed PCR assay that targeted the rearranged immunoglobulin heavy chain (IGH) and T cell receptor beta (TCRB) receptor locus. Deep DNA sequencing allowed us to characterize the TCRB and IGH immune repertoire from mature T and B cells in a large cohort of healthy adults. Random chance predicts that less than one-third of somatic rearrangements at the TCRB and IGH loci will lead to transcripts with key motifs in-frame and no premature stops (i.e. a productive antigen receptor). Selection during lymphocyte maturation ensures that all mature T and B cells carry at least one rearrangement coding for a productive receptor, with a second allele rearranging in some cells that can be out of frame or include a V, D or J pseudogene segment (i.e. non-productive receptor). We classified each V, D, and J gene segment as functional or pseudogene based on the proportion of in-frame rearrangements and on 3-nt periodicity in the length of the CDR3 hypervariable coding region. Based on these data we were able to conclusively annotate the functional status of each gene segment in the complex TCRB and IGH immune receptor loci. In TCRB, we found 2 presumed-functional gene segments that are clearly pseudogenes; in IgH we found 3 presumed-functional genes that are actually pseudogenes and 1 annotated pseudogene that is clearly functional. In both loci we identified numerous cases of gene segment functional/pseudogene status segregating among healthy subjects, indicating that genotype at the TCR and IGH loci may be a source of substantial differences in the naive antigen receptor repertoire between individuals. These results have helped us to more accurately characterize the genetic landscape underpinning somatic rearrangement of antigen receptors, a seminal event in the generation of robust antigen-specific immune responses. Disclosures: Emerson: Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Rieder:Adaptive Biotechnologies: Employment, Equity Ownership.

Differential Effects of Milatuzumab On Human Antigen-Presenting Cells in Comparison to Malignant B Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2744-2744
Author(s):  
Xiaochuan Chen ◽  
Rhona Stein ◽  
Chien-Hsing Chang ◽  
David M. Goldenberg
Keyword(s):  
T Cell ◽  
B Cells ◽  
Cell Lines ◽  
Hairy Cell ◽  
Employment Equity ◽  
Cross Presentation ◽  
Equity Ownership ◽  
Antigen Presenting

Abstract Abstract 2744 Poster Board II-720 Introduction: The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, is in clinical evaluation as a therapeutic mAb for non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and multiple myeloma after preclinical evidence of activity in these tumor types. In addition to its expression in malignant cells, CD74 is also expressed in normal B cells, monocytes, macrophages, Langerhans cells, follicular and blood dendritic cells. A question therefore arises whether milatuzumab is toxic to or affects the function of these immune cells. This has important implications, not only for safe therapeutic use of this mAb, but also for its potential application as a novel delivery modality for in-vivo targeted vaccination. Methods: We assessed the binding profiles and functional effects of milatuzumab on human antigen-presenting cell (APC) subsets. Studies on the effect of milatuzumab on antigen presentation and cross-presentation are included. In addition, binding and cytotoxicity on a panel of leukemia/lymphoma cell lines and CLL patient cells were tested to demonstrate the range of malignancies that can be treated with this mAb. Results: Milatuzumab bound efficiently to different subsets of blood dendritic cells, including BDCA-1+ myeloid DCs (MDC1), BDCA-2+ plasmacytoid DCs (PDC), BDCA-3+ myeloid DCs (MDC2), B lymphocytes, monocytes, and immature DCs derived from human monocytes in vitro, but not LPS-matured DCs, which correlated well with their CD74 expression levels. In the malignant B-cells tested, milatuzumab bound to the surface of 2/3 AML, 2/2 mantle cell (MCL), 4/4 ALL, 1/1 hairy cell leukemia, 2/2 CLL, 7/7 NHL, and 5/6 multiple myeloma cell lines, and cells of 4/6 CLL patient specimens. Significant cytotoxicity (P<0.05) was observed in 2/2 MCL, 2/2 CLL, 3/4 ALL, 1/1 hairy cell, 2/2 NHL, and 2/2 MM cell lines, and 3/4 CD74-positive CLL patient cells, but not in the AML cell lines following incubation with milatuzumab. In contrast, milatuzumab had minimal effects on the viability of DCs or B cells that normally express CD74. The DC maturation and DC-mediated T-cell functions were not altered by milatuzumab treatment, which include DC-induced T-cell proliferation, CD4+CD25+FoxP3+ Treg expansion, and CD4+ naïve T-cell polarization. Moreover, milatuzumab had little effect on CMV-specific CD8- and CD8+ T cell interferon-g responses of peripheral blood mononuclear cells stimulated in vitro with CMV pp65 peptides or protein, suggesting that milatuzumab does not influence antigen presentation or cross-presentation. Conclusion: These results demonstrate that milatuzumab is a highly specific therapeutic mAb against B-cell malignancies with potentially minimal side effects. It also suggests that milatuzumab may be a promising novel delivery mAb for in vivo targeted vaccinations, given its efficient binding, but lack of cytotoxicity and functional disruption on CD74-expressing normal APCs. (Supported in part by NIH grant PO1-CA103985.) Disclosures: Chang: Immunomedics Inc.: Employment, Equity Ownership, Patents & Royalties. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Sequencing-Based Minimal Residual Disease Assessment In T-Cell Malignancies: T-Cell Prolymphocytic Leukemia Case Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1392-1392
Author(s):  
Katherine Sutherland ◽  
Katherine Kong ◽  
Aaron C. Logan ◽  
Malek Faham ◽  
David B. Miklos
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Cell Receptor ◽  
Employment Equity ◽  
Prolymphocytic Leukemia ◽  
Blood Samples ◽  
Leukemia Cutis ◽  
Equity Ownership

Abstract Background The prognostic significance of minimal residual disease (MRD) quantification in the post-transplant setting has been demonstrated in multiple lymphoid malignancies, including acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Previous studies support the clinical utility of molecular MRD quantification of tumor burden after allogeneic hematopoietic cell transplantation (allo-HCT) (Logan et al, Leukemia 2013). We have developed the ClonoSIGHT™ test, which is based on the deep sequencing of immunoglobulin and T-cell receptor rearrangements and has a detection limit of one cancer cell per million leukocytes in peripheral blood or bone marrow (Faham et al, Blood 2012; Armand et al, Brit J Haematol 2013). In this report, we will discuss the technical performance of the ClonoSIGHT test for routine MRD quantification after allo-HCT and present a case study on a patient with T-cell prolymphocytic leukemia (T-PLL). Methods A 55 year old female presented with T-PLL including symptomatic CNS disease, received 12 weeks of Alemtuzumab therapy and then 12 weeks following her last Alemtuzumab treatment received an unrelated donor myeloablative allo-HCT using Fludarabine, BCNU and Melphalan conditioning with antithymoglobulin, Mycophenolate mofetil and cyclosporine primary immune prophylaxis. Peripheral blood samples were collected for MRD assessment before and serially after allo-HCT. Using universal primer sets, we amplified T-cell receptor beta (TRB), delta (TRD) and gamma (TRG) variable, diversity, and joining gene segments from genomic DNA isolated from peripheral blood mononuclear cells (PBMC). Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination, and leukemia-specific clonotypes were identified based on their frequency within the T-cell repertoire (>5%). The leukemia-specific clonotype was then quantified in serial peripheral blood samples and reported as the absolute number of leukemic-specific clones among total leukocytes. Results A single clonal TRG gene rearrangement accounting for 26.1% WBC in the pre-transplant sample was identified and quantified in serial peripheral blood samples. A 4-log decline in MRD levels occurred post allo-HCT (Figure 1) thru 56 days following graft infusion; however, serial MRD monitoring demonstrated increasing levels of leukemia-specific clonotypes in the peripheral blood over time (Figure 1). Immunosuppression tapering strategies were employed in response to clinical events and MRD levels. Specifically, the patient developed an EBV+ post-transplant lymphoproliferative disease (PTLD) 60 days post allo-HCT, and cyclosporine was tapered in addition to instituting anti-CD20 rituximab treatment. As per institutional practice, a bone marrow biopsy 84 days post-HCT showed full donor engraftment with normal cellularity and no evidence of PLL was detected by flow cytometry when ClonoSIGHT detected 0.013% PLL in the patient's blood. Unfortunately, in the setting of immune suppression taper at 100 days post allo-HCT, the patient developed Grade II skin GVHD and was treated with 0.5mg/kg prednisone daily and tapered as indicated. At 160 days post allo-HCT, the patient presented with new skin papules suspected to be leukemia cutis. The PLL clonotype was detected in the skin biopsy; however, it was present at lower frequency in the TRG repertoire than in the blood, thus not supporting a diagnosis of leukemia cutis. In agreement, skin pathology revealed Verruca Vulgaris (warts). However, the patient's MRD continued to increase in the blood while immunosuppression was tapered and stopped completely 6 months post-HCT. Conclusions MRD assessment can be used to monitor a patient's disease progression after immune cellular therapy and aids immune suppression management following allo-HCT. Further, as presented in this case study, ClonoSIGHT detection of the leukemia clone in the blood compared with other tissues can sensitively and specifically assess extramedullary relapse. Disclosures: 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 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 Predominant T cell receptor V gene usage in patients with abnormal clones of B cells

Blood ◽  
1991 ◽  
Vol 77 (8) ◽  
pp. 1776-1780 ◽  
Author(s):  
CH Janson ◽  
J Grunewald ◽  
A Osterborg ◽  
H DerSimonian ◽  
MB Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Cell Receptor ◽  
Gene Segment ◽  
Cd8 T Cell ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Gene Usage ◽  
V Gene

We have examined alpha/beta V gene segment usage of peripheral blood CD4+ and CD8+ T cells, respectively, from patients with multiple myeloma and monoclonal gammopathy of undetermined significance, by using T cell receptor (TCR) for antigen monoclonal antibodies (MoAbs). In 7 of 16 patients we found an increase in the usage of various TCR V gene segments. The expansion was confined to either the CD4+ or the CD8+ T-cell subset, except for one patient where an abnormal pattern was observed both within the CD4+ and CD8+ T-cell subsets. In one patient 47%, and in another patient 30% of the CD8+ lymphocytes reacted with alpha V12.1 and beta V6.7 antibodies, respectively. In two other patients 29% and 40% of the CD4+ lymphocytes reacted with beta V6.7 and beta V8.1 antibodies, respectively. We conclude that T cells with a predominant V gene usage is a frequent feature in patients with abnormal clonal B cells of malignant or benign types. T- and B-cell populations are normally clonally linked in regulatory circuits. An abnormal proliferation of B cells might therefore induce, or be regulated by, an expansion of clonal T cells, as suggested by the present results.

Reduction Of Immune Repertoire Diversity Through DNA Sequence Constraints At VDJ Junctions

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4944-4944
Author(s):  
Bryan Howie ◽  
Harlan Robins ◽  
Christopher S Carlson
Keyword(s):  
Immune System ◽  
T Cell ◽  
T Cell Receptor ◽  
Adaptive Immune System ◽  
Cell Receptor ◽  
Immune Repertoire ◽  
Adaptive Immune ◽  
Repertoire Diversity ◽  
Equity Ownership ◽  
Junctional Diversity

Abstract B and T lymphocytes are effector cells of the adaptive immune system. These cells express surface receptors that bind a huge variety of antigens, and together they comprise a person’s immune repertoire. A diverse repertoire is essential for mounting robust immune responses against a wide range of pathogens, and repertoire diversity affects the probability that DNA sequencing can uniquely tag a clonally expanded population of cells for the detection of minimum residual disease (MRD) during cancer treatment. Immune repertoire diversity arises partly through the combinatorial splicing of gene segments from the variable (V), diversity (D), and joining (J) regions of a B or T cell receptor locus. Much additional diversity is created through the stochastic insertion and deletion of nucleotides at the splice junctions, and by somatic hypermutation (SHM) in maturing lymphocytes. The generation of junctional diversity is an important part of this process, but it may be constrained by the underlying biological mechanisms. To explore the landscape of junctional diversity among immune receptor loci, we developed a likelihood model that can annotate VDJ junctions in the presence of SHM and compute the probability that a given receptor sequence was generated only once in a person’s repertoire, which is essential for tracking MRD. Using high-throughput sequencing data from several individuals and a range of receptor loci, we identify mechanistic constraints that limit B and T cell receptor diversity. For example, we show that the usual variability in CDR3 length is reduced at the immunoglobulin kappa (IgK) locus, and we connect this finding to sequence motifs that constrain nucleotide deletion at the ends of IgK gene segments. Our findings will inform future genetic studies of the adaptive immune system, and they provide quantitative guidance for deciding which cancer clones can be tracked for reliable MRD detection. Disclosures: Howie: Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

The transcription factor E2A activates multiple enhancers that drive Rag expression in developing T and B cells

2020 ◽  
Vol 5 (51) ◽  
pp. eabb1455 ◽  
Author(s):  
Kazuko Miyazaki ◽  
Hitomi Watanabe ◽  
Genki Yoshikawa ◽  
Kenian Chen ◽  
Reiko Hidaka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
T Cell ◽  
B Cells ◽  
Cell Lineage ◽  
Cell Receptor ◽  
Regulatory Elements ◽  
Mouse Strains ◽  
Specific Gene ◽  
T And B Cells

Cell type–specific gene expression is driven by the interplay between lineage-specific transcription factors and cis-regulatory elements to which they bind. Adaptive immunity relies on RAG-mediated assembly of T cell receptor (TCR) and immunoglobulin (Ig) genes. Although Rag1 and Rag2 expression is largely restricted to adaptive lymphoid lineage cells, it remains unclear how Rag gene expression is regulated in a cell lineage–specific manner. Here, we identified three distinct cis-regulatory elements, a T cell lineage–specific enhancer (R-TEn) and the two B cell–specific elements, R1B and R2B. By generating mice lacking either R-TEn or R1B and R2B, we demonstrate that these distinct sets of regulatory elements drive the expression of Rag genes in developing T and B cells. What these elements have in common is their ability to bind the transcription factor E2A. By generating a mouse strain that carries a mutation within the E2A binding site of R-TEn, we demonstrate that recruitment of E2A to this site is essential for orchestrating changes in chromatin conformation that drive expression of Rag genes in T cells. By mapping cis-regulatory elements and generating multiple mouse strains lacking distinct enhancer elements, we demonstrate expression of Rag genes in developing T and B cells to be driven by distinct sets of E2A-dependent cis-regulatory modules.

scholarly journals Expression and Function of Tec, Itk, and Btk in Lymphocytes: Evidence for a Unique Role for Tec

2004 ◽  
Vol 24 (6) ◽  
pp. 2455-2466 ◽  
Author(s):  
Michael G. Tomlinson ◽  
Lawrence P. Kane ◽  
Jennifer Su ◽  
Theresa A. Kadlecek ◽  
Marianne N. Mollenauer ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Th2 Cells ◽  
Receptor Signaling ◽  
T And B Cells ◽  
Nfat Activation ◽  
Cell Receptor Signaling

ABSTRACT The Tec protein tyrosine kinase is the founding member of a family that includes Btk, Itk, Bmx, and Txk. Btk is essential for B-cell receptor signaling, because mutations in Btk are responsible for X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice, whereas Itk is involved in T-cell receptor signaling. Tec is expressed in both T and B cells, but its role in antigen receptor signaling is not clear. In this study, we show that Tec protein is expressed at substantially lower levels in primary T and B cells relative to Itk and Btk, respectively. However, Tec is up-regulated upon T-cell activation and in Th1 and Th2 cells. In functional experiments that mimic Tec up-regulation, we find that Tec overexpression in lymphocyte cell lines is sufficient to induce phospholipase Cγ (PLC-γ) phosphorylation and NFAT (nuclear factor of activated T cells) activation. In contrast, overexpression of Btk, Itk, or Bmx does not induce NFAT activation. Tec-induced NFAT activation requires PLC-γ, but not the adapters LAT, SLP-76, and BLNK, which are required for Btk and Itk to couple to PLC-γ. Finally, we show that the unique effector function for Tec correlates with a unique subcellular localization. We hypothesize that Tec functions in activated and effector T lymphocytes to induce the expression of genes regulated by NFAT transcription factors.

scholarly journals Resistance of Natural Killer and T Cells to Venetoclax Allows for Combination Treatment with Cancer Immunotherapy Agents

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1118-1118 ◽  
Author(s):  
Elisabeth A Lasater ◽  
An D Do ◽  
Luciana Burton ◽  
Yijin Li ◽  
Erin Williams ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Immune Cell ◽  
Single Agent ◽  
Employment Equity ◽  
Equity Ownership

Abstract Introduction: Intrinsic apoptosis is regulated by the BCL-2 family of proteins, which consists of both anti-apoptotic (BCL-2, BCL-XL, MCL-1) and pro-apoptotic (BIM, BAX, BAK, BAD) proteins. Interaction between these proteins, as well as stringent regulation of their expression, mediates cell survival and can rapidly induce cell death. A shift in balance and overexpression of anti-apoptotic proteins is a hallmark of cancer. Venetoclax (ABT-199/GDC-0199) is a potent, selective small molecule BCL-2 inhibitor that has shown preclinical and clinical activity across hematologic malignancies and is approved for the treatment of chronic lymphocytic leukemia with 17p deletion as monotherapy and in combination with rituximab. Objective: To investigate the effects of BCL-2 inhibition by venetoclax on viability and function of immune-cell subsets to inform combinability with cancer immunotherapies, such as anti-PD-L1. Methods and Results: B cells, natural killer (NK) cells, CD4+ T cells, and CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from healthy donors (n=3) were exposed to increasing concentrations of venetoclax that are clinically achievable in patients, and percentage of live cells was assessed by flow-cytometry using Near-IR cell staining. B cells were more sensitive to venetoclax (IC50 of ~1nM) than CD8+ T cells (IC50 ~100nM), NK cells (IC50 ~200nM), and CD4+ T cells (IC50 ~500nM) (Figure A). CD8+ T-cell subset analysis showed that unstimulated naive, but not memory cells, were sensitive to venetoclax treatment (IC50 ~30nM and 240nM, respectively). Resistance to venetoclax frequently involves compensation by other BCL-2 family proteins (BCL-XL and MCL-1). As assessed by western blot in PBMCs isolated from healthy donors (n=6), BCL-XL expression was higher in NK cells (~8-fold) and CD4+ and CD8+ T cells (~2.5-fold) than in B cells (1X). MCL-1 protein expression was higher only in CD4+ T cells (1.8-fold) relative to B cells. To evaluate the effect of venetoclax on T-cell function, CD8+ T cells were stimulated ex vivo with CD3/CD28 beads, and cytokine production and proliferation were assessed. Venetoclax treatment with 400nM drug had minimal impact on cytokine production, including interferon gamma (IFNg), tumor necrosis factor alpha (TNFa), and IL-2, in CD8+ effector, effector memory, central memory, and naïve subsets (Figure B). CD8+ T-cell proliferation was similarly resistant to venetoclax, as subsets demonstrated an IC50 >1000nM for venetoclax. Taken together, these data suggest that survival of resting NK and T cells in not impaired by venetoclax, possibly due to increased levels of BCL-XL and MCL-1, and that T-cell activation is largely independent of BCL-2 inhibition. To evaluate dual BCL-2 inhibition and PD-L1 blockade, the syngeneic A20 murine lymphoma model that is responsive to anti-PD-L1 treatment was used. Immune-competent mice bearing A20 subcutaneous tumors were treated with clinically relevant doses of venetoclax, murine specific anti-PD-L1, or both agents. Single-agent anti-PD-L1 therapy resulted in robust tumor regression, while single-agent venetoclax had no effect. The combination of venetoclax and anti-PD-L1 resulted in efficacy comparable with single-agent anti-PD-L1 (Figure C), suggesting that BCL-2 inhibition does not impact immune-cell responses to checkpoint inhibition in vivo. These data support that venetoclax does not antagonize immune-cell function and can be combined with immunotherapy targets. Conclusions: Our data demonstrate that significant venetoclax-induced cell death at clinically relevant drug concentrations is limited to the B-cell subset and that BCL-2 inhibition is not detrimental to survival or activation of NK- or T-cell subsets. Importantly, preclinical mouse models confirm the combinability of BCL-2 and PD-L1 inhibitors. These data support the combined use of venetoclax and cancer immunotherapy agents in the treatment of patients with hematologic and solid tumor malignancies. Figure Figure. Disclosures Lasater: Genentech Inc: Employment. Do:Genentech Inc: Employment. Burton:Genentech Inc: Employment. Li:Genentech Inc: Employment. Oeh:Genentech Inc: Employment. Molinero:Genentech Inc: Employment, Equity Ownership, Patents & Royalties: Genentech Inc. Penuel:Genentech Inc: Employment. Sampath:Genentech Inc: Employment. Dail:Genentech: Employment, Equity Ownership. Belvin:CytomX Therapeutics: Equity Ownership. Sumiyoshi:Genentech Inc: Employment, Equity Ownership. Punnoose:Roche: Equity Ownership; Genentech Inc: Employment. Venstrom:Genentech Inc: Employment. Raval:Genentech Inc: Consultancy, 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 CD20-TCB, a Novel T-Cell-Engaging Bispecific Antibody, Induces T-Cell-Mediated Killing in Relapsed or Refractory Non-Hodgkin Lymphoma: Biomarker Results From a Phase I Dose-Escalation Trial

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5319-5319 ◽  
Author(s):  
Ann-Marie E Bröske ◽  
Ian James ◽  
Anton Belousov ◽  
Enrique Gomez ◽  
Marta Canamero ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Board Of Directors ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Activation ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Introduction: CD20-TCB (RG6026) is a novel T-cell-engaging bispecific (TCB) antibody with a '2:1' molecular format that comprises two fragment antigen binding regions that bind CD20 (on the surface of B cells) and one that binds CD3 (on the surface of T cells). CD20-TCB offers the potential for increased tumor antigen avidity, rapid T-cell activation, and enhanced tumor cell killing versus other bispecific formats. The safety, tolerability, pharmacokinetics, biomarkers, and antitumor activity of CD20-TCB are currently being investigated in a multicenter Phase I dose-escalation trial (NP30179; NCT03075696). We recently presented preliminary clinical data demonstrating promising clinical activity in relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) patients with indolent or aggressive disease (Dickinson et al. ICML 2019). Here, we present preliminary blood and tissue biomarker analyses to explore modes of action, support optimal biological dose selection, and identify potential outcome predictors. Methods: For biomarker analyses, we performed immune profiling of peripheral blood by flow cytometry, analyzed plasma cytokine levels by ELISA, and characterized baseline and on-treatment tumor biopsies by immunohistochemistry/immunofluorescence assays and RNA sequencing. Biomarker data were obtained from 122 patients dosed with 0.005-25mg CD20-TCB. Results: CD20-TCB infusion led to a rapid and transient reduction in T cells in the peripheral circulation (T-cell margination) in all patients. T-cell margination reached nadir 6 hours after the first CD20-TCB infusion, and showed a strong association with CD20-TCB dose and receptor occupancy (RO%; as determined by Djebli et al. ASH 2019). Interestingly, rebound of T cells 160 hours after the first CD20-TCB infusion was associated with response to treatment. Responding patients showed long-term T-cell activation after the first infusion of CD20-TCB at doses from 0.6mg and above. T-cell activation was demonstrated by 2-4-fold elevation of T-cell activation markers such as Ki67, HLA-DR, PD-1, ICOS, OX40, and 4-1BB, which was sustained up to Cycle 5 (105 days). Analysis of paired pre- and on-treatment tumor biopsies (n=6) obtained before and 2-3 weeks after the first dose of CD20-TCB showed evidence of T-cell-mediated tumor cell killing. Analysis of archival and pre-treatment tumor biopsies (n=80) revealed that clinical responses were achieved irrespective of the amount of tumor T-cell infiltration at baseline. In contrast, preliminary baseline bulk tumor RNA sequencing data (n=46) showed upregulation of gene signatures associated with cell proliferation/Myc and T-cell subsets (effector vs exhausted-like) in non-responding patients. Conclusions: In this study, we demonstrated the mode of action of CD20-TCB, a novel bispecific antibody with promising clinical activity in R/R NHL. We also demonstrated that biomarker data on T-cell activation can support dose finding in conjunction with pharmacokinetics. Additional analysis is ongoing to evaluate response predictors and better characterize the population that will benefit most from T-cell mediated therapies. Disclosures Bröske: Roche: Employment, Equity Ownership. James:A4P Consulting Ltd: Consultancy. Belousov:Roche: Employment. Gomez:F. Hoffmann-La Roche Ltd: Employment. Canamero:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Ooi:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Grabole:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Wilson:F. Hoffmann-La Roche Ltd: Employment. Korfi:F. Hoffmann-La Roche Ltd: Consultancy. Kratochwil:F. Hoffmann-La Roche Ltd: Employment. Morcos:Roche: Employment, Equity Ownership. Ferlini:Roche: Employment, Equity Ownership. Thomas:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Dimier:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Moore:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Bacac:Roche: Employment, Equity Ownership, Patents & Royalties: Patents, including the one on CD20-TCB. Weisser:Pharma Research and Early Development Roche Innovation Center Munich: Employment, Equity Ownership, Patents & Royalties. Dickinson:Merck Sharpe and Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: 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, Speakers Bureau. OffLabel Disclosure: CD20-TCB (also known as RG6026, RO7082859) is a full-length, fully humanized, immunoglobulin G1 (IgG1), T-cell-engaging bispecific antibody with two fragment antigen binding (Fab) regions that bind to CD20 (on the surface of B cells) and one that binds to CD3 (on the surface of T cells) (2:1 format). The 2:1 molecular format of CD20-TCB, which incorporates bivalent binding to CD20 on B cells and monovalent binding to CD3 on T cells, redirects endogenous non-specific T cells to engage and eliminate malignant B cells. CD20-TCB is an investigational agent.

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