Pharmacokinetics (PK) of blinatumomab and its clinical implications.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3048-3048 ◽  
Author(s):  
Benjamin Wu ◽  
Youssef Hijazi ◽  
Andreas Wolf ◽  
Christian Brandl ◽  
Yu-Nien Sun ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Target Cells ◽  
Clinical Implications ◽  
Single Chain ◽  
Disease Heterogeneity ◽  
Kidney Involvement ◽  
All Treatment

3048 Background: Blinatumomab is an investigational, bispecific, single-chain T cell engaging (BiTE) antibody of 55 kD that targets CD19 on B cells and CD3 on T cells. Blinatumomab induces polyclonal T cell activation and proliferation, resulting in redirected lysis of CD19+target cells. Comprehensive analysis of its PK and the clinical implications is presented. Methods: PK data from 131 patients enrolled in 3 phase 1 or 2 studies of non-Hodgkin lymphoma (NHL; N=76), relapsed/refractory acute lymphoblastic leukemia (r/r ALL; N=36), and ALL with minimal residual disease (MRD; N=19) were analyzed. Blinatumomab was given by constant IV (cIV) infusion at 0.5, 1.5, 3, 5, 10, 15, 30, 60 and 90 μg/m2/d over 4 weeks/cycle. Serum blinatumomab concentrations were assessed using a validated bioassay. PK parameters, including volume of distribution (V), half-life (t1/2) and clearance (CL), were analyzed with noncompartmental methods. CL was derived by dividing infusion rate by blinatumomab concentration at steady state (Css). Mean CL from each patient was used to determine disease heterogeneity and for covariate analyses. Effective dose was assessed with PK, in vitroand clinical B cell data. Results: At the doses tested, blinatumomab had linear PK that was stable over time. V (~5 L) approximated that of monoclonal antibodies. Blinatumomab had a t1/2 of ~2 h, with systemic CL of ~2 L/h. Minute blinatumomab concentrations were detected in the urine of 3 (of 13) NHL patients at a dose of 60 μg/m2/d. There was no trend of NHL, r/r ALL or ALL with MRD; CrCL, age, gender, weight, or body surface area (BSA) influencing CL. Preliminary analysis showed comparable mean CL values in patients with CrCL ≥30 mL/min, ranging from 1.9 to 2.6 L/h. To achieve Css above the in vitro EC90 value for leukemia cell lines (470 pg/mL) and complete B cell suppression in patients, 15 μg/m2/d of blinatumomab given as cIV over 4 weeks/cycle is desired for ALL treatment. Conclusions: Blinatumomab PK was linear, stable, and independent of NHL, r/r ALL or ALL with MRD. Kidney involvement in its excretion was limited at the investigated doses. cIV administration is required due to the short t1/2. Flat or BSA-based dosing can be used in adults. A cIV dose of ≥15 μg/m2/d provides adequate exposure for ALL treatment. Clinical trial information: NCT00274742, NCT01209826, NCT00560794.

Construction and Pre-Clinical Evaluation of An Anti-CD19 Chimeric Antigen Receptor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4623-4623 ◽  
Author(s):  
James Kochenderfer ◽  
Steven Feldman ◽  
Yangbing Zhao ◽  
Hui Xu ◽  
Mary Black ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Costimulatory Molecule ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Target Cells ◽  
Single Chain ◽  
Cell Counts

Abstract Adoptive T cell immunotherapy can cause regression of established malignancy. One promising approach is to transfer genes encoding chimeric antigen receptors (CARs) that specifically recognize tumor-associated antigens to T cells before the T cells are adoptively transferred to patients. We have constructed a CAR that consists of an anti-CD19 single chain variable region (scFv) that is coupled to a portion of the CD28 costimulatory molecule and the signaling component of the CD3-zeta chain. CD19 is a promising target for immunotherapy because most malignant B cell express CD19, but the only normal cells that express CD19 are B cells, B cell precursors, and perhaps follicular dendritic cells. We have demonstrated that gamma-retroviruses encoding the anti-CD19 CAR can be used to transduce human T cells and that these transduced T cells specifically recognize CD19+ targets. To transduce T cells, we stimulated T cells with the anti-CD3 monoclonal antibody OKT3 on day 0 then conducted sequential retroviral transductions on day 2 and on day 3. Transductions were performed by spin-loading retroviruses onto RetroNectin (Takara) coated culture plates followed by overnight incubation of the OKT3- stimulated T cells on the plates. Forty-five to sixty-seven percent of T cells expressed the anti-CD19 CAR as measured by flow cytometry 7–8 days after transduction (n=8). Anti-CD19-CAR-transduced CD8+ and CD4+ T cells produced IFNg and IL-2 specifically in response to stimulation with CD19+ target cells. The transduced T cells specifically killed primary chronic lymphocytic leukemia (CLL) cells. T cells from CLL patients that were either untreated or previously treated with fludarabine plus rituximab could be transduced and induced to proliferate sufficiently to provide enough cells for clinical adoptive T cell transfer. In addition, we adapted this protocol for use in CLL patients with very high peripheral blood leukemia cell counts by depleting CD19+ cells using magnetic bead sorting prior to OKT3 stimulation. In preparation for a clinical trial that will enroll patients with advanced B cell malignancies, we have generated a producer cell clone that produces GALV (Gibbon ape leukemia virus)-enveloped gamma-retroviruses encoding the anti-CD19 CAR, and we have produced sufficient retroviral supernatant for the proposed clinical trial under good manufacturing practice (GMP) conditions.

scholarly journals Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 810-810 ◽  
Author(s):  
Haiying Qin ◽  
Sang M Nguyen ◽  
Sneha Ramakrishna ◽  
Samiksha Tarun ◽  
Lila Yang ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Single Chain ◽  
Dual Targeting ◽  
Single Antigen ◽  
Therapeutic Function

Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

Immunopharmacodynamic response to blinatumomab in patients with relapsed/refractory B-precursor acute lymphoblastic leukemia (ALL).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 7020-7020 ◽  
Author(s):  
Andrea Schub ◽  
Virginie Nägele ◽  
Gerhard Zugmaier ◽  
Christian Brandl ◽  
Youssef Hijazi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Lymphoblastic Leukemia ◽  
Granzyme B ◽  
Significant Proportion ◽  
Target Cells ◽  
Cell Counts

7020 Background: Blinatumomab is an anti-CD19/anti-CD3 bispecific T cell engager (BiTE) that induces target cell-dependent, polyclonal T cell activation and proliferation, resulting in redirected lysis of CD19+ target cells. Methods: In a phase 2 study, adult patients (N=36) with relapsed/refractory B-precursor ALL received continuous blinatumomab IV infusion for 28 days in ≤5 treatment/consolidation cycles. Whole blood and serum samples were collected throughout treatment and analyzed for lymphocyte subpopulations, cytokines, granzyme B, and blinatumomab serum concentrations. Results: Lymphocytes in all patients responded in a similar fashion. After infusion start, peripheral B cell counts dropped to ≤1 B cell/μL in <1 week and remained undetectable throughout treatment. Peripheral T cells showed a redistribution characterized by swift disappearance within the first 2-6 hrs and subsequent recovery to baseline within several days. Otherwise, T cell counts remained at least stable in most patients. In some patients even an expansion of the T cell compartments was observed, most likely due to specific proliferation of activated T cells but could not be defined as prerequisite for treatment efficacy. During the first infusion days, a significant proportion of T cells newly expressed the activation marker CD69, and the T cell effector molecule granzyme B was detectable in serum. Additionally, a transient cytokine release dominated by IL-10, IL-6 and IFN-γ was observed in most patients shortly after first infusion start, which was alleviated or absent in subsequent cycles. Blinatumomab serum steady state concentrations (mean±SD) were 198±61 pg/mL and 694±236 pg/mL at doses of 5 and 15 μg/m²/d, respectively, which is comparable to those from previous studies. Conclusions: Immunopharmacodynamic response to blinatumomab was characterized by B cell depletion, T cell activation and redistribution, and release of granzyme B and cytokines, suggesting T cell engagement according to the expected BiTE mode of action. The tested pharmacodynamic markers did not allow for predictive differentiation between patients achieving a hematologic response and those who did not. Clinical trial information: NCT01209286.

scholarly journals Redirecting T cells to hematological malignancies with bispecific antibodies

Blood ◽  
2018 ◽  
Vol 131 (1) ◽  
pp. 30-38 ◽  
Author(s):  
Mireya Paulina Velasquez ◽  
Challice L. Bonifant ◽  
Stephen Gottschalk
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Clinical Studies ◽  
Hematological Malignancies ◽  
Lymphoblastic Leukemia ◽  
Bispecific Antibodies ◽  
Special Focus ◽  
Single Chain ◽  
Median Response

Abstract There is a need to improve outcomes for patients with recurrent and/or refractory hematological malignancies. Immunotherapy holds the promise to meet this need, because it does not rely on the cytotoxic mechanism of conventional therapies. Among different forms of immunotherapy, redirecting T cells to hematological malignancies with bispecific antibodies (BsAbs) is an attractive strategy. BsAbs are an “off-the-shelf” product that is easily scalable in contrast to adoptive T-cell therapies. Among these, the bispecific T-cell engager blinatumomab has emerged as the most successful BsAb to date. It consists of 2 single-chain variable fragments specific for CD19 present on B-cell malignancies and CD3 expressed on almost all T cells. Blinatumomab has shown potent antitumor activity as a single agent, particularly for acute lymphoblastic leukemia, resulting in its US Food and Drug Administration approval. However, although successful in inducing remissions, these are normally short-lived, with median response durations of <1 year. Nevertheless, the success of blinatumomab has reinvigorated the BsAb field, which is bustling with preclinical and clinical studies for not only B-cell–derived lymphoblastic leukemia and lymphoma but also acute myeloid leukemia and multiple myeloma. Here, we will review the successes and challenges of T-cell–targeted BsAbs for the immunotherapy of hematological malignancies with special focus on conducted clinical studies and strategies to improve their efficacy.

Generation of autologous cytotoxic and helper T-cell responses against the B-cell leukemia–associated antigen HB-1: relevance for precursor B-ALL–specific immunotherapy

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 2885-2891 ◽  
Author(s):  
Björn de Rijke ◽  
Hanny Fredrix ◽  
Agnes Zoetbrood ◽  
Frank Scherpen ◽  
Henry Witteveen ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Lymphoblastic Leukemia ◽  
Specific Antigen ◽  
T Cell Responses ◽  
Treatment Modalities ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Cell Responses

Abstract Tumor relapses in patients with precursor B-cell acute lymphoblastic leukemia (BALL) occur frequently after primary treatment. Therefore, development of additional treatment modalities to eliminate residual tumor cells is needed. Active immunotherapy using dendritic cells (DCs) loaded with tumor-associated antigens is a promising approach to induce specific T-cell immunity in patients with cancer. In previous studies, we described HB-1 as a B-cell lineage-specific antigen that is recognized by donor-derived cytotoxic T lymphocytes (CTLs) on allogeneic B-ALL tumor cells. Here, we investigated the potential use of the HB-1 antigen as an autologous T-cell vaccine target. To determine whether HB-1–specific CTL precursors are present within the T-cell repertoire, we induced expansion of CD8+ T cells using mature monocyte-derived DCs pulsed with the previously identified HB-1.B44 antigenic peptide. In 6 of 8 donors, CD8+ CTL lines have been generated that exert cytotoxicity against target cells exogenously pulsed with peptide or endogenously expressing the HB-1 antigen. From one of these HB-1–specific T-cell lines, we isolated a CD8+ CTL that produces interferon-γ on stimulation with B-ALL tumor cells. Interestingly, the HB-1 antigen also induced CD4+ T-helper responses on activation with protein-loaded mature monocyte-derived DCs. We identified 2 novel epitopes recognized in the context of HLA-DR4 and HLA-DR11 with the use of HB-1–specific CD4+ T-cell clones generated from different donors. These present data, that HB-1 induces both helper and cytotoxic T-cell responses, indicate that the HB-1 antigen is a candidate target to induce T-cell–mediated antitumor immunity in patients.

scholarly journals CSPG4-Specific CAR T Cells for High-Risk Childhood B Cell Precursor Leukemia

2019 ◽  
Vol 20 (11) ◽  
pp. 2764 ◽  
Author(s):  
Dennis C. Harrer ◽  
Gerold Schuler ◽  
Jan Dörrie ◽  
Niels Schaft
Keyword(s):  
T Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Target Cells ◽  
Activation Markers ◽  
Car T Cells ◽  
Car T ◽  
B Cell Leukemia

The advent of CD19-specific chimeric antigen receptor (CAR) T cells has proven to be a powerful asset in the arsenal of cancer immunotherapy of acute lymphoblastic leukemia and certain B cell lymphomas. However, a sizable portion of patients treated with CD19-CAR T cells relapse with CD19-negative cancer cells, necessitating the quest for back-up antigens. Chondroitin sulfate proteoglycan 4 (CSPG4) expression has been reported on leukemic blasts bearing the ill-fated MLL 11q23 rearrangement. We aimed at exploring the use of CSPG4-specific CAR T cells against mixed-lineage leukemia (MLL)-rearranged leukemic blasts, using the precursor B cell leukemia cell line KOPN8 (MLL–MLLT1 translocation) as a model. First, we confirmed CSPG4 expression on KOPN8 cells. Bulk T cells electroporated with mRNA encoding a CSPG4-specific CAR upregulated activation markers and secreted the Th1 cytokines TNF and IFNγ in an antigen-specific manner upon co-culture with KOPN8 cells. More importantly, CSPG4-specific CAR T cells evinced specific degranulation towards KOPN8 cells and specifically lysed KOPN8 target cells in chromium lysis experiments. CSPG4 is a well-established CAR target in cutaneous melanoma. Here, we provide proof-of-principle data for the use of CSPG4-specific CAR T cells against MLL-translocated leukemias.

Epigenetic Inactivation of Notch Signaling Target Genes HES in B Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3372-3372
Author(s):  
Shaoqing Kuang ◽  
Patrick Zweidler-McKay ◽  
Hui Yang ◽  
Zhi Hong Fang ◽  
Weigang Tong ◽  
...  
Keyword(s):  
Dna Methylation ◽  
T Cell ◽  
B Cell ◽  
Notch Signaling ◽  
Cell Lines ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Cell Acute Lymphoblastic Leukemia

Abstract The Notch signaling pathway has been implicated in multiple functions during normal hemato-lymphoid development. It also plays critical roles in T-cell leukemogenesis through influencing T-cell proliferation, differentiation and survival. In contrast, we have previously reported a tumor suppressor role in B-cell leukemias, where Notch signaling leads to growth inhibition and apoptosis. The Notch target genes Hairy/Enhancer of Split (HES1-7) encode transcriptional repressors with basic helix-loop-helix (bHLH) domains. Functional and phenotypic analyses of some of the HES family members have been reported, however, expression and epigenetic regulation of the HES family in leukemia is largely unknown. Using Methylated CpG Island Amplification (MCA) / DNA promoter microarray, we identified several HES family genes as hypermethylated in B cell acute lymphoblastic leukemia (B ALL). We further investigated the comprehensive methylation profiles of HES family genes in a panel of leukemia cell lines and ALL patient samples by bisulfite pyrosequencing. Aberrant DNA methylation of HES2, HES4, HES5 and HES6 was detected in most B ALL cell lines including B-JAB, RS4:11, REH, Raji and Ramos but not in normal B cell controls. In contrast, in T cell leukemia cell lines such as Molt4, PEER, T-ALL1 and J-TAG, these genes were generally unmethylated. In B ALL patient samples, the frequencies of DNA methylation in the promoter regions of these genes were 25% for HES2, 50% for HES4, 76% for HES5 and 71% for HES6. Expression analysis of HES4, HES5 and HES6 in leukemia cell lines by real-time PCR further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with DNA methyltransferase inhibitor 5’-aza-2’-deoxycytidine resulted in HES gene re-expression. Finally, forced re-expression of HES5 and HES6 in methylation silenced Rs4 and REH cell lines inhibited cell growth. These results suggest that the Notch/HES signaling pathway is epigenetically-inactivated in B ALL. These data support the role of the HES family as tumor suppressors in pre-B ALL and establish epigenetic modulation as a novel mechanism of Notch pathway regulation. We anticipate that therapies capable of activating Notch/HES signaling may have therapeutic potential in B cell leukemias.

scholarly journals Tumor staging in a Beagle dog with concomitant large B-cell lymphoma and T-cell acute lymphoblastic leukemia

2021 ◽  
pp. 104063872110110
Author(s):  
Alessandro Ferrari ◽  
Marzia Cozzi ◽  
Luca Aresu ◽  
Valeria Martini
Keyword(s):  
Bone Marrow ◽  
Lymph Node ◽  
T Cell ◽  
B Cell ◽  
Cell Lymphoma ◽  
Tumor Staging ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Cell Acute Lymphoblastic Leukemia

An 8-y-old spayed female Beagle dog was presented with peripheral lymphadenomegaly. Lymph node cytology and flow cytometry led to the diagnosis of large B-cell lymphoma (LBCL). We detected minimal percentages of LBCL cells in peripheral blood and bone marrow samples. However, a monomorphic population of neoplastic cells different from those found in the lymph node was found in the bone marrow. T-cell acute lymphoblastic leukemia was suspected based on flow cytometric immunophenotyping. PCR for antigen receptor rearrangement (PARR) revealed clonal rearrangement of both B-cell and T-cell receptors, and the presence of both neoplastic clones in the lymph node, peripheral blood, and bone marrow. The dog was treated with multi-agent chemotherapy but died 46 d following diagnosis. Tumor staging and patient classification are needed to accurately establish a prognosis and select the most appropriate therapeutic protocol.

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