scholarly journals Treating B Cell Lymphomas with Anti CD81 Antibodies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4180-4180
Author(s):  
Felipe Vences-Catalan ◽  
Chiung-Chi Kuo ◽  
Ranjani Rajapaksa ◽  
Caroline Duault ◽  
Ronald Levy ◽  
...  
Keyword(s):  
Tumor Growth ◽  
B Cell ◽  
Scid Mice ◽  
Comparable Efficacy ◽  
B Cell Lymphomas ◽  
In Vivo Bioluminescence Imaging ◽  
Human Igg1 ◽  
Mouse Igg2a

Abstract The tetraspanin CD81 associates with CD19 on B cells; this molecular complex functions as co-receptor to lower the threshold of BCR-initiated B cell activation. Recently we have shown the importance of CD81 in tumor growth and metastasis of solid tumors (Vences-Catalan et al., 2015). However, the role of CD81 in lymphoid malignancies has not been explored. Previous studies demonstrated anti-proliferative effects of anti-CD81 antibodies on human B cell lymphomas using in vitro assays (Oren et al., 1990). Here we tested the therapeutic effect of an anti-human CD81 antibody in vivo against Raji and SUP-B8 B cell lymphomas using a xenograft model in SCID mice. Our studies demonstrated that our anti-human CD81 antibody (mouse IgG1) had therapeutic effect comparable to Rituximab (human IgG1) (Figure 1A). Yet, the two antibodies differ in their ability to mediate antibody-dependent cell cytotoxicity (ADCC), Rituximab is known to be highly effective, whereas the mouse IgG1 anti-CD81 antibody is not expected to mediate ADCC. To enhance the anti-CD81-mediated ADCC, we class switched the hybridoma to mouse IgG2a; we also engineered a chimeric antibody containing human IgG1ADCC-HIGH Fc constant region. Indeed, mouse IgG2a and the chimeric human IgG1 anti-CD81 mAb showed a remarkable increase in NK cell-mediated ADCC as well as complement-dependent cytotoxicity (CDC) when compared to Rituximab in vitro (data not shown) and in vivo (Figure 1B). These results suggest that CD81 can be a potential therapeutic target on B cell lymphomas by virtue of both its direct cytotoxic effect and as a mediator of ADCC and CDC. The humanized IgG1 version is being developed as a therapeutic candidate. Comparable efficacy of anti-CD81 to Rituximab. SCID mice were I.V.-injected with 1.5x106 Raji-GFP-Luc cells, tumors growth proceeded for 5 days before IP injection of 4 weekly doses of 100 ug of the indicated antibodies. (A) Survival of Raji-GFP-Luc bearing SCID mice given anti CD81 (n=30), Rituximab (n=20) or control MsIgG1 (n=30). (B) In vivo bioluminescence imaging of tumor growth in mice injected (left to right) with control mouse IgG1; anti-CD81 (MsIgG1); anti-CD81 MsIgG2a; chimeric anti-CD81 (HuIgG1) and Rituximab. Comparable efficacy of anti-CD81 to Rituximab. SCID mice were I.V.-injected with 1.5x106 Raji-GFP-Luc cells, tumors growth proceeded for 5 days before IP injection of 4 weekly doses of 100 ug of the indicated antibodies. (A) Survival of Raji-GFP-Luc bearing SCID mice given anti CD81 (n=30), Rituximab (n=20) or control MsIgG1 (n=30). / (B) In vivo bioluminescence imaging of tumor growth in mice injected (left to right) with control mouse IgG1; anti-CD81 (MsIgG1); anti-CD81 MsIgG2a; chimeric anti-CD81 (HuIgG1) and Rituximab. Disclosures Levy: Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

scholarly journals Tumor dormancy and cell signaling. II. Antibody as an agonist in inducing dormancy of a B cell lymphoma in SCID mice.

1995 ◽  
Vol 181 (4) ◽  
pp. 1539-1550 ◽  
Author(s):  
E Racila ◽  
R H Scheuermann ◽  
L J Picker ◽  
E Yefenof ◽  
T Tucker ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Growth ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Scid Mice ◽  
Tumor Dormancy ◽  
Cellular Mechanisms

Tumor dormancy can be induced in a murine B cell lymphoma (BCL1) by immunizing BALB/c mice with the tumor immunoglobulin (Ig) before tumor cell challenge. In this report, we have investigated the immunological and cellular mechanisms underlying the induction of dormancy. BCL1 tumor cells were injected into SCID mice passively immunized with antibody against different epitopes on IgM or IgD with or without idiotype (Id)-immune T lymphocytes. Results indicate that antibody to IgM is sufficient to induce a state of dormancy. Antibodies against other cell surface molecules including IgD and CD44 (Pgp1) had no effect on tumor growth. Id-immune T cells by themselves also had no effect on tumor growth in SCID mice. However, simultaneous transfer of anti-Id and Id-immune T cells enhanced both the induction and duration of the dormant state. In vitro studies indicated that antibody to IgM induced apoptosis within several hours and cell cycle arrest by 24 h. Hyper cross-linking increased apoptosis. The Fc gamma RII receptor played little or no role in the negative signaling. Antibodies that did not negatively signal in vitro did not induce dormancy in vivo. The results suggest that anti-IgM plays a decisive role in inducing tumor dormancy to BCL1 by acting as an agonist of IgM-mediated signal transduction pathways.

scholarly journals Anti-CD19 inhibits the growth of human B-cell tumor lines in vitro and of Daudi cells in SCID mice by inducing cell cycle arrest

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1329-1336 ◽  
Author(s):  
MA Ghetie ◽  
LJ Picker ◽  
JA Richardson ◽  
K Tucker ◽  
JW Uhr ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Cycle Arrest ◽  
Inhibitory Effect ◽  
Scid Mice ◽  
Cell Tumor ◽  
Cycle Arrest ◽  
A Chain

Abstract In this report, we extend our previous findings that IgG or F(ab′)2 fragments of HD37 anti-CD19 antibody (Ab) in combination with the immunotoxin (IT), RFB4-anti-CD22-deglycosylated ricin A chain (dgA) (but neither reagent alone), prolonged the survival of SCID mice with disseminated human Daudi lymphoma (SCID/Daudi mice) to 1 year at which time they still remained tumor-free. We explored the mechanisms by which the HD37 Ab exerts antitumor activity in vivo by studying its activity in vitro. We found that it has antiproliferative activity (IC50 = 5.2 - 9.8 x 10(-7) mol/L) on three CD19+ Burkitt's lymphoma cell lines (Daudi, Raji, and Namalwa) but not on a weakly CD19-positive (CD19lo) pre-B cell tumor (Nalm-6). The inhibitory effect was manifested by cell cycle arrest, but not apoptosis. Results using three additional anti-CD19 Abs, suggest that the affinity of the antibody and possibly the epitope which it recognizes may effect its capacity to transmit a signal that induces cell cycle arrest. Hence, therapeutically useful Abs may exert anti-tumor activity by a variety of mechanisms, each of which should be evaluated before undertaking clinical trials in humans.

scholarly journals P03.20 A murine, myc-driven lymphoma model expressing human CD22 enables testing of targeted therapies and their effects on tumor immune microenvironment

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A30.1-A30
Author(s):  
F Gsottberger ◽  
C Brandl ◽  
S Petkovic ◽  
L Nitschke ◽  
A Mackensen ◽  
...  
Keyword(s):  
Tumor Growth ◽  
B Cell ◽  
Targeted Therapies ◽  
Immune Cell ◽  
Myeloid Cells ◽  
B Cell Lymphoma ◽  
Primary Lymphoma ◽  
Murine Lymphoma

BackgroundThe tumor microenvironment (TME) is composed of various cell types which closely interact via cell cell contacts and cytokines leading to tumor promotion, immune cell inhibition and drug resistance. TME is increasingly recognized for its role in cancer immunotherapies. In B-cell malignancies, myeloid cells play a central role in supporting tumor growth and immune suppression (Roussel et al., 2017, Cancer Immunol Immunother). Despite the importance of a syngeneic TME, preclinical studies with novel drugs have mainly been performed in models lacking a functional immune system. Therefore, we developed an immune competent murine lymphoma model transgenic to human CD22 to study effects of targeted therapies on TME.Materials and MethodsA chimeric CD22 consisting of human extracellular and murine intracellular CD22 (h/mCD22) was introduced in BL6 mice (BL6h/mCD22). Crossbreeding with BL6λ-myc lead to spontaneous development of murine lymphoma that were serially transplanted. Tumor infiltration and TME was characterized by flow cytometry. Mice were treated with Moxetumomab pasudotox, a CD22 targeted immunotoxin and Doxorubicin.ResultsSpontaneously developed tumors in lymphoid organs from BL6h/mCD22 x λ-myc consist of a monomorphic population of h/mCD22+ murine B cells. Three primary lymphoma subclones were isolated from distinct mice and serially transplanted in syngeneic mice. Stable tumor growth was established after subcutaneous (sc) and intravenous (iv) injection. However, TME of sc tumors was infiltrated by less than 1% immune cells, while myc-driven lymphoma in humans usually show substantial immune infiltration. In contrast to sc tumors, systemically growing lymphoma in murine bone marrow (BM) are infiltrated by 30% myeloid cells and 1% T-cells and in murine spleen by 10% and 30%, respectively. Myeloid cells found in these tumors were shown to suppress T cell proliferation in vitro. To test functionality of the h/mCD22 transgene, lymphoma-bearing mice were treated with Moxetumomab, which reduced BM lymphoma infiltration by 20 to 100-fold and infiltration in spleen by 5 to 20-fold in the three lymphoma models. Effects of treatment on TME were analyzed after treatment with Doxorubicin which is known to activate myeloid cells in vivo. Compared to untreated controls, Doxorubicin increased CD11b+ cells in spleen by 1.5-fold. Among these cells, Ly6G+ granulocytic cells increased most substantially.ConclusionsWe established primary, myc-driven h/mCD22+ B-cell lymphoma which stably engraft in syngeneic mice with a TME mimicking myc-driven lymphoma in men. The model responds well to CD22-targeted therapy and Doxorubicin induces expected immunologic changes. Therefore, our unique model provides a platform to test CD22-targeting treatment strategies in an immune competent background.Disclosure InformationF. Gsottberger: None. C. Brandl: None. S. Petkovic: None. L. Nitschke: None. A. Mackensen: None. F. Müller: None.

TRU-016, An Anti-CD37 SMIP™ Biologic, In Combination with Other Therapeutic Drugs In Models of Non-Hodgkin's Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3931-3931 ◽  
Author(s):  
Paul A. Algate ◽  
Jennifer Wiens ◽  
Christy Nilsson ◽  
Mien Sho ◽  
Debra T. Chao ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Lymphoma ◽  
Combination Index ◽  
Growth Delay ◽  
Tumor Growth Delay ◽  
B Cell Malignancies

Abstract Abstract 3931 Background: CD37 is a 50–55 kDa heavily glycosylated member of the tetraspanin superfamily of molecules. This cell surface protein is expressed on normal and transformed B-cells, and has been implicated in diverse processes including cellular activation and proliferation, cell motility, and cell-cell adhesion. TRU-016 is a novel humanized anti-CD37 SMIP™ protein. Pre-clinical studies have demonstrated that anti-CD37 SMIP™ protein mediates caspase-independent direct killing of normal and malignant B-cells, a mechanism of action that appears to be different than CD20 therapies. In addition, TRU-016 results in indirect killing through NK cell mediated SMIP-protein directed cellular cytotoxicity (SDCC). The therapeutic potential of TRU-016 against several subsets of B-cell malignancies is currently being investigated in the clinic. Methods: The ability of TRU-016 to interact and increase cell killing with established therapeutics rituximab (anti-CD20 antibody), bendamustine (bi-functional alkylating agent/nucleoside analog), LY294002 (PI3K inhibitor) and temsirolimus (mTOR inhibitor) was investigated in vitro using the Rec-1 (mantle cell lymphoma) and SU-DHL-6 (diffuse large B cell lymphoma) cell lines. Individual drugs were tested in combination with TRU-016 as well as in a multiple drug cocktail. Combination index analyses were performed for drug combinations over the 20–90% effect levels. To determine whether in vitro synergy could be recapitulated in vivo, DoHH-2 (follicular lymphoma) xenografts were treated with TRU-016, bendamustine, and the combination of TRU-016 and bendamustine with or without rituximab. Furthermore, the effect of the dosing schedule with the combination of TRU-016 and rituximab was explored by comparing the treatment over a short time period to an extended (maintenance) dosing regimen. CD37 expression on the tumor xenografts was evaluated post different treatment by immunohistochemistry. Results: Combination index analyses determined that the killing effects of TRU-016 was synergistic with rituximab, bendamustine and temsirolimus in NHL models. Furthermore, TRU-016 provided additional efficacy when added to the combination of rituximab and bendamustine. In vivo results demonstrated that the in vitro synergy results were applicable to a more complex in vivo disease model. The combination of TRU-016 with bendamustine or rituximab resulted in increased tumor growth delay compared to that attained with the individual drugs. The addition of TRU-016 to the combination of bendamustine and rituximab resulted in increased tumor growth delay compared to the two drugs alone. The observed efficacy of the combination of TRU-016 and rituximab could be extended with repeated (maintenance) dosing with tumor free survival being observed beyond the 35 days of dosing. The combination of TRU-016 with temsirolimus also resulted in a reduction of tumor growth compared to either molecule alone. CD37 target expression was detected in the xenograft tumors post-treatment with all drugs tested. Conclusions: TRU-016 in combination with rituximab, bendamustine or temsirolimus increased cell killing of NHL cells in vitro over that observed for each agent alone. Furthermore, the triple combination of TRU-016 with rituximab, bendamustine or temsirolimus displayed greater anti-tumor activity in vivo than each of the agents alone against a follicular lymphoma tumor model. The addition of TRU-016 to a combination of rituximab and bendamustine resulted in increased killing in vitro and in vivo. The combinatorial activity of TRU-016 and rituximab in vivo was increased when the drugs were administered over a longer period. These results provide preclinical rationale for the potential different combinations of TRU-016 with several established therapeutics for the treatment of NHL and related B-cell malignancies. Disclosures: Algate: Trubion Pharmaceuticals: Employment. Wiens:Trubion Pharmaceuticals: Employment. Nilsson:Trubion Pharmaceuticals: Employment. Sho:Facet/Abbott: Employment. Chao:Facet/Abbott: Employment. Starling:Facet/Abbott: Employment. Gordon:Trubion Pharmaceuticals: Employment.

Superior Immunogenicity of Idiotype Fab Fragments As Compared to Entire Immunoglobulin for Active Lymphoma Immunotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1633-1633
Author(s):  
Marcelo A. Navarrete ◽  
Benjamin Kisser ◽  
Hendrik J. Veelken
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
B Cell Lymphomas ◽  
Cd8 Cells ◽  
Fab Fragments ◽  
The Individual

Abstract Abstract 1633 Introduction: The individual collection of epitopes within the variable regions of the unique immunoglobulin expressed by every mature B-cell lymphoma (idiotype, or Id) represents a tumor-specific antigen and lends itself as a target for therapeutic vaccination strategies. Immunization with tumor Id has the capacity to elicit polyclonal antibody responses as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented on class I and class II HLA molecules, respectively. Due to a perceived low immunogenicity of lymphoma-derived Id, most Id vaccines tested in clinical trials so far have been formulated as conjugates with the strongly immunogenic carrier keyhole limpet hemocyanin (KLH). In contrast, we have consistently observed high rates of humoral and cellular anti-Id immune responses in consecutive trials of active immunization with unconjugated recombinant Fab fragments of Id in indolent B-cell lymphomas (Bertinetti et al., Cancer Res. 2006; Navarrete et al., BLOOD 2011). We therefore hypothesized that Id Fab fragment might be intrinsically more immunogenic than entire Id Ig and tested this hypothesis by comparative in vitro experiments. Methods: Monocyte-derived dendritic cells (DC) where loaded with human monoclonal IgG, papain-digested Fab fragments, Fc fragments, or recombinant lymphoma-derived Fab fragments. Functional DC phenotypes were assessed by flow cytometry of crucial maturation and activation markers. IL-10 and IL-12 was measured in DC culture supernatants by ELISA. Antigen-loaded DC where subsequently used for priming of CFSE-labeled autologous peripheral blood mononuclear cells. Stimulated T cell populations were analyzed by multicolor flow cytometry. Results: Loading of DC with Fab, Fc, IgG, or mixtures of Fab and Fc fragments did not alter surface expression of CD11c, CD80, CD83, CD86, HLA-DR, PDL-1 and PDL-2 on DC. Likewise, the various antigens did not influence the cytokine release by DC during the loading or maturation process. DC loaded with isolated Fab fragments induced significantly higher proliferation of both CD4+ and CD8+ T cells than undigested IgG. The mean proliferation rate of CD4+ cells stimulated with Fab fragments was 18.5% versus 5.6% for undigested IgG stimulation (p=0.021); proliferation rates of CD8+ cells were 14.2% versus 6.2% (p=0.034). These results were reproduced for 4 different monoclonal IgGs tested on 4 different donors. The addition of Fc fragments to Fab reduced the proliferation rates of CD4+ and CD8+ cells to 10.2% and 8.6% respectively. In addition, DC loaded with undigested IgG induced a relative increase in the number of CD25high/FoxP3+ regulatory T cells compared with Fab stimulation (8.2% versus 1.4%; p<0.01). Conclusions: Isolated Fab fragments, i.e. the Id portions that contain the individual candidate antigenic epitopes of B-cell lymphomas, prime autologous T cells in vitro more efficiently than entire IgG. This finding is consistent with the high immune response rate against recombinant unconjugated Fab fragments observed in vivo in our clinical vaccination trials. Peptide sequences shared between Ig molecules that are predominantly located in the IgG Fc fragment appear to exert an inhibitory effect on T-cell priming. In accordance with our recent in vivo data in a syngeneic mouse model of Id vaccination (Warncke et al., Cancer Immunol. Immunother. 2011), this effect may be mediated by effective activation of Treg. Fab fragments therefore appear to be the more immunogenic and therefore preferable Ig antigenic format for active anti-Id immunotherapy. Furthermore, the inhibitory effects of IgG Fc offers a potential explanation for the recently reported lack of efficacy of Id vaccination in IgG-expressing follicular lymphomas in a randomized phase III trial, in which patients with IgM-expressing lymphomas, in contrast, had a significant benefit from Id vaccination in intention-to-treat analyses (Schuster et al., JCO 2011). Disclosures: No relevant conflicts of interest to declare.

scholarly journals TC-PTP is required for the maintenance of MYC-driven B-cell lymphomas

Blood ◽  
2009 ◽  
Vol 114 (24) ◽  
pp. 5016-5023 ◽  
Author(s):  
Ryan M. Young ◽  
Avital Polsky ◽  
Yosef Refaeli
Keyword(s):  
B Cells ◽  
B Cell ◽  
Protein Tyrosine Phosphatases ◽  
Molecular Target ◽  
Cell Tumor ◽  
Tumor Proliferation ◽  
B Cell Lymphomas ◽  
Tumor Maintenance

Abstract We sought to determine the contributions of protein tyrosine phosphatases (PTPs) to the pathogenesis of B-cell lymphomas. We found that T-cell PTP (TC-PTP) was overexpressed in transformed B cells. We hypothesized that TC-PTP may be a tumor-promoting gene that is regulated by MYC overexpression in B cells. Knockdown of TC-PTP in murine tumors resulted in decreased cell viability in vitro because of an arrest in the G1 phase of the cell cycle. Furthermore, cells with reduced TC-PTP expression were unable to either engraft or expand in vivo. Taken together, these data indicate that TC-PTP is required for B-cell tumor maintenance. Our data also suggested a correlation between TC-PTP expression and MYC overexpression. To investigate this further, we used malignant murine B cells that contain a doxycycline-repressible MYC transgene. We found that repression of MYC overexpression with doxycycline reduced TC-PTP expression. Moreover, enforced expression of TC-PTP showed partial rescue of the expansion of tumor cells after suppression of MYC overexpression. These results suggest that MYC overexpression induces TC-PTP overexpression, which in turn promotes tumor proliferation, implicating TC-PTP as an important effector of the MYC-driven proliferation program in B-cell lymphomas. Thus, TC-PTP may be a suitable molecular target for the treatment of B-cell lymphomas.

scholarly journals Development of Multi-Engineered iPSC-Derived CAR-NK Cells for the Treatment of B-Cell Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1729-1729
Author(s):  
Luis Borges ◽  
Mark A Wallet ◽  
Chiamin-Liao Bullaughey ◽  
Michael F Naso ◽  
Buddha Gurung ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Growth Inhibition ◽  
B Cell ◽  
Nk Cells ◽  
Stock Options ◽  
Tumor Growth Inhibition ◽  
B Cell Malignancies ◽  
Privately Held

Abstract Induced-pluripotent stem cells (iPSCs) can be differentiated into various somatic cells, including different immune cell types. We have engineered iPSC-derived NK cells with multiple features to generate therapeutic candidates designed to eliminate cancer cells while avoiding recognition by the host immune system. The unlimited replication capacity of iPSCs facilitates the engineering of several genetic modifications without the risk of driving cells to exhaustion as in the case of cell products derived from fully differentiated immune cells. Once all edits are completed, our cells are single-cell cloned and each clone is genetically characterized to select clones without off-target insertions or deletions. Following the genetic characterization, selected clones are differentiated and tested in vitro and in vivo to identify the final clinical candidate. The use of a single-cell iPSC clone enables the generation of a master cell bank producing a highly uniform cell product that can be made available off-the-shelf at any clinical site. CNTY-101 is an iPSC-derived CAR-NK clinical candidate for the treatment of B-cell malignancies. It incorporates six gene edits designed to improve persistence and functionality as well as safety. These modifications include edits to reduce graft rejection due to alloreactivity, the expression of a homeostatic cytokine to improve functionality and persistence, the introduction of a chimeric antigen receptor (CAR) targeting CD19 to mediate tumor cell engagement and killing, as well a safety switch to eliminate the cells, if ever necessary. To prevent rejection by the patient's CD8 T cells, the beta-2-microbulin (ß2M) gene was disrupted with simultaneous insertion of a transgene encoding the HLA-E protein tethered with ß2M and a peptide. HLA-E was introduced to prevent NK cell cytotoxicity against the engineered cells, which lack HLA-I. For resistance to CD4 T cell-mediated allogenic immune rejection, the class II major histocompatibility complex transactivator (CIITA) gene was disrupted with simultaneous insertion of a transgene encoding the extra-cellular and transmembrane domains of EGFR, and the NK cell growth factor IL-15. EGFR provides an elimination tag that can be engaged by clinically approved anti-EGFR antibodies, such as cetuximab. Finally, the CAR transgene targeting the CD19 antigen was inserted into the AAVS1 safe harbor locus. Our data indicates that CNTY-101 iNK cells have strong antitumor activity against lymphoma cell lines both in vitro and in vivo. In vitro, CNTY-101 eliminates lymphoma cell lines through multiple rounds of killing without reaching exhaustion. Clones expressing higher levels of IL-15 tend to have better persistence and functionality, with some clones showing robust cytotoxicity for over fifteen rounds of serial killing. In vivo, the clones that demonstrated better in vitro serial killing tend to mediate the best anti-tumor activity in lymphoma xenograft models. Upon 3 weekly doses, the most active candidate clone demonstrated significant tumor growth inhibition after administration of fresh (91 % tumor growth inhibition) or cryopreserved cells (76 % tumor growth inhibition). The efficacy of the EGFR-safety switch was also investigated both in vitro and in vivo. In vitro, addition of cetuximab to co-cultures of IL-2-activated PBMC and cells mediated antibody-dependent cellular cytotoxicity (ADCC) in a concentration-dependent fashion, with an EC50 of 2 ng/ml. In vivo, there was a 96% reduction in the number of iPSC-derived CAR-NK cells in the lungs and a 95% reduction in the number of CAR-NK cells in the blood of mice that received cetuximab versus PBS-treated mice. In summary, CNTY-101 is a novel, multi-engineered, allogeneic CAR-iNK product candidate for the treatment of B-cell malignancies. It includes multiple immune evasion features to prevent recognition by the patient's immune system and expression of IL-15 to facilitate persistence and functionality. We have initiated GMP manufacturing of CNTY-101 and plan to enter clinical trials in 2022. Disclosures Borges: Century Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wallet: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bullaughey: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Naso: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Gurung: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Keating: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Carton: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Wheeler: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Campion: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Mendonca: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Jessup: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Beqiri: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Chin: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Millar Quinn: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Morse: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company.

scholarly journals Targeted Inhibition of PI3K α/δ Is Synergistic with BCL-2 Blockade in Genetically Defined Subtypes of DLBCL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 39-39
Author(s):  
Kamil Bojarczuk ◽  
Kirsty Wienand ◽  
Jeremy A. Ryan ◽  
Linfeng Chen ◽  
Mariana Villalobos-Ortiz ◽  
...  
Keyword(s):  
Tumor Growth ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Research Report ◽  
Genetic Bases

Abstract Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that is transcriptionally classified into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. A subset of both GCB- and ABC-DLBCLs are dependent on B-cell receptor (BCR) signaling. Previously, we defined distinct BCR/PI3K-mediated survival pathways and subtype-specific apoptotic mechanisms in BCR-dependent DLBCLs (Cancer Cell 2013 23:826). In BCR-dependent DLBCLs with low baseline NF-κB activity (GCB tumors), targeted inhibition or genetic depletion of BCR/PI3K pathway components induced expression of the pro-apoptotic HRK protein. In BCR-dependent DLBCLs with high NF-κB activity (ABC tumors), BCR/PI3K inhibition decreased expression of the anti-apoptotic NF-κB target gene, BFL1. Our recent analyses revealed genetic bases for perturbed BCR/PI3K signaling and defined poor prognosis DLBCL subsets with discrete BCR/PI3K/TLR pathway alterations (Nat Med 2018 24:679). Cluster 3 DLBCLs (largely GCB tumors) exhibited frequent PTEN deletions/mutations and GNA13 mutations. Cluster 5 DLBCLs (largely ABC tumors) had frequent MYD88L265P and CD79B mutations that often occurred together. These DLBCL subtypes also had different genetic mechanisms for deregulated BCL2 expression - BCL2 translocations in Cluster 3 and focal (18q21.33) or arm level (18q) BCL2 copy number gains in Cluster 5. These observations prompted us to explore the activity of PI3K inhibitors and BCL2 blockade in genetically defined DLBCLs. We utilized a panel of 10 well characterized DLBCL cell line models, a subset of which exhibited hallmark genetic features of Cluster 3 and Cluster 5. We first evaluated the cytotoxic activity of isoform-specific, dual PI3Kα/δ and pan-PI3K inhibitors. In in vitro assays, the PI3Kα/δ inhibitor, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. We next assessed the transcriptional abundance of BCL2 family genes in the DLBCLs following copanlisib treatment. In BCR-dependent GCB-DLBCLs, there was highly significant induction of the pro-apoptotic HRK. In BCR-dependent ABC-DLBCLs, we observed significant down-regulation of the anti-apoptotic BFL1 protein and another NF-κB target gene, BCLxL (the anti-apoptotic partner of HRK). We then used BH3 profiling, to identify dependencies on certain BCL2 family members and to correlate these data with sensitivity to copanlisib. BCLxL dependency significantly correlated with sensitivity to copanlisib. Importantly, the BCLxL dependency was highest in DLBCL cell lines that exhibited either transcriptional up-regulation of HRK or down-regulation of BCLxL following copanlisib treatment. In all our DLBCL cell lines, PI3Kα/δ inhibition did not alter BCL2 expression. Given the genetic bases for BCL-2 deregulation in a subset of these DLBCLs, we next assessed the activity of the single-agent BCL2 inhibitor, venetoclax, in in vitro cytotoxicity assays. A subset of DLBCL cell lines was partially or completely resistant to venetoclax despite having genetic alterations of BCL2. We postulated that BCR-dependent DLBCLs with structural alterations of BCL2 might exhibit increased sensitivity to combined inhibition of PI3Kα/δ and BCL2 and assessed the cytotoxic activity of copanlisib (0-250 nM) and venetoclax (0-250 nM) in the DLBCL cell line panel. The copanlisib/venetoclax combination was highly synergistic (Chou-Talalay CI<1) in BCR-dependent DLBCL cell lines with genetic bases of BCL2 deregulation. We next assessed copanlisib and venetoclax activity in an in vivo xenograft model using a DLBCL cell line with PTENdel and BCL2 translocation (LY1). In this model, single-agent copanlisib did not delay tumor growth or improve survival. Single-agent venetoclax delayed tumor growth and improved median survival (27 vs 51 days, p<0.0001). Most notably, we found that the combination of copanlisib and venetoclax delayed tumor growth significantly longer than single-agent venetoclax (p<0.0001). Additionally, the combined therapy significantly increased survival in comparison with venetoclax alone (median survival 51 days vs not reached, p<0.0013). Taken together, these results provide in vitro and in vivo pre-clinical evidence for the rational combination of PI3Kα/δ and BCL2 blockade and set the stage for clinical evaluation of copanlisib/venetoclax therapy in patients with genetically defined relapsed/refractory DLBCL. Disclosures Letai: AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; AstraZeneca: Consultancy, Other: Lab research report. Shipp:AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

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