Target cell killing effects of CD20 targeting chimeric antigen receptor T cells derived from the type II anti-CD20 antibody.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e14548-e14548 ◽  
Author(s):  
Yihong Yao ◽  
Xin Yao ◽  
Shigui Zhu ◽  
Wei Zhu ◽  
Zhiyuan Li ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Cell Activation ◽  
Type I ◽  
Type Ii ◽  
Hydrophobic Residues ◽  
Car T ◽  
Small Loop ◽  
Cd20 Antibody ◽  
Anti Cd20

e14548 Background: Chimeric Antigen Receptor T cells (CAR-Ts) targeting CD19 have shown very promising clinical outcomes in treatment of B-cell linage hematological malignancies. However, many patients with relapsed diseases were found to have down-regulated/loss of CD19 surface expression after CD19 CAR-T therapy. To solve this issue of CD19 single-targeting escape, we explored the application of another B-cell antigen, CD20, for targeted CAR-T therapy. Methods: We constructed four CD20 targeting CARs (all with 4-1BB co-stimulatory signaling) base on single-chain variable fragments (scFV) derived from four well-studied CD20 specific antibodies: Leu16, Rituximab, Obinutuzumab, and Ofatumumab. Leu16, Rituximab, and Obinutuzumab belong to the type I anti-CD20 antibody family and appear to bind to different epitopes located on the large loop of CD20, whereas Ofatumumab is the type II anti-CD20 antibody which has been shown to interact with the hydrophobic residues on the small loop surrounding a deep binding cleft. Results: All four CAR-T cells can specifically recognized CD20 positive target cells in our pre-clinical studies. They all showed up-regulated antigen-specific cell activation and high level of IFN-g release upon CD20 stimulation, and CAR-T20-Ofatumumab cells appeared to have significantly higher cell activation and more than 2-fold increase in IFN-g release compared to the other three CAR-T20 cells with their scFVs deriving from type I anti-CD20 antibodies. CAR-T20-Ofatumumab cells also showed higher degranulation upon stimulation, and it displayed ~50% of increase in ability to kill CD20 positive cells in cytotoxicity assays. Conclusions: Our data suggested that CAR-T20-Ofatumumab has better in vitro function and appears to be a CAR superior to those derived from other three antibodies. A possible explanation for this observation is that Ofatumumab interacts with the hydrophobic residues on the small loop, which is very close to cell membrane and confers more extensive binding to the small loop with striking slow off-rate. Our results suggest that CAR-Ts targeting CD20 with the scFVs from the type II anti-CD20 antibody may have superior cell killing effects.

Differential Patterns of Gene Expression and CD20 Localisation by the Type II Anti-CD20 Antibody Obinutuzumab (GA101) Compared with the Type I Antibody Rituximab Suggests Binding to Functionally Distinct CD20 Subpopulations on B-Cell Lymphoma Cell Lines,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3721-3721
Author(s):  
Gerhard Niederfellner ◽  
Olaf Mundigl ◽  
Alexander Lifke ◽  
Andreas Franke ◽  
Ute Baer ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mechanisms Of Action ◽  
Type I ◽  
Type Ii ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract Abstract 3721 The anti-CD20 antibody rituximab has become central to the treatment of B-cell malignancies over the last decade. Recently, it has been shown that anti-CD20 antibodies can be divided into two types based on their mechanisms of action on B cells. Rituximab is a type I antibody that redistributes CD20 into lipid rafts and promotes complement-dependent cytotoxicity (CDC), while the type II, glycoengineered antibody GA101 has lower CDC activity but higher antibody-dependent cellular cytotoxicity and direct cell death activity. In preclinical studies GA101 was superior to rituximab in B-cell killing in vitro, depletion of B cells from whole blood, and inhibition of tumour cell growth in lymphoma xenograft models. GA101 is currently being evaluated in Phase II/III trials, including comparative studies with rituximab. To investigate the differences in direct effects of GA101 and rituximab on B-cell lymphoma signaling, we have analysed the effects of antibody binding on gene expression in different B-cell lines using a GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). Rituximab and GA101 rapidly induced gene expression changes in SUDHL4 and Z138 cells, including regulation of genes associated with B-cell-receptor activation such as EGR2, BCL2A1, RGS1 and NAB2. The effects on gene expression differed markedly between different cell lines and between the two antibodies. SUDHL4 cells showed pronounced changes in the gene expression pattern to rituximab treatment, while Z138 cells, which represent a different B-cell stage, showed less pronounced changes in gene expression. The reverse was true for GA101, suggesting not only that the signaling mediated by CD20 differs in different cell lines, but also that in a given cell line the two types of antibodies bind CD20 molecules with different signaling capacity. For each cell line, gene expression induced by other type I antibodies (LT20, 2H7, MEM97) was more like rituximab and that induced by other type II antibodies (H299/B1, BH20) was more like GA101 in terms of the number of genes regulated and the magnitude of changes in expression. Unbiased hierarchical clustering analysis of gene expression in SUDHL4 could discriminate type I from type II antibodies, confirming that the two classes of antibody recognised CD20 complexes with inherently different signalling capacities. By confocal and time-lapse microscopy using different fluorophores, rituximab and GA101 localised to different compartments on the membrane of lymphoma cells. GA101/CD20 complexes were relatively static and predominantly associated with sites of cell–cell contact, while rituximab/CD20 complexes were highly dynamic and predominantly outside areas of contact. These findings suggest that type II antibodies such as GA101 bind distinct subpopulations of CD20 compared with type I antibodies such as rituximab, accounting for the differences in mechanisms of action and anti-tumour activity between these antibodies. Disclosures: Niederfellner: Roche: Employment. Mundigl:Roche: Employment. Lifke:Roche: Employment. Franke:Roche: Employment. Baer:Roche: Employment. Burtscher:Roche: Employment. Maisel:Roche: Employment. Belousov:Roche: Employment. Weidner:Roche: Employment. Umana:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Effect of Rituximab On the Activity of T Cells Expressing CD20-Specific Chimeric Antigen Receptors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4222-4222
Author(s):  
Gregory A. Rufener ◽  
Michael C. Jensen ◽  
Shaunda Brouns ◽  
Lihua E. Budde ◽  
David G. Maloney ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Cell Function ◽  
Cytokine Secretion ◽  
Lymphoma Cells ◽  
Car T Cells ◽  
Car T ◽  
Anti Cd20

Abstract Abstract 4222 BACKGROUND: Adoptive cellular therapy using autologous T cells that have been genetically modified to express a chimeric antigen receptor (CAR) has emerged as a promising therapy for lymphoma. Clinical trials for lymphoid malignancies to date have primarily targeted either the CD19 or CD20 antigens. While CD20 has a more established track record as an immunotherapy target, one potential drawback of targeting CD20 with CAR+ T cells is the theoretical possibility that residual levels of circulating anti-CD20 antibodies (Ab) from prior chemoimmunotherapy regimens could partially or completely block CAR-antigen interactions. This could negatively impact the efficacy of CD20-targeted CAR+ T cells. However, previous data from our group and others indicate that CD20 CAR+T cell function is only partially blocked by anti-CD20 Ab, and T cell function in the setting of anti-CD3 × anti-CD20 bispecific Ab is not blocked by rituximab (R) levels of up to 100 μg/ml. Collectively, these data suggest that a very low number of available CD20 binding sites may be sufficient to trigger CAR signaling and T cell activation. METHODS: We tested the effect of different levels of R on in vitro function of polyclonal T cells from healthy donors negatively selected by MACS, activated with anti-CD3/CD28 beads, and transduced with epHIV7 lentiviral vectors encoding 1st or 3rdgeneration (αCD20-ζ or αCD20-CD28–41BB-ζ) anti-CD20 CARs. T cells were re-stimulated 1 week after initial activation by co-culture with antigen presenting cells (APCs) that had been pre-incubated for 30 minutes with varying concentrations of R (ranging from 0 to 800 μg/ml). APCs were K562 cells transduced to express CD80 with or without CD20 (denoted “K80” and “K80-20”), or Ramos lymphoma cells. Proliferation, cytokine secretion, and cytotoxicity were then assessed as discussed below. RESULTS: We first used flow cytometry to test whether varying concentrations of R blocked binding of the Leu16 Ab and, as expected, found a dose-dependent blockade of CD20 on each cell line, with 50 μg/ml and 200 μg/ml causing near-complete blockade of K80-20 and Ramos cells, respectively. However, despite this apparent blockade, proliferation was largely unimpaired in CFSE-labeled 1st or 3rd generation CAR+ T cells cultured with K80-20 or Ramos cells pre-incubated with R concentrations of up to 400 μg/ml. We concurrently measured cytokine secretion of these T cells using Luminex assays and found that IL-2 and IFN-γ secretion decreased with increasing R levels, but 50–85% of baseline levels were still achieved at R concentrations of up to 100 μg/ml. Cytotoxicity against K80-20 and Ramos target cells in standard 51Cr-release assays by 1st and 3rd generation CAR+ T cells was largely preserved at low R concentrations, and 50–75% of cytolytic activity was retained at 100 μg/ml. Nonspecific proliferation, cytokine secretion, and cytotoxicity were excluded in these experiments by using CAR+ T cells incubated with K80 cells lacking CD20 expression, or T cells transduced with an empty vector as negative controls. Mouse xenograft experiments are currently ongoing to test the effect of serum R levels on the in vivo anti-tumor efficacy of CD20-CAR T cells. CONCLUSIONS: These in vitro results suggest that despite apparent blockade of the CD20 antigen, CAR+ T cells targeting CD20 retain significant activity in the presence of R concentrations of up to 100 ug/ml. Patients receiving 2–3 cycles of R-chemotherapy have serum R trough levels in the range of 30–70 μg/ml. We therefore predict that residual serum R levels will not present a significant impediment to CD20-targeted adoptive T cell therapy given after salvage R-chemotherapy. Disclosures: Jensen: ZetaRx: Equity Ownership, Patents & Royalties. Maloney:Roche: Consultancy; Genentech: Consultancy. Off Label Use: Lentiviral vector encoding a CD20-specific chimeric antigen receptor, used to re-direct autologous T cells to recognize B cell lymphoma cells.

scholarly journals Optimizing Ex-Vivo Expanded NK Cell- Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) Combined with NKTR-255 in Chronic Lymphocytic Leukemia (CLL), Follicular Lymphoma (FL), and Burkitt Lymphoma (BL)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 23-24
Author(s):  
Yaya Chu ◽  
Susiyan Jiang ◽  
Jian Jiang ◽  
Meijuan Tian ◽  
Dean Anthony Lee ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Type I ◽  
Type Ii ◽  
Advisory Committees ◽  
Anti Cd20

Background: The CD20 molecule is universally expressed by normal B cells in all stages of development, from the pre-B cell up to the mature plasma cell as well as by most B cell malignancies including CLL, FL and BL (Chu/Cairo, BJH, 2016). Rituximab, a monoclonal chimeric anti-CD20 antibody, has been widely used as a chemoimmunotherapeutic regimen in the frontline therapy for patients with CD20+ BL and diffuse large B-cell lymphoma. The addition of rituximab to the CHOP backbone or to standard FAB/LMB therapy has greatly improved outcomes without significantly increasing toxicity in patients with B-NHL (Goldman/Cairo, Leukemia, 2013, Coiffier et al, NEJM, 2002). However, patients who relapse have a poor clinical response to rituximab retreatment. Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity. It has lower complement-dependent cytotoxicity than rituximab but greater ADCC, phagocytosis and direct B-cell killing effects (Chu/Cairo, BJH, 2018). Obinutuzumab has been successfully utilized in front-line therapy in FLL (Marcus, et al, NEJM, 2017) and CLL (Goede, et al, NEJM, 2014; Moreno, et al, Lancet, 2019). Our group has successfully expanded functional and active peripheral blood NK cells PBNKwith irradiated feeder cells to target B-NHL (Chu/Cairo, et al, Can Imm Res 2015). We previously demonstrated that obinutuzumab has significantly enhanced expanded PBNK mediated cytotoxicity against BL and pre-B-ALL cell lines compared to rituximab (Tiwari/Cairo et al, BJH, 2015). NKTR-255 is an IL-15 receptor agonist designed to activate the IL-15 pathway and expand natural killer (NK) cells and promote the survival and expansion of memory CD8+ T cells without inducing suppressive regulatory T cells (Kuo/Zalevsky, Cancer Res. 2017). NKTR-255 stimulates proliferation and survival of NK, CD8+ T cells, and enhances long-term immunological memory which may lead to sustained anti-tumor immune response. Objective: To investigate the effects of NKTR-255 on the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL. Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Dean Lee, PLoS One, 2012). Expanded PBNK cells were isolated using Miltenyi NK cell isolation kit. NKTR-255 was generously provided by Nektar Therapeutics. In vitro cytotoxicity was examined using luminescence reporter-based assays. IFNg, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, ASH, 2018). MEC-1 (CLL), PGA-1 (CLL), DOHH2 (FL) and Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Results: NKTR-255 significantly enhanced the in vitro cytotoxicity of expanded NK cells when combined with rituximab against MEC-1 (E:T=3:1, p<0.001), PGA-1 (E:T=3:1, p<0.001), and DOHH2 (E:T=3:1, p<0.001) as compared to the control groups (Fig.1A). NKTR-255 also significantly enhanced granzyme and perforin release from expanded NK cells when combined with rituximab against MEC-1 (granzyme: p<0.05; perforin: p<0.001), PGA-1(granzyme: p<0.05; perforin: p<0.05), DOHH2 (granzyme: p<0.05; perforin: p<0.001) as compared to controls. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab agains rituximab-resistant BL cells like Raji-2R (E:T=3:1, p <0.01), and Raji-4RH (E:T=3:1, p<0.01) as compared to the control groups (Fig.1B). NKTR-255 also significantly enhanced IFN-g, granzyme and perforin release from expanded NK cells when combined with obinutuzumab against Raji-2R (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.001 and perforin: p<0.001) and Raji-4RH (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.01 and perforin: p<0.01) as compared to controls. Conclusion: We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells in vitro with enhanced IFN-g, granzyme B and perforin release. The in vivo effects of NKTR-255 with expanded NK cells and anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells using humanized NSG models are under investigation. Disclosures Lee: Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Madakamutil:Nektar Therapeutics: Current Employment. Marcondes:Nektar Therapeutics: Current Employment. Klein:Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Cairo:Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding.

scholarly journals Reduction of Mitochondrial Membrane Potential Leads to Enhancement of Type-II CD20-Antibody Cytotoxicity in Diffuse Large B-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1763-1763 ◽  
Author(s):  
Emma Vilventhraraja ◽  
Tetyana Klymenko ◽  
Jennifer Edelmann ◽  
John Gribben ◽  
Andrejs Ivanov
Keyword(s):  
Oxidative Phosphorylation ◽  
Cell Lines ◽  
Mitochondrial Function ◽  
Type I ◽  
Type Ii ◽  
Antibody Treatment ◽  
Respiratory Capacity ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Cd20 Antibodies

Abstract Diffuse Large B Cell Lymphoma (DLBCL) is the most prevalent non-Hodgkin lymphoma (NHL) in adults. Since the addition of the Type I anti-CD20 antibody Rituximab to chemotherapy, the overall survival of NHL patients has improved dramatically compared to the pre-Rituximab era. DLBCL however, has the worst survival rates out of all NHLs with an average 5-year survival of 55%. Unfortunately 40% of all DLBCL patients relapse within 2 years, and those that relapse or have refractory disease tend not to respond well to antibody-based salvage therapies. Since the discovery and utilisation of Rituximab, many have tried to enhance the efficacy of anti-CD20 antibodies in order to improve first-line treatment of DLBCL, leading to the evolution of Type II humanised anti-CD20 antibodies. The complete biological role of CD20 remains unclear, however it has been shown to act as part of an ion channel complex that is a component of the store operated calcium (Ca2+) system. This complex has the ability to facilitate mitochondrial membrane permeabilisation, resulting in reduced mitochondrial function. In order to investigate the effect of Type I- and Type II- anti-CD20 antibodies on mitochondrial function, we established a panel of 4 DLBCL cell lines. We used the XF Seahorse Mito Stress Test to reveal bioenergetic profiles of the cell lines before and after treatment with a panel of Type I and Type II anti-CD20 antibodies (2 Type-I and 2 Type-II anti-CD20 antibodies for each cell line). Basal oxidative phosphorylation (OxPhos), ATP production, and maximal and spare respiratory capacity of each sample were calculated as a measure of mitochondrial function. Next we used Metformin, a well-established inhibitor of oxidative phosphorylation to reduce the mitochondrial membrane potential (MMP) across our panel of cell lines. We confirmed MMP reduction by staining cells with JC-1, a chameleon dye used as an indicator of MMP and analysed samples using flow cytometry. We then used the XF Seahorse Mito Stress Test, this time to assess how combining each CD20-antibody with an OxPhos inhibitor effects mitochondrial function (10 conditions for each cell line). Finally, we used the same conditions to conduct clonogenic survival assays to see whether cytotoxicity of Type-I or Type-II anti-CD20 antibodies could be enhanced. We have observed that treatment with anti-CD20 antibodies results in a significant increase in the maximal respiratory capacity of our panel of cell lines. Conversely, pharmacological inhibition of oxidative phosphorylation causes a significant reduction in basal oxidative phosphorylation as well as a reduction in the maximal respiratory capacity of the cell lines in our panel. We also show that treatment combining an OxPhos inhibitor with either Type-I or Type-II CD20-antibodies prevents the increase in maximal respiratory capacity observed with CD20-antibody treatment alone. When analysing the clonogenic survival of cell lines we have found that only the cytotoxicity of Type-II anti-CD20 antibodies is enhanced by simultaneously treating cell lines with Metformin. We also used Annexin V/PI staining to assess cell death and show that inhibiting oxidative phosphorylation in conjunction with CD20-antibody treatment does not result in a significant increase in cell death across our panel of cell lines. Our data indicate for the first time that when cells are treated with CD20-antibodies they increase their maximal mitochondrial respiratory capacity to compensate for reduced basal mitochondrial function. We also show that inhibition of oxidative phosphorylation disables the cells from being able to compensate for the reduced mitochondrial function that is caused by CD20-antibody treatment. Importantly our data show that the reduction of mitochondrial function caused by combining Metformin with Type-II CD20 antibodies leads to a significant reduction in clonogenicity. We believe that understanding the mechanism of the inhibition of mitochondrial function will allow us to establish effective treatment combinations to significantly improve the efficacy of anti-CD20 antibody therapy in DLBCL. Disclosures No relevant conflicts of interest to declare.

Crystal Structure Analysis Reveals That the Novel Type II Anti-CD20 Antibody GA101 Interacts with a Similar Epitope as Rituximab and Ocrelizumab but in a Fundamentally Different Way.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3726-3726 ◽  
Author(s):  
Gerhard J Niederfellner ◽  
Alfred Lammens ◽  
Manfred Schwaiger ◽  
Guy Georges ◽  
Kornelius Wiechmann ◽  
...  
Keyword(s):  
Research Funding ◽  
Type I ◽  
The Novel ◽  
Type Ii ◽  
Employment Equity ◽  
Equity Ownership ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Point Mutagenesis ◽  
Cd20 Antibodies

Abstract Abstract 3726 Poster Board III-662 CD20 is a specific cell surface marker found on normal as well as malignant B cells. Rituximab, a monoclonal antibody directed against CD20, has a major impact on treatment of malignant lymphomas. Although all therapeutic CD20 antibodies are directed against the two relatively small extracellular loops of CD20, such antibodies can be classified into Type I CD20 antibodies like Rituximab, Ofatumumab or Ocrelizumab or Type II CD20 antibodies like the novel glycoengineered humanized CD20 antibody GA101 or the murine antibody Tositumumab. Type I and Type II antibodies differ significantly in their mode of action and mechanisms of killing malignant B-cells. The molecular basis of this is not understood. We use data from epitope mapping, X-ray crystallography, isothermal titration calorimetry, and point mutagenesis i) to accurately map the epitopes of different anti-CD20 antibodies, in particular GA101, and ii) to compare the molecular interactions involved in their binding. Although the epitope regions of these antibodies largely overlap, the crystal structure shows that GA101 binds CD20 in a completely different orientation from Rituximab, Ocrelizumab and Ofatumumab and that its binding also involves a larger surface area. In agreement with predictions based on the crystallographic data, point mutagenesis of single amino acid residues confirmed that exchanges at certain positions in CD20 affect binding of Rituximab and GA101 differently. Our data suggest that engagement of CD20 by these antibodies favors different conformations of CD20, which could form the molecular basis for the observed differences in cellular signals triggered by the respective antibodies. Disclosures: Niederfellner: Roche: Employment. Schwaiger:Roche: Employment. Georges:Roche: Employment. Wiechmann:Roche: Research Funding. Franke:Roche: Research Funding. Schaefer:Roche: Employment. Jenewein:Roche: Employment. Slootstra:Pepscan: Employment, Patents & Royalties. Moessner:Glycart: Employment, Equity Ownership, Patents & Royalties. Umana:Glycart: Employment, Equity Ownership, Patents & Royalties. Hopfner:Roche: Research Funding. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Repurposing endogenous immune pathways to tailor and control chimeric antigen receptor T cell functionality

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Mohit Sachdeva ◽  
Brian W. Busser ◽  
Sonal Temburni ◽  
Billal Jahangiri ◽  
Anne-Sophie Gautron ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Cell Activation ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T ◽  
Immune Pathways ◽  
And Control

Abstract Endowing chimeric antigen receptor (CAR) T cells with additional potent functionalities holds strong potential for improving their antitumor activity. However, because potency could be deleterious without control, these additional features need to be tightly regulated. Immune pathways offer a wide array of tightly regulated genes that can be repurposed to express potent functionalities in a highly controlled manner. Here, we explore this concept by repurposing TCR, CD25 and PD1, three major players of the T cell activation pathway. We insert the CAR into the TCRα gene (TRACCAR), and IL-12P70 into either IL2Rα or PDCD1 genes. This process results in transient, antigen concentration-dependent IL-12P70 secretion, increases TRACCAR T cell cytotoxicity and extends survival of tumor-bearing mice. This gene network repurposing strategy can be extended to other cellular pathways, thus paving the way for generating smart CAR T cells able to integrate biological inputs and to translate them into therapeutic outputs in a highly regulated manner.

A Phase I/II Study of RO5072759 (GA101) in Patients with Relapsed/Refractory CD20+ Malignant Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 234-234 ◽  
Author(s):  
Gilles Andre Salles ◽  
Franck Morschhauser ◽  
Guillaume Cartron ◽  
Thierry Lamy ◽  
Noel-Jean Milpied ◽  
...  
Keyword(s):  
Phase I ◽  
Malignant Disease ◽  
Single Agent ◽  
Tumor Lysis Syndrome ◽  
T Cell Subsets ◽  
Type I ◽  
Type Ii ◽  
Cell Counts ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract RO5072759 (GA101) is the first humanized and glycoengineered monoclonal anti-CD20 antibody to enter clinical trials. Glycoengineeering results in a significantly increased antibody-dependent cytotoxicity (ADCC) compared to rituximab as shown in in vitro models. Additionally, RO5072759 binds with high affinity to a type II epitope on CD20 and is characterized by reduced complement-dependent cytotoxicity (CDC) and strongly enhanced direct cell death compared to type I antibodies. In preclinical in vivo lymphoma models RO5072759 has shown superior efficacy compared to rituximab. In this Phase I/IIa study RO5072759 was administered as a single agent to patients with CD20+ malignant disease for whom no therapy of higher priority was available. The aim of which was to determine the safety and tolerability of RO5072759, any dose-limiting toxicity (DLT), its pharmacokinetics and to establish the recommended phase II dose. Patients were treated with RO5072759 by intravenous infusion (premedication with acetaminophen and anti-histamines) administered as a flat dose on days 1, 8 and 22 and subsequently every 3 weeks for a total of 9 infusions. The dose of the first infusion was 50% that of subsequent infusions. The dose was escalated based on the safety in a 3+3 design. Since September 2007 twenty four patients have been treated with RO5072759 at doses from 50 mg to 2000 mg. Complete data, presented herein, is available on the first 12 patients, median age 59 yrs (39–83), from the first 4 cohorts (50 mg–800 mg). Most patients had follicular NHL (9) others include DLBCL (1) CLL (1) Waldenstrom’s macroglobulinemia (1). All were previously exposed to rituximab and had received a median of 4 (range 1–7) prior regimens. RO5072759 was well tolerated with no DLTs observed. The most common adverse events are Grade 1 or 2 (CTCAE V3.0) infusion related reactions, characterized by fever, chills, hypo/hypertension, nausea and vomiting limited in the main, to the first infusion. These responded well to slowing or interruption (5 pts) of the infusion and steroids (1 pt). No tumor lysis syndrome has been observed. Only 6 minor infections (4 upper respiratory track, 1 urinary track and 1 oral herpes) were observed to-date. Measurement of plasma cytokines and complement during and immediately after the first infusion showed an increase in IL6 and IL8 with a smaller increase in IL10 and TNFα. No change in complement fractions (C3, C3a, C4a, C5, C5a, Bb) was observed. Concurrent to cytokine increase was apparent a decrease in T-cell subsets (CD3, CD4 and CD8 subsets) and NK cell counts in the peripheral blood. This decrease, as with the cytokine increase, was transient and levels were at or near baseline by day 8. No similar changes, for the majority of patients, were seen with subsequent infusions. Circulating B-cell (CD19) depletion occurs rapidly and is sustained. The pharmacokinetics of RO5072759 are broadly similar to those of rituximab and show a dose-dependent increase in exposure, but with significant inter- and intra-patient variability. Time dependent clearance was noted which is consistent with a reduction in target-mediated antibody clearance with increasing duration of treatment. To-date 7 of 12 patients have responded by day 85 to treatment [3 CR (25%), 4 PR (33%) ORR (58%)], 1 patient has SD at 8 months, 3 patients have PD (2 have since died) and 1 patient died from an event unrelated to treatment (cerebrovascular accident). Responses have occurred at all dose levels. In conclusion, RO5072759 is a novel ADCC-enhanced type II anti-CD20 antibody that has shown a similar safety profile to rituximab and promising efficacy in this difficult-to-treat patient population. Dose finding is continuing and data from all 24 patients will be presented.

scholarly journals Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR)

2019 ◽  
Author(s):  
Rui Dong ◽  
Kendra A. Libby ◽  
Franziska Blaeschke ◽  
Alexander Marson ◽  
Ronald D. Vale ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Intracellular Signaling ◽  
Cell Activation ◽  
Antigen Receptor ◽  
Downstream Signaling ◽  
Car T Cells ◽  
Car T

AbstractThe chimeric antigen receptor (CAR) directs T cells to target and kill specific cancer cells. Despite the success of CAR T therapy in clinics, the intracellular signaling pathways that lead to CAR T cell activation remain unclear. Using CD19 CAR as a model, we report that, similar to the endogenous T cell receptor (TCR), antigen-engagement triggers the formation of CAR microclusters that transduce downstream signaling. However, CAR microclusters do not coalesce into a stable central supramolecular activation cluster (cSMAC). Moreover, LAT, an essential scaffold protein for TCR signaling, is not required for microcluster formation, immunological synapse formation, and actin remodeling following CAR activation. Meanwhile, CAR T cells still require LAT for the normal production of the cytokine IL-2. Together, these data show that CAR T cells can bypass LAT for a subset of downstream signaling outputs, thus revealing a rewired signaling pathway as compared to native T cells.

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