Development of Novel Non-Immunoglobulin Centyrin-Based Cars (CARTyrins) Targeting Human Bcma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4557-4557 ◽  
Author(s):  
Burton Earle Barnett ◽  
Xinxin Wang ◽  
David L. Hermanson ◽  
Yening Tan ◽  
Eric M. Osertag ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
B Cell ◽  
Tumor Cells ◽  
Surface Expression ◽  
Domain Swapping ◽  
Target Cells ◽  
High Quality ◽  
Car T

Abstract Chimeric-antigen receptor (CAR)-T cell immunotherapy is a promising type of cancer therapy and substantial progress has been made in developing adoptive T cell approaches for B cell malignancies. B cell maturation antigen (BCMA) is an attractive target for patients with multiple myeloma (MM) due to its high level of expression on tumor cells and restricted expression on normal tissues. Traditionally, the antigen-binding domain of a CAR is a single chain variable fragment (scFv) comprised of heavy chain (HC) and light chain (LC) variable fragments joined by a flexible linker that has been derived from a non-human monoclonal Ab (mAb). However, there are a number of disadvantages to scFv-based CARs including the limited availability of scFv, their potential to elicit antibody responses, and their association with tonic signaling due, in part, to inherent instability and flexibility of the structure and the potential for both HC/LC domain swapping and multimer formation through framework region interactions. Thus, replacement with alternative binding technologies may improve CAR-T efficacy in the clinic. Centyrins are alternative scaffold molecules that bind protein targets with high affinity and specificity, similar to scFv molecules. However, unlike scFv, Centyrins are smaller, derived from human consensus tenascin FN3 domains and are predicted to have decreased immunogenicity. Additionally, a monomeric Centryin in CAR format (i.e. CARTyrin molecule) is less likely to engage in domain swapping or interact with other Centyrins at the cell surface, thereby limiting the potential for the tonic signaling that drives the functional exhaustion of CAR T cells. Centyrins can be isolated against virtually any antigen through ex vivo panning of an extensive Centyrin library, yielding many distinct binders with a range of affinities and target epitopes. Panning with soluble BCMA protein yielded a large pool of BCMA-specific Centyrins, from which 11 distinct monomeric binders and 1 non-monomeric binder were selected for further study in CAR format. In addition, we tested numerous signal peptides, linkers, transmembrane domains and signaling domains to determine optimal configuration. We then created all CARTyrins by fusing each Centyrin with a CD8a leader peptide, spacer and transmembrane domain, as well as an intracellular signaling domain derived from both 4-1BB and CD3ζ. High quality mRNA of each CARTyrin construct was produced in order to rapidly screen CARTyrin cell surface expression and functionality in human pan T cells against BCMA+ targets. We also constructed scFv-based CARs against CD19 and BCMA for comparison. Previously CD3/CD28-stimulated T cells were electroporated (EP) with mRNA encoding each of the 12 anti-BCMA CARTyrins and, the following day, analyzed for surface expression of CARTyrin and their ability to degranulate against BCMA+ tumor cells. All 12 CARTyrins were detected on the cell surface and the 11 monomeric CARTyrins imparted BCMA-specific killing capacity to T cells. Notably, in these assays, CARTyrins were functionally comparable to scFv-based CARs against BCMA or to CD19-specific scFv-based CARs in a parallel assay with CD19+ tumor cells. The 11 functional anti-BCMA CARTyrins were further characterized for functional avidity by determining their activity against a panel of target cells with titrated levels of surface BCMA expression. To create this panel, various amounts of high quality BCMA mRNA were electroporated into BCMA- K562 tumor cells. After 4 hours of co-culture with the panel of BCMA expressing cells, CARTyrin+ T cell activity was measured as a function of CD107a expression. We observed a range of activities by each CARTyrin and show that this assay can be utilized to determine the minimal effective dose of BCMA needed to induce killing by CARTyrin+ cells. Furthermore, we establish that certain BCMA-specific CARTyrins are responsive to target cells with extremely low levels of surface BCMA expression. These results confirm that Centyrins are viable replacements for scFv in the construction of functional CARs and establish their potential utility in generating novel BCMA-specific CAR molecules, as well as other novel targetable tumor antigens. Disclosures Barnett: Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Hermanson:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.

CD19-Targeted Donor T Cells Exert Potent Graft Versus Lymphoma Activity and Attenuated Gvhd

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 451-451 ◽  
Author(s):  
Arnab Ghosh ◽  
Marco L. Davila ◽  
Lauren F. Young ◽  
Christopher Kloss ◽  
Gertrude Gunset ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Acute Gvhd ◽  
Tcr Signaling ◽  
Car T Cells ◽  
Donor T Cells ◽  
Car T

Abstract Abstract 451 Chimeric antigen receptors (CAR) represent a potent strategy to target T cells against selected tumor antigens. Ongoing clinical trials indicate that autologous T cells expressing CARs targeting CD19, a B cell-associated antigen, can induce complete remission and B cell aplasia in patients with B cell malignancies. Donor CD19-CAR+ T cells could potentially be used to treat recipients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the risk of alloreactivity mediated by endogenous T cell receptors (TCR) triggering an acute GVHD is not known. This is partly due to the absence of in vivo models to study the relative effects of CAR and endogenous TCR signaling. For the first time, we have evaluated the relative effects of CD19-targeted donor T cells on the elimination of CD19+ B cells and endogenous TCR-mediated alloreactivity in mouse models of allo-HSCT. We generated a panel of retroviral vectors encoding mouse CD19-specific CARs: as a control, CD19-delta, a tail-less CAR lacking the CD3ζ signaling domain; CD19z1, which signals through its CD3ζ endodomain; and CD19-28z, which signals through CD28 and CD3ζ (Figure 1A). CD19z1+ and CD19-28z+ T cells mediated specific lysis of CD19-expressing tumors in vitro, while CD19-delta+ T cells did not. In order to assess the anti-tumor capacity of CD19-CAR+ T cells in vivo, we transferred the transduced B6 donor T cells into lethally irradiated BALB/c recipients that were administered T cell-depleted allografts and CD19+ lymphoma A20-TGL (B6–> BALB/c+A20-TGL). CD19-CAR+ T cells (CD19z1 and CD19-28z) mediated clearance of A20 tumor cells visualized by in vivo imaging of luciferase-expressing tumor cells (Figure 1B and data not shown) and significantly improved tumor free survival. CD19-CAR+ B6 T cells could sustain prolonged B cell hypoplasia when adoptively transferred into lethally irradiated haploidentical CBF1 recipients of T cell-depleted allografts (B6–> CBF1, Figure 1C). These data indicate that under alloreactive conditions, donor CD19-CAR+ T cell signaled through the CAR leading to specific elimination of CD19+ tumors and B lineage cells. In order to determine the risk of GVHD, we transferred the donor CD19-CAR+ T cells into haploidentical HSCT recipients. Interestingly, CD19-CAR+ T cells mediated significantly less acute GVHD, resulting in improved survival and lower GVHD scores (Figure 1D). Donor CD19-delta+ T cells however mediated lethal GVHD, indicating that the endogenous TCR mediated strong alloreactivity in the absence of CAR signaling. Similar results were obtained from experiments using MHC-mismatched (B6–> BALB/c) models. It is known that signaling through endogenous TCR is accompanied by down-regulation of surface TCR expression. We found significant decreases in surface CD3ϵ, TCRβ and CD90 expressions in donor CD19-delta+ T cells under alloreactive conditions. In contrast, donor CD1928z+ T cells failed to down-regulate surface TCR expression under similar conditions, suggesting that endogenous TCR function was altered in CAR-activated T cells. In the context of allo-HSCT, preferential CAR signaling at the expense of alloreactive endogenous TCR signaling may thus lead to reduced alloreactivity and attenuation of GVHD. These results provide the first pre-clinical evidence suggesting that CAR-modified, unselected donor T cells may be safely applied in an allogeneic context. Disclosures: No relevant conflicts of interest to declare.

A New Platform For Trivalent Bispecific Antibodies Used For T-Cell Redirected Killing Of B-Cell Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3078-3078
Author(s):  
Diane L Rossi ◽  
Edmund A Rossi ◽  
David M Goldenberg ◽  
Chien-Hsing Chang
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cell Surface ◽  
B Cell ◽  
Tumor Cells ◽  
Stock Option ◽  
B Cell Malignancies ◽  
Hla Dr ◽  
Close Proximity

Abstract Background Various formats of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise both pre-clinically and clinically. The scFv-based constructs, including BiTE and DART, which bind monovalently to CD3 on T cells and to the target antigen on tumor cells, exhibit fast blood clearance and neurological toxicity due to their small size (∼55 kDa). Herein, we describe the generation of novel T-cell redirecting trivalent bsAbs comprising an anti-CD3 scFv covalently conjugated to a stabilized F(ab)2. The design was initially characterized with a prototype construct designated (19)-3s, which specifically targets CD19 on B cells. A panel of trivalent bsAbs was evaluated for their potential use in targeted T-cell immunotherapy of various B-cell malignancies. Potential advantages of this design include bivalent binding to tumor cells, a larger size (∼130 kDa) to preclude rapid renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity. Methods The DOCK-AND-LOCKTM (DNLTM) method was used to generate a panel of B-cell targeting bsAbs, (19)-3s, (20)-3s, (22)-3s, and (C2)-3s, which target CD19, CD20, CD22, and HLA-DR, respectively. This was achieved by combining a stabilized anti-X F(ab)2 with an anti-CD3-scFv, resulting in a homogeneous covalent structure of the designed composition, as shown by LC-MS, SE-HPLC, ELISA, SDS-PAGE, and immunoblot analyses. Each construct can mediate the formation of immunological synapses between T cells and malignant B cells, resulting in T-cell activation. At an E:T ratio of 10:1, using isolated T cells as effector cells, the bsAbs induced potent T-cell-mediated cytotoxicity in various B-cell malignancies, including Burkitt lymphomas (Daudi, Ramos, Namalwa), mantle cell lymphoma (Jeko-1), and acute lymphoblastic leukemia (Nalm-6). A non-tumor binding control, (14)-3s, induced only moderate T-cell killing at >10 nM. The nature of the antigen/epitope, particularly its size and proximity to the cell surface, appears to be more important than antigen density for T-cell retargeting potency (Table 1). It is likely that (20)-3s is consistently more potent than (19)-3s and (C2)-3s, even when the expression of CD19 or HLA-DR is considerably higher than CD20, as seen with Namalwa and Jeko-1, respectively. This is likely because the CD20 epitope comprises a small extracellular loop having close proximity to the cell surface. When compared directly using Daudi, (22)-3s was the least potent. Compared to CD19 and CD20, CD22 is expressed at the lowest density, is a rapidly internalizing antigen, and its epitope is further away from the cell surface; each of these factors may contribute to its reduced potency. Finally, sensitivity to T-cell retargeted killing is cell-line-dependent, as observed using (19)-3s, where Raji (IC50 >3 nM) is largely unresponsive yet Ramos (IC50 = 2 pM) is highly sensitive, even though the former expresses higher CD19 antigen density. Conclusions (19)-3s, (20)-3s, (22)-3s, and (C2)-3s can bind T cells and target B cells simultaneously and induce T-cell-mediated killing in vitro. The modular nature of the DNL method allowed the rapid production of several related conjugates for redirected T-cell killing of various B-cell malignancies, without the need for additional recombinant engineering and protein production. The close proximity of the CD20 extracellular epitope to the cell surface results in the highest potency for (20)-3s, which is an attractive candidate bsAb for use in this platform. We are currently evaluating the in vivo activity of these constructs to determine if this novel bsAb format offers additional advantages. Disclosures: Rossi: Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties. Chang:Immunomedics, Inc: Employment, Stock option Other; IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other.

Selective targeting of HER2-overexpressing solid tumors with a next-generation CAR-T cell therapy.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3041-3041
Author(s):  
Jenny Mu ◽  
Justin Edwards ◽  
Liubov Zaritskaya ◽  
Jeffrey Swers ◽  
Ankit Gupta ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Antigen Receptor ◽  
Target Antigen ◽  
Target Cells ◽  
Car T

3041 Background: Conventional chimeric antigen receptor T cell (CAR-T) therapies have achieved limited clinical success in the treatment of solid tumors, in part due to the challenges of identifying tumor antigen(s) that are uniquely expressed on tumor cells. The dearth of such targets requires that current CAR-T therapies be re-engineered to preferentially target tumor cells thereby mitigating potential on-target off-tumor toxicity to normal cells. Herein we describe a novel cell therapy platform comprising Antigen Receptor Complex T (ARC-T) cells that are readily activated, silenced, and reprogrammed in vivo by administration of a novel tumor-targeting soluble protein antigen-receptor X-linker (sparX). The formation of the ARC-T, sparX, and tumor complex is required for the ARC-T to kill the tumor. Because ARC-T activity is entirely dependent on the dose of sparX administered, therapeutic doses of sparX may be defined that preferentially target cells over-expressing a target antigen and thus limit coincident kill of normal cells expressing lower levels of target antigen. Methods: We have created a library of sparX that bind different cell surface antigens, including HER2. The HER2 sparX was tested as both monovalent and bivalent constructs in vitro by assessing ARC-T cell activation, cytokine release and target cell cytotoxicity. In vivo efficacy models utilized NSG mice and incorporated tumor volume measurements and histopathologic assessments to evaluate tumor clearance. Results: In vitro studies demonstrate that co-culture of ARC-T cells, sparX-HER2 and HER2-expressing target cells drives T cell activation, expansion, cytokine secretion and cytotoxicity of target cells in a dose-dependent manner. Furthermore, by affinity tuning the HER2 binding domain and bivalent formatting of sparX-HER2, we achieved selective killing of HER2-overexpressing breast cancer cells with minimal effect on cells expressing HER2 levels representative of normal tissues. In vivo proof-of-principal studies with ARC-T/sparX-HER2 similarly demonstrate complete eradication of HER2-overexpressing solid tumor cells. Conclusions: These results demonstrate that a single intravenous dose of ARC-T cells can traffic to a solid tumor site and induce tumor eradication upon systemic administration and co-localization of tumor-targeting sparX in a mouse model. Bivalent formatting of sparX-HER2 further enabled ARC-T sensitivity to target antigen density to avoid the on-target off-tumor toxicity that has hindered conventional monovalent CAR-T treatments.

IL-18 Secreting CAR T Cells Enhance Cell Persistence, Induce Prolonged B Cell Aplasia and Eradicate CD19+ Tumor Cells without Need for Prior Conditioning

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 816-816 ◽  
Author(s):  
Mauro P. Avanzi ◽  
Dayenne G. van Leeuwen ◽  
Xinghuo Li ◽  
Kenneth Cheung ◽  
Hyebin Park ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Cytokine Analysis ◽  
Car T ◽  
Ifn Γ

Abstract Chimeric antigen receptor (CAR) T cell therapy has consistently shown significant results against acute lymphoblastic leukemia (ALL) in clinical trials1. However, results with other hematological or solid malignancies have been far more modest2. These disparate outcomes could be partially due to an inhibitory tumor microenvironment that suppresses CAR T cell function3. Thus, in order to expand the anti-tumor CAR T cell applications, a novel strategy in which these cells are capable of overcoming the hostile tumor microenvironment is needed. The cytokine interleukin-18 (IL-18) induces IFN-γ secretion, enhances the Th1 immune response and activates natural killer and cytotoxic T cells4. Early phase clinical trials that utilized systemic administration of recombinant IL-18 for the treatment of both solid and hematological malignancies have demonstrated the safety of this therapy5. We hypothesize that CAR T cells that constitutively secrete IL-18 could enhance CAR T cell survival and anti-tumor activity, and also activate cells from the endogenous immune system. To generate CAR T cells that constitutively secrete IL-18, we modified SFG-1928z and SFG-19m28mz CAR T cell constructs and engineered bicistronic human and murine vectors with a P2A element to actively secrete the IL-18 protein (1928z-P2A-hIL18 and 19m28mz-P2A-mIL18, respectively). Human and mouse T cells were transduced with these constructs and in vitro CAR T cell function was validated by coculturing the CAR T cells with CD19+ tumor cells and collecting supernatant for cytokine analysis. Both human and mouse CAR T cells secreted increased levels of IL-18, IFN-γ and IL-2. Proliferation and anti-tumor cytotoxic experiments were conducted with human T cells by coculturing CAR T cells with hCD19+ expressing tumor cells. 1928z-P2A-hIL18 CAR T cells had enhanced proliferation over 7 days and enhanced anti-tumor cytotoxicity over 72 hours when compared to 1928z CAR T cells (p=0.03 and 0.01, respectively) Next, the in vivo anti-tumor efficacy of the IL-18 secreting CAR T cell was tested in xenograft and syngeneic mouse models. Experiments were conducted without any prior lympho-depleting regimen. In the human CAR T cell experiments, Scid-Beige mice were injected with 1x106 NALM-6 tumor cells on day 0 and 5x106 CAR T cells on day 1. Survival curves showed a significant improvement in mouse survival with the 1928z-P2A-hIL18 CAR T cell treatment when compared to 1928z CAR T cell (p=0.006). Subsequently, to determine if IL-18 secreting CAR T cells could also improve anti-tumor efficacy in immunocompetent mice, we tested the murine 19m28mz-P2A-mIL18 CAR T cells in a syngeneic mouse model. The C57BL/6 hCD19+/- mCD19+/- mouse model was utilized and injected with 1x106 EL4 hCD19+ tumor cells on day 0 and 2.5 x106 CAR T cells on day 1. Mice treated with 19m28mz-P2A-mIL18 CAR T cells had 100% long-term survival, when compared to 19m28mz (p<0.0001). 19m28mz-P2A-mIL18 CAR T cells were detected in peripheral blood for up to 30 days after injection, whereas the 19m28mz CAR T cells were not detectable at any time point. In addition, 19m28mz-P2A-mIL18 CAR T cells were capable of inducing B cell aplasia for greater than 70 days, whereas 19m28mz treatment was not capable of inducing B cell aplasia. In vivo serum cytokine analysis demonstrated that 19m28mz-P2A-mIL18 CAR T cells, as compared to 19m28mz, significantly increased the levels of IFN-γ and TNF-α in the peripheral blood for up to 14 days after injection (p<0.0001 and 0.01, respectively). Despite the increase in IFN-γ and TNF-α cytokines, there was no increase in IL-6 levels. Our findings demonstrate that anti-CD19 CAR T cells that constitutively secrete IL-18 significantly increase serum cytokine secretion, enhance CAR T cell persistence, induce long-term B cell aplasia and improve mouse survival, even without any prior preconditioning. To our knowledge, this is the first description of an anti-CD19 CAR T cell that constitutively secretes IL-18 and that induces such high levels of T cell proliferation, persistence and anti-tumor cytotoxicity. We are currently investigating other mechanisms by which this novel CAR T cell functions, its interactions with the endogenous immune system, as well as testing its applicability in other tumor types. We anticipate that the advances presented by this new technology will expand the applicability of CAR T cells to a wider array of malignancies. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Distribution of chimeric antigen receptor-modified T cells against CD19 in B-cell malignancies

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhitao Ying ◽  
Ting He ◽  
Xiaopei Wang ◽  
Wen Zheng ◽  
Ningjing Lin ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Target Cells ◽  
B Cell Malignancies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Over Time

Abstract Background The unprecedented efficacy of chimeric antigen receptor T (CAR-T) cell immunotherapy of CD19+ B-cell malignancies has opened a new and useful way for the treatment of malignant tumors. Nonetheless, there are still formidable challenges in the field of CAR-T cell therapy, such as the biodistribution of CAR-T cells in vivo. Methods NALM-6, a human B-cell acute lymphoblastic leukemia (B-ALL) cell line, was used as target cells. CAR-T cells were injected into a mice model with or without target cells. Then we measured the distribution of CAR-T cells in mice. In addition, an exploratory clinical trial was conducted in 13 r/r B-cell non-Hodgkin lymphoma (B-NHL) patients, who received CAR-T cell infusion. The dynamic changes in patient blood parameters over time after infusion were detected by qPCR and flow cytometry. Results CAR-T cells still proliferated over time after being infused into the mice without target cells within 2 weeks. However, CAR-T cells did not increase significantly in the presence of target cells within 2 weeks after infusion, but expanded at week 6. In the clinical trial, we found that CAR-T cells peaked at 7–21 days after infusion and lasted for 420 days in peripheral blood of patients. Simultaneously, mild side effects were observed, which could be effectively controlled within 2 months in these patients. Conclusions CAR-T cells can expand themselves with or without target cells in mice, and persist for a long time in NHL patients without serious side effects. Trial registration The registration date of the clinical trial is May 17, 2018 and the trial registration numbers is NCT03528421.

scholarly journals CAR T cell therapy in B-cell acute lymphoblastic leukaemia: Insights from mathematical models

2020 ◽  
Author(s):  
Odelaisy León-Triana ◽  
Soukaina Sabir ◽  
Gabriel F. Calvo ◽  
Juan Belmonte-Beitia ◽  
Salvador Chulián ◽  
...  
Keyword(s):  
Mathematical Model ◽  
T Cells ◽  
T Cell ◽  
Side Effects ◽  
B Cell ◽  
Target Cells ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

AbstractImmunotherapies use components of the patient immune system to selectively target cancer cells. The use of CAR T cells to treat B-cell malignancies – leukaemias and lymphomas– is one of the most successful examples, with many patients experiencing long-lasting complete responses to this therapy. This treatment works by extracting the patient’s T cells and adding them the CAR group, which enables them to recognize and target cells carrying the antigen CD19+, that is expressed in these haematological tumors.Here we put forward a mathematical model describing the time response of leukaemias to the injection of CAR T-cells. The model accounts for mature and progenitor B-cells, tumor cells, CAR T cells and side effects by incorporating the main biological processes involved. The model explains the early post-injection dynamics of the different compartments and the fact that the number of CAR T cells injected does not critically affect the treatment outcome. An explicit formula is found that provides the maximum CAR T cell expansion in-vivo and the severity of side effects. Our mathematical model captures other known features of the response to this immunotherapy. It also predicts that CD19+ tumor relapses could be the result of the competition between tumor and CAR T cells analogous to predator-prey dynamics. We discuss this fact on the light of available evidences and the possibility of controlling relapses by early re-challenging of the tumor with stored CAR T cells.

scholarly journals Targeting B-Cell Maturation Antigen (BCMA) with CC-93269, a 2+1 T Cell Engager, Elicits Significant Apoptosis in Diffuse Large B-Cell Lymphoma Preclinical Models

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1580-1580 ◽  
Author(s):  
Patrick R. Hagner ◽  
Michelle Waldman ◽  
Falon D. Gray ◽  
Renee Yura ◽  
Sarah Hersey ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Surface Expression ◽  
Employment Equity ◽  
Car T ◽  
Equity Ownership

B-cell maturation antigen (BCMA), a member of the TNF receptor superfamily, serves as the cell surface receptor for B-cell activating factor (BAFF). Upon binding of BAFF to BCMA, an intracellular signaling cascade is initiated resulting in upregulation of JNK pathway signaling events and NFkB mediated transcription. The canonical expression pattern of BCMA begins on germinal center B-cells and becomes maximally expressed on mature cells such as plasma cells. Given the high degree of expression of BCMA on multiple myeloma (MM) cells, the malignant counterpart to plasma cells, it has become a target of interest for CAR T and antibody mediated modalities such as antibody drug conjugates and bispecific molecules. Recent clinical data from clinical trials employing BCMA targeted CAR T cells or T cell engager (TCE) antibodies have demonstrated significant responses in heavily pretreated myeloma patients with overall response rates ranging from 70% to 80%. As BCMA is known to be expressed in earlier B-cell lineages, we sought to explore the expression of BCMA in non-Hodgkin lymphoma (NHL) and its sensitivity to CC-93269, a 2+1 TCE currently being clinically investigated in MM. NHL is a heterogeneous collection of lymphomas that can be classified into two major subgroups; aggressive lymphomas of which diffuse large B-cell (DLBCL) is the most prevalent subtype and indolent lymphomas of which follicular lymphoma is the largest constituent. We first sought to directly quantitate cell surface expression of BCMA utilizing a flow cytometry system based on a logarithmic dilution of phycoerythrin beads of a known quantity. In a panel of 43 NHL cell lines, we determined that BCMA expression ranged from 43 to 17,048 molecules per cell (median, 420). An isogenic pair of K562 that is null for BCMA expression and K562 constitutively overexpressing BCMA (K562-BCMA) (15,866 molecules/cell) served as negative and positive controls, respectively. Additionally, the MM cell line H929 was profiled to serve as an additional control with a BCMA expression level of 7,065 molecules/cell. Next, utilizing quantitative PCR we found that relative BCMA mRNA expression in the lymphoma cell lines ranged from 0.001 to 0.17-fold when normalized to the H929 MM cell line. Furthermore, we were able to determine that in the lymphoma cells there is a poor correlation between protein expression (mean fluorescent intensity) and mRNA expression (r2, 0.33). We next examined if there was any correlation between BCMA surface expression and T-cell mediated cytotoxicity after administration of CC-93269 in a co-culture assay. We selected 11 DLBCL cell lines with a surface expression ranging from 45 molecules to 17,000 molecules per cells and incubated them in a co-culture system with a defined 1:5 target:effector ratio with CC-93269 (0-200 ng/ml) for five days. Significant apoptosis as measured by annexin V and ToPro-3 staining of CFSE positive target cells was observed in 10 of the 11 cell lines profiled with an IC50 of 0.1 to 38 ng/ml for CC-93269. As controls, the K562 isogenic pair were also profiled with the K562-BCMA cell line exhibiting an IC50 of 0.5 ng/ml and no activity observed against the parental K562 cell line. Additionally, a bispecific antibody where the two binding domains for BCMA was altered to target HEL (hen egg lysozyme) demonstrated no activity against any of the cell lines profiled at a defined dose of 200 ng/ml. No association between CC-93269 activity and BCMA expression or cell of origin was found. To determine the expression of BCMA in primary DLBCL biopsy samples, we developed a novel monoclonal BCMA immunohistochemistry antibody (clone: G12). The antibody and IHC staining protocol were validated to have good on-target specificity in both cell lines and tissues, including MM and DLBCL biopsies, with a range of stain intensity (1-3+) observed in both the golgi and on the plasma membrane. A proof of concept study on a cohort of 110 commercial DLBCL samples is currently underway. Cumulatively, our data demonstrate that BCMA is expressed on the cell surface of a broad panel of NHL cell lines and in primary DLBCL lymph node biopsies. Additionally, the expression levels of BCMA in these preclinical cell line models was sufficient to elicit significant CC-93269 mediated cytotoxicity. These data highlight the potential for the treatment of DLBCL patients with a 2+1 T-cell engager targeting BCMA. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Waldman:Celgene: Employment, Equity Ownership, Patents & Royalties. Gray:Celgene: Employment, Equity Ownership. Yura:Celgene: Employment, Equity Ownership. Hersey:Celgene: Employment, Equity Ownership. Chan:Celgene: Employment, Equity Ownership. Zhang:Celgene: Employment, Equity Ownership. Boss:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Preclinical Development of a T-Cell ALL CAR Demonstrates That Differences in CAR Membrane Distribution May Impact Efficacy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4019-4019
Author(s):  
Haneen Shalabi ◽  
Haiying Qin ◽  
Kelsey Wanhainen ◽  
Jillian Smith ◽  
Rimas Orentas ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Surface Expression ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Car T Cells ◽  
Car T

Abstract Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an uncommon childhood leukemia that has been associated with very poor clinical outcomes in some studies. ETP-ALL cells arrest at a more immature differentiation stage than other T-lymphoblasts, and are hypothesized to retain multi-lineage differentiation potential, which may contribute to chemoresistance with standard lymphoid-directed therapy. Based on the recent clinical success of chimeric antigen receptor (CAR)-modified T-cells in children with B-ALL, we sought to identify potential surface protein targets on ETP lymphoblasts using differential gene expression analysis combined with a bioinformatic algorithm to predict surface expression. Methods: Cell-surface targets on ETP-ALL were predicted by identifying overexpressed transcripts based on gene expression and a bioinformatic algorithm to predict surface expression. Using several gene expression platforms and reference databases, (Oncogenomics website-Pediatric Oncology Branch, NCI, Gene Expression Omnibus, Gene Ontology, Human Protein References Database) ETP-ALL samples were compared to peripheral blood mononuclear cell (PBMC) controls on an individual transcript basis. A list of the top 25 transcripts was generated based on cell surface proteins, and the resultant list ordered by the degree of difference from PBMC controls. We next used human leukemia cells from six established ETP-ALL patient-derived xenograft (PDX) models using flow cytometry to evaluate for cell surface expression of proteins encoded by the overexpressed transcripts. Additionally, since CD7 and CD33 expression on ETP-ALL patient samples is universal with minimal normal tissue distribution, we developed two new second-generation anti-CD7 or anti-CD33 CAR constructs using a 41-BB/CD3ζ backbone. Results: Multiple gene transcripts encoding cell surface proteins potentially amenable to CAR T-cell targeting were overexpressed in ETP-ALL cells in comparison to PBMC controls. Many of these proteins are involved in cell signaling, cell adhesion, and metastasis, and thus potentially important for leukemic cell survival. TSPAN7 (also known as TALLA-1) was the strongest differentially expressed transcript. Despite identification of several transcripts, we did not detect increased surface expression of multiple antigens that were identified as top 25 transcripts, including TALLA-1, MCAM, EPHB6, or TSLPR. Interestingly, TALLA-1 was expressed on the more mature T-cell ALL lines, JURKAT and HPB-AU, suggesting that the surface expression of TALLA protein may be developmentally regulated. Although a new target could not be identified, given the universal expression of CD7 and CD33 on ETP-ALL, we proceeded with development of CARs targeting these antigens. CD33 CAR T-cells had excellent in vitro activity in human AML cell line MOLM-14 with minimal anti-leukemia activity in six tested ETP-ALL PDX models, perhaps due to their lower CD33 expression. We next tested T-cells transduced with a bicistronic CD7-redirected CAR with a truncated EGFR (EGFRt) to facilitate measurement of transduction efficiency and to provide a CAR deletion method. Despite high EGFRt surface expression in transduced T-cells, these CD7 CAR T-cells did not demonstrate in vitro activity against ETP-ALL or mature T-ALL samples despite high CD7 surface expression on all leukemia cell lines. We postulated that abnormal CAR distribution within the T-cell itself could be a potential factor in the observed lack of CD7 CAR T-cell activity. Using fluorescent-labeling to assess CAR surface membrane distribution, we detected high intracellular expression of the CD7 CAR, and noted that it did not traffic to the cell surface. Conclusions: We applied multimodal techniques to evaluate for cell surface expression on ETP-ALL that could serve as a target for immunotherapy. Although novel targets could not be identified, we were able to design an active anti-CD33 CAR. Further studies are in progress to evaluate what degree of antigen expression is needed to be amenable to targeted therapy. Additionally, ongoing studies are assessing whether optimization of CAR design can enhance cell surface trafficking and thereby potentially improve the anti-leukemia efficacy of CD7 CAR T-cells. Disclosures Orentas: Lentigen Technology, Inc.: Employment. Maude:Novartis: Consultancy. Teachey:Novartis: Research Funding.

scholarly journals CD19/CD22 Dual-Targeted Chimeric Antigen Receptor T-Cell Therapy for Relapsed/Refractory Aggressive B-Cell Lymphoma: a Safety and Efficacy Study

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-34
Author(s):  
Yongxian Hu ◽  
Yanlei Zhang ◽  
Houli Zhao ◽  
Yiyun Wang ◽  
Arnon Nagler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Target Cells ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T ◽  
Aggressive B Cell Lymphoma

Introduction Chimeric antigen receptor (CAR)-T-cell therapy has revolutionized the treatment of relapsed/refractory (R/R) B-cell hematological malignancies, primarily acute lymphoblastic leukemia (ALL), and B-cell non-Hodgkin lymphoma (NHL). CD19 CAR-T cells have been extensively studied and have been shown to yield complete remission (CR) rates of about 90% in R/R ALL, but substantially lower (50%) rates in R/R NHL. Moreover, persistence is usually limited, and antigen escape-mediated relapse is a major limitation. Dual CAR-T cells targeting both CD19 and CD22 may address these limitations. Patients and methods We developed a bispecific CAR-T cells that could concomitantly recognize CD19- and CD22-expressing targets by incorporating both CD19 and CD22 single-chain variables in a single CAR construct (Figure 1A). We designed a prospective study to assess the safety and efficacy profiles of the dual CAR-T therapy in patients with R/R aggressive B-cell lymphoma. Results The preclinical cytotoxicity evaluation of the CD19/CD22 dual-targeted CAR-T cells was performed in comparison with mono-specific CD19-BB-002 and CD22-BB-002 CAR-T cells in HeLa cells that were engineered to express CD19, CD22, or both antigens. The dual-antigen specific CAR-T cells performed equally well when compared with the mono-specific CAR-T cells when there was only a single antigen present on the target cells; better performance was observed when both antigens were present on target cells (Figure 1B). In addition, the dual-antigen specific CAR-T cells induced equal amounts of interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)-γ, when compared with the two mono-specific CAR-T cells (Figure 1C). Furthermore, the CD19 CAR-T cells induced more IL-2 and tumor necrosis factor (TNF)-α than the CD22 CAR-T cells and dual-antigen CAR-T cells. However, in the presence of both CD19 and CD22 antigens, the dual-specific CAR-T cell tended to produce more granzyme B, which may explain the higher degree of cytotoxicity when compared with the two mono-specific CAR-T cells (Figure 1D). Twenty-four patients were screened. Of the 16 eligible patients 14 (87.5%) achieved objective response (RR), with 10 (62.5%) achieving complete response (CR). The 2-year overall survival (OS) and progression-free survival (PFS) rates were 77.3% and 40.2%, respectively (Figure 2A). Achieving CR (HR: 0.017, 95% CI: 0.000-0.935; P=0.046) and number of prior lines of chemotherapy (n=2) (HR:135.784, 95% CI: 1.069-17248.110, P=0.047) were found as independent prognostic factors associated with favorable PFS. The 2-year OS and PFS of the CR patients were higher than those of the non-CR patients (100% versus 41.7%, P=0.015; 66.7% versus 0%, P &lt; 0.001), respectively (Figure 2B). The 2-year PFS in patients received 2 prior lines of chemotherapy was higher as compared to those that received more than 2 lines of chemotherapy (68.6% versus 16.7%, P=0.049) whereas the OS in the 2 groups did not differ significantly (83.3% and 71.1%, P=0.613) (Figure 2C). Severe grade 3 cytokine release syndrome (CRS) was observed in only one patient, while 4 had grade one and 11 had grade 2, respectively. No patient developed neurotoxicity. Conclusions Immunotherapy with a novel CD19/CD22 dual targeted CAR-T cells yields a potent and durable anti-lymphoma response with no neurotoxicity or severe CRS. Bispecific CD19/CD22 CAR-T cells represent a safe and potent anti-lymphoma cellular based targeted immunotherapy. Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals SILAC phosphoproteomics reveals unique signaling circuits in CAR-T cells and the inhibition of B cell-activating phosphorylation in target cells

2021 ◽  
Author(s):  
Alijah A. Griffith ◽  
Kenneth P. Callahan ◽  
Nathan Gordo King ◽  
Qian Xiao ◽  
Xiaolei Su ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Target Cells ◽  
Tcr Signaling ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

AbstractChimeric antigen receptor (CAR) is a single-pass transmembrane receptor designed to specifically target and eliminate cancers. While CARs prove highly efficacious against B cell malignancies, the intracellular signaling events which promote CAR T cell activity remain elusive. To gain further insight into both CAR T cell signaling and the potential signaling response of cells targeted by CAR, we analyzed phosphopeptides captured by two separate phopshoenrichment strategies from third generation CD19-CAR T cells cocultured with SILAC labeled Raji B cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we report that CD19-CAR T cells upregulated several key phosphorylation events also observed in canonical T cell receptor (TCR) signaling while Raji B cells exhibited a significant decrease in B cell receptor-signaling related phosphorylation events in response to coculture. Our data suggest that CD19-CAR stimulation activates a mixture of unique CD19-CAR-specific signaling pathways and canonical TCR signaling while global phosphorylation in Raji B cells is reduced after association with the CD19-CAR T cells.

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