scholarly journals Lenalidomide Enhances the Function of CS1 Chimeric Antigen Receptor Redirected-T Cells Against Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 812-812 ◽  
Author(s):  
Xiuli Wang ◽  
Ryan Urak ◽  
Miriam Walter ◽  
Lihong Weng ◽  
Laura Lim ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Memory T Cells ◽  
Central Memory ◽  
Central Memory T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Multiple myeloma (MM) is an incurable malignancy of plasma cells even with great advances in treatment. Chimeric Antigen Receptor (CAR) directed T cell therapy, which can specifically recognize tumor associated antigens and kill tumor cells in an MHC independent manner, is a promising approach for hematological malignancy. There are several candidate antigens for CAR T cell targeting of multiple myeloma, including BCMA and CS1. Our goal is to develop novel CARs for the treatment of MM and explore the potential benefits of combinatorial therapy of CAR T cells and immunomodulatory drugs (IMiDs) such as lenalidomide. In the present study, we redirected central memory T cells to express second-generation CARs specific for either CS1 or BCMA that incorporate CD28 signaling moieties. Central memory T cells were activated by CD3/CD28 bead stimulation, transduced with lentivirus encoding the CAR construct, and expanded ex vivo. The engineered and expanded CS1 and BCMA CAR T cells exhibited similar phenotypes and comparable in vitro effector function. However, once adoptively transferred into MM tumor-bearing NOD/Scid IL2RγCnull (NSG) mice by intravenous injection of 1x10^6 CAR T cells, CS1 CAR T cells exhibited superior antitumor activity over BCMA CART cells and significantly prolonged mouse survival (P<0.01). To further improve the anti-MM activity of CAR T cell therapy, we investigated the effects of lenalidomide on CS1 CAR T cell function against MM. Central memory T cells were activated and transduced with lentivirus encoding CS1 CAR and then expanded in vitro in the presence of 0, 1 or 10mM lenalidomide for 3-4 weeks and then effector function was evaluated. We found that CD8+ CAR T cells were preferentially expanded over CD4+ CAR T cells in a dose-dependent manner. Lenalidomide-treated CAR T cells secreted higher levels of Th1 cytokines such as IFN-gamma, TNF-alpha, and IL-2, but reduced Th2 cytokines such as IL-4 and IL-10 upon antigen stimulation as compared with untreated CAR T cells. Meanwhile we observed that lenalidomide greatly improved the maintenance of T cell memory markers (CD62L, CD28, and CD27) in the culture and enhanced the formation of immune synapses between CAR T cells and MM cells. RNA-seq analysis revealed that more than 600 genes were differentially expressed among the lenalidomide treated and un-treated CD8+CAR+ T cells. Among those, expression of immune synapse related genes such as cell junction and biological assembly is significantly increased with lenalidomide treatment. Moreover, lenalidomide results in elevated gene transcrips characteristic of memory, homing and cytolytic function of CAR T cells. To test the synergistic effects, MM bearing mice were treated with a single infusion of 1x10^6 CS1 CAR T cells (i.v) on day 0 and/or 5-7.5mgkg-1 of lenalidomide daily (i.p.) initiating on day 0 for 30 days. CS1 CAR T cells and lenalidomide exhibited synergistic anti-MM activity in vivo when MM mice received combinatorial treatment. The combination therapy significantly inhibited tumor growth in vivo, prolonged mouse survival (P<0.01) and improved CAR T cell persistence in mice as compared to single-agent treatment. Taken together, these findings indicate that lenalidomide plays a co-stimulatory role in immune modulation of CAR T cells and strengthens the anti-tumor activity of CS1 CAR T cells in vivo. Rational combination of these immunotherapeutic regimens is an effective strategy and the planned clinical trial will use a combination of lenalidomide and CS1 CAR T cells for increasing treatment efficacy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Polarizing CD8+ Central Memory T Cells and Th1 Cells By Lenalidomide Contributes to the Antitumor Function of CD19 CAR-T Cells in Killing Diffused Large B Cell Lymphoma in Vitro

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4190-4190
Author(s):  
Zhen Jin ◽  
Han Liu ◽  
Molly Allen ◽  
Xiaoyang Li ◽  
Rufang Xiang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Memory T Cells ◽  
Central Memory ◽  
Central Memory T Cells ◽  
Car T Cells ◽  
Car T

Abstract Background CD19-CAR T cells with costimulatory ligand of CD28 or 4-1BB have acquired well response in ALL and CLL, whereas it shows less effective in B-cell NHL. The microenvironment of lymphomas is much more complicated than that of leukemia, which containing physical barriers and higher immunosuppression levels preventing lymphoma cells from T cell attack. To overcome such T cell toleration, one can optimize T cell fitness by adding co-stimulatory domain or polarizing T cell differentiation. Some pre-clinical studies have reported the 3rd generation of CD19-CAR T cells with CD28 and 4-1BB domain in treating ALL, but the results were in controversy. Lenalidomide has been proved to have direct anti-tumor effects in killing DLBCL cell lines except its immunomodulatory functions. Therefore, we did preliminary investigation in vitro to seek whether the combination of lenalidomide and CD19 CAR-T cells with both CD28 and 4-1BB costimulatory domain could acquire better effects Method We first verified the proliferation inhibition of lenalidomide in treating both ABC-DLBCL cell lines (Su-DHL2 and OCI-Ly3) and GCB-DLBCL cell line OCI-Ly1. CY cell was primary cells isolated from GCB-DLBCL patients in Rui-jin Hospital. Under the maximum observed plasma concentration of lenalidmomide (2.2¦ÌM), the growth inhibition in both GCB-CY and OCI-Ly1 cell line was minimal, whereas the impact on ABC-DLBCL cell lines was more obvious. We further examined the efficiency of lenalidomide in vivo using a patient-derived mouse model. The primary lymphoma cells were obtained from a ABC-DLBCL patient and subcutaneously transplanted into NOD/SCID mouses. However, daily treated with lenalidomide could not delay the tumor growth (p>0.05) (Fig A, B, C). We next isolated CD3+ T cells from healthy donors, expanded with CD3/CD28 beads. The pLenti-EF1¦Á-CD19-28-BB-¦Æ-mcherry lentiviral vectors was generated and transduced in the expanded T cells to generate CD19 CAR-T cells. T cells transduced with pLenti-EFI¦Á-Actin-mcherry lentiviral vector were used as control. CD19-CAR T cells and T cells transdued with Actin-mcherry were pretreated with 2¦ÌM lenalidomide for 72 hours. LDH assay was then performed to identify the cytotoxicity of CD19-CAR T cells against CY in 7 hours. We found that lenalidomide substantially enhanced the anti-tumor function of CD19 CAR T cells and it also promoted the CD19-CAR T cells proliferation to some extent (Fig D, E). We therefore used three DLBCL patients CAR-T cells to identify the cytokine secretion. It was found that lenalidomide promoted Th1-biased cytokines secretion (IL-2, IFN-¦Ã, TNF-¦Á) and decreased Th2-biased cytokines (IL-6, IL-10). Interestingly, CAR-T cells secreted less IFN-¦Ã and TNF-¦Á but more IL-6 and IL-10 in killing OCI-Ly3 compared with OCI-Ly1 and CY (Fig F). The results leaded us to next determine the CD19-CAR T cell differentiation. A comparable increase of CD8+CD45RA-CD62L+ CD19 CAR T cells was observed as well as the CD4+CCR6-CCR4-CXCR3+ subset, indicating lenalidomide could induce CD19 CAR T cells differentiate to CD8+ central memory T cells and Th1 cells (Fig G). As the central memory T cells are more likely to home to the lymph nodes, we found that lenalidomide considerably increased the CD19-CAR T cell migration toward CCL21 and CCL19 in transwell system (Fig H). Conclusion In conclusion, our results indicate that lenalidomide could polarize CD19-CAR T cells to CD8 TCM and Th1 subset, which might contribute to the enhanced antitumor function of CD19 CAR-T cells. Meanwhile, by overexpressed CD62L, lenalidomide could promote the migrating capability of CD19 CAR-T cells. More in-vivo work shall be done to determine the combination therapy in the future. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Optimal Dual-Targeted CAR Construct Simultaneously Targeting Bcma and GPRC5D Prevents Bcma-Escape Driven Relapse in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 136-136 ◽  
Author(s):  
Carlos Fernandez de Larrea ◽  
Mette Staehr ◽  
Andrea Lopez ◽  
Yunxin Chen ◽  
Terence J Purdon ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Car T Cells ◽  
Car T Cell ◽  
Dual Targeting ◽  
Car T ◽  
Previously Treated

Multiple myeloma (MM) remains generally incurable, calling for the development of novel treatment strategies such as chimeric antigen receptor (CAR) T cell therapy. Most clinically tested CAR T cell therapies for MM target B cell maturation antigen (BCMA), but despite high response rates, many patients relapse (Raje N. NEJM 2019). BCMA negative-low MM cells are implicated as a reservoir preceding relapse (Brudno J. JCO 2018; Cohen A. JCI 2019). Our aims are to (1) evaluate whether upfront simultaneous targeting of an additional antigen such as G protein-coupled receptor class C group 5 member D (GPRC5D; Smith EL. Sci Trans Med 2019) can mitigate BCMA escape-mediated relapse in MM, and (2) compare dual targeting strategies to identify an optimal approach. Dual targeting for CD19/CD22 malignancies has been investigated, and multiple approaches are feasible; however, approaches have yet to be comprehensively compared head to head. Here, we compare 2 parallel production and 3 single-vector dual targeting strategies (Fig. 1A). To enhance clinical translatability, all strategies are built on the BCMA(125)/4-1BBζ CAR (BCMA scFv 125; Smith EL. Mol Ther 2018), which is currently under multi-center clinical investigation (NCT03430011; Mailankody S. ASH 2018). We confirmed that all dual targeted approaches lyse, proliferate, and secrete polyfunctional cytokines specifically in response to BCMA and GPRC5D mono- and dual-positive cell lines and/or primary patient MM aspirate samples. Activity in vivo was confirmed using the bone marrow-tropic OPM2 MM model (endogenously BCMA+GPRC5D+). In all experiments MM cells (2 x 106) were injected IV into NSG mice and engrafted/expanded for 14 days before treatment. A high dose of all dual targeted CAR T cell approaches (3 x 106 CAR+) induced long-term disease control (median overall survival (mOS) BCMA(125) non-signaling del control 32d vs other groups mOS not reached; p &lt; 0.05). Prevention of latent BCMA escape-mediated relapse was evaluated by re-challenge of previously treated long-surviving mice with 2 x 106 OPM2 BCMA CRISPR KO (OPM2BCMA KO) cells at day 100 without re-treatment. While mice previously treated with BCMA(125)/41BBζ CAR T cells succumbed to OPM2BCMA KO disease, dual targeted approaches prevented OPM2BCMA KO growth (mOS BCMA mono-targeted arm 37d post re-challenge vs other groups mOS not reached; p &lt; 0.05). To better recapitulate human MM and distinguish among dual targeting approaches, we modeled established BCMA heterogeneous disease by spiking 5-10% OPM2BCMA KO into bulk OPM2WT cells for injection. Each OPM2 population was modified to express distinct luciferases for simultaneous in vivo monitoring by bioluminescent imaging (BLI). Treatment with a moderate (5 x 105) dose of CAR T cells eradicated OPM2WT cells in all groups, but anti-GPRC5D CARs with CD28 co-stimulation, whether included within a mixed T cell population or in a bicistronic construct (Fig. 1A ii, iv), failed to control OPM2BCMA KO cells (Fig. 1B). Correspondingly, 4-1BB-only containing CAR T cells had increased in vivo expansion (2.1-4.1-fold increase CAR T cell BLI at day 7 over CD28 containing groups; p &lt; 0.05). As this result is likely from greater activation-induced cell death in the CD28-containing approaches that was not rescued by 4-1BB, we later compared 4-1BB-only containing approaches (Fig. 1A i, iii, v). These 3 dual targeting approaches effectively controlled OPM2WT disease at moderate (1 x 106 CAR+) and low (2.5 x 105 CAR+) doses. However, when using a sub-therapeutic dose (2.5 x 105 CAR+) in the OPM2BCMA KO-spiked model, the tandem scFv-single stalk design was least effective in controlling OPM2BCMA KO disease (Fig 1C). At a dose that is sub-therapeutic to control OPM2WT disease (1 x 105 CAR+), the bicistronic dual 4-1BB design (Fig. 1A iii) was more effective in eradicating tumor compared with the parallel production approach (6-fold difference tumor BLI at day 28; p &lt; 0.05). These results indicate that upfront dual targeting of BCMA/GPRC5D with CAR T cells can mitigate BCMA escape-mediated relapse in a model of MM. While parallel infusion of separate BCMA- and GPRC5D-targeted CAR T cells is effective, a single bicistronic vector encoding two 4-1BB-containing CARs avoids the practical challenges of parallel manufacturing, and uniquely may provide superior anti-MM efficacy. Figure Disclosures Fernandez de Larrea: Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Brentjens:JUNO Therapeutics: Consultancy, Patents & Royalties, Research Funding; Celgene: Consultancy. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy.

scholarly journals CD19-CAR Therapy Using Naive/Memory or Central Memory T Cells Integrated into the Autologous Stem Cell Transplant Regimen for Patients with B-NHL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 610-610 ◽  
Author(s):  
Leslie Popplewell ◽  
Xiuli Wang ◽  
Suzette Blanchard ◽  
Jamie Wagner ◽  
Araceli Naranjo ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Research Funding ◽  
Memory T Cells ◽  
Central Memory ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Autologous stem cell transplantation (ASCT) remains an important consolidative therapy for patients with recurrent non-Hodgkin lymphoma (NHL), but is limited by the high incidence of NHL relapse. We report a Phase I clinical trial of ASCT followed by CD19-specific CD28-costimulatory chimeric antigen receptor (CD19:28z-CAR) T cells, with the goal of reducing NHL relapse rates. Safety and feasibility were the primary objectives, with CAR T cell persistence and expansion in the myeloablative ASCT setting as secondary objectives. This study examines safety and feasibility for two manufacturing platforms that differed in the T cell subset composition used for CAR engineering. Initially, the T cell population for CAR transduction was central memory (Tcm)-enriched: participants' peripheral blood mononuclear cells (PBMC) were depleted for CD14+ monocytes, CD25+ Tregs, and CD45RA+ naïve and stem-memory T cells, after which they were selected for CD62L+ Tcm (Wang et al. Blood;127:2980). Based on comparative preclinical data, a second arm was added to the trial to evaluate a Tn/mem-derived manufacturing platform that enriched central memory, naïve, and stem memory T cells (no CD45RA+ depletion). Either Tcm- or Tn/mem-enriched T cells were activated with CD3/CD28 beads, transduced with lentiviral vector encoding the CD19:28z-CAR, and expanded ex vivo. This phase I trial used the toxicity equivalence range design (Blanchard and Longmate. Contemp Clin Trials; 32;114) with an equivalence range for DLTs of 0.20-0.35 and a target toxicity rate of 0.25. The first 3 participants on each arm were followed one at a time, with later accrual in cohorts of 3. Twenty-three participants underwent ASCT and received CD19:28z-CAR T cells 2 days post stem cell infusion at the assigned dose level (DL): 17 on the Tcm arm (DL 50 million [M] CAR+ T cells [n=3], 200 M [n=5], 600 M [n=9]); 6 on the Tn/mem arm (DL 200 M). Participants were followed for dose limiting toxicity (DLT) for 28 days. Table 1 shows results by arm and DL. Both arms demonstrated safety and feasibility. There was no delayed hematopoietic reconstitution on either arm. On the Tcm arm, the only DLT was at DL 600 M (1 of 9 at 600 M). The Tn/mem arm was opened at 200 M and 6 participants were treated with no DLTs. The dose was not escalated as the protocol management team had seen activity at the 200M level in 2 other trials using the Tn/mem product. Tcm Arm: Fourteen of 17 participants (82%) had a diagnosis of diffuse large B-cell lymphoma (DLBCL) and 3 had mantle cell lymphoma. The mean age of the participants on the Tcm arm was 57 (35-75). The median number of prior chemotherapy regimens was 2 (1-5). The median progression-free survival (PFS) was 34.6 months 95% CI [21.8, undefined]. Seven of 17 participants (41%) have progressed, 1 died in remission of unrelated intracranial hemorrhage (6%), 7 (41%) remain in CR and are still in follow-up, and 2 are lost to follow-up (12%). All 17 participants achieved a CR or a continuing CR after ASCT and T cells. Tn/mem arm: Five of 6 participants (83%) had a DLBCL diagnosis, and 1 was NHL not otherwise specified. The mean age of the participants was 50 (40-72). The median number of prior chemotherapy regimens was 2.5 (1-3). The median follow-up time for the Tn/mem arm was 12 months, with median PFS not yet reached. One of 6 (17%) has progressed, 4 (66%) remain in CR and are still in follow-up, and 1 is lost to follow-up (17%). Five of 6 (83%) participants achieved a best response of CR or continuing CR after therapy. Several differences were observed between the manufacturing platforms. Since the Tn/mem production platform has fewer depletion steps, it resulted in a higher product yield, which shortened the ex vivo expansion period by 4.1 days (95% CI [1.5%, 6.6%]) from 18.9 days (15-24) for Tcm to 14.8 days (12-18) for Tn/mem (P<0.005). Notably in the ASCT minimal disease burden setting, the Tn/mem-derived CD19:28z-CAR T cell products exhibited significantly higher in vivo CAR T cell expansion compared to Tcm products at the 200M DL (Figure 1). We conclude that although both Tcm- and Tn/mem-enriched CD19CAR T cell therapies are safe, the Tn/mem product is more promising due to its 1) shorter production time, 2) higher cell yield, and 3) better in vivo expansion, despite the low antigen drive in these patients post-salvage and ASCT therapy. Longer follow-up for the 2-year PFS secondary objective will indicate if improved Tn/mem expansion impacts tumor control. Disclosures Wang: Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Budde:Mustang Therapeutics: Consultancy, Other: Licensing Agreement, Patents & Royalties, Research Funding. Brown:Mustang Therapeutics: Consultancy, Other: Licensing Agreement, Patents & Royalties, Research Funding. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.

scholarly journals Developing a Safer Anti-CD44v6 Chimeric Antigen Receptor T Cell Against Hematological Cancers By Mitigating on-Target Off-Tumor Toxicity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2796-2796
Author(s):  
Dennis Awuah ◽  
Lawrence Stern ◽  
Ian Schrack ◽  
Tae Yoon Kim ◽  
Joseph Cohen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Soluble Scfv

Abstract Adoptively transferred chimeric antigen receptor (CAR) T cells have shown significant promise in targeted immunotherapy against hematological tumors, despite concerns with safety and antigen escape. The CD44 adhesion molecule, which binds components of ECM such as collagen and hyaluronan, has been implicated in growth and survival as well as metastasis of tumor initiating stem cells (TSCs). A splice variant of CD44, CD44v6 is broadly expressed on malignant tumors including AML, CML and multiple myeloma (MM), where it aids tumor migration and predicts poor disease prognosis 1. Moreover, the expression of CD44v6 on healthy tissues including keratinocytes, presents a significant challenge in 'on-target, off-tumor' toxicity 2,3. We have developed a second generation, anti-CD44v6 CAR T cell (CD44v6CAR) that mediates potent cytotoxic and effector function against AML and MM in-vitro. Additionally, we observed lesser potency of our CD44v6CAR T cells against MM in comparison with AML in-vivo. Interestingly, higher levels of soluble CD44v6 antigen were also detected in mice engrafted with MM.1S compared to THP1. Possibly, the presence of soluble CD44v6 in mouse serum culminated in reduced anti-tumor activity against MM in-vivo, most likely due to CAR scFv blockade with antigen. Here, we provide an alternative strategy for improving the CD44v6 CAR T cell therapy and mitigating on-target off-tumor toxicity using CD44v6 CAR T cells that secrete a soluble protease-susceptible version of the CAR-expressed CD44v6 scFv that will block CAR binding in healthy tissue, but will be cleaved by cancer-specific proteases in the tumor site, allowing for CAR T cell binding and activation (Figure 1). We leveraged the presence of matrix metalloproteinase (MMP)-2, which is significantly overexpressed in multiple myeloma tumor microenvironment 4 to develop a modified CAR T construct (sCD44v6CAR) with engineered MMP-2 cut site in the linker between the heavy and light chains of the soluble CD44v6scFv. Kinetic analysis with varying MMP-2 concentration and digestion time showed moderate proteolytic susceptibility of our scFv construct, with digestion efficiency increasing in a dose-dependent manner. Furthermore, our engineered protease-liable scFv demonstrated higher affinity for CD44v6 antigen binding in titration assays compared to CAR scFv suggesting that engineered scFv are able to 1) bind and neutralize soluble antigen and 2) bind antigen on healthy tissues with high affinity to mitigate off-tumor toxicity. As expected, both conventional (CD44v6CAR) and modified sCD44v6 CAR T cells showed effective cytotoxicity against AML in vitro. Interestingly, cytotoxic activity against MM.1S using the modified T cells (sCD44v6CAR) was significantly suppressed, likely resulting from secretion of soluble scFv. The addition of recombinant MMP-2 in co-culture assays cleaved soluble scFvs, rescuing CAR-mediated tumor killing. Taken together, the data confirms our proof-of-concept hypothesis and highlights the protective capacity of engineered sCD44v6CAR T cells, with its ability to potentially neutralize off-target toxicity and improve anti-MM activity in future studies, which has impact on the CAR T cell therapy as a general strategy. Figure 1: Schematic representation of CAR T Cell mitigating off-disease recognition. Presence of protease-susceptible linker in soluble scFv is efficiently cleaved by tumor-specific proteases in tumor microenvironment enabling CAR binding and activation. Lack of specific proteases in healthy tissues leads to high affinity, soluble scFv-target binding and effective blocking. References 1. Heider, K. H., Kuthan, H., Stehle, G. & Munzert, G. CD44v6: a target for antibody-based cancer therapy. Cancer Immunol Immunother 53, 567-579, (2004). 2. Casucci, M. et al. CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma. Blood 122, 3461-3472, (2013). 3. Riechelmann, H. et al. Phase I trial with the CD44v6-targeting immunoconjugate bivatuzumab mertansine in head and neck squamous cell carcinoma. Oral Oncology 44, 823-829, (2008). 4. Shay, G. et al. Selective inhibition of matrix metalloproteinase-2 in the multiple myeloma-bone microenvironment. Oncotarget 8, 41827-41840, (2017). Figure 1 Figure 1. Disclosures Forman: Allogene: Consultancy; Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company; Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company. Wang: Pepromene Bio, Inc.: Consultancy.

scholarly journals CS-1 Re-Directed Central Memory T Cell Therapy for Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1114-1114 ◽  
Author(s):  
Xiuli Wang ◽  
ChingLam W Wong ◽  
Ryan Urak ◽  
Wen-Chung Chang ◽  
Elizabeth E. Budde ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
The United States ◽  
Central Memory ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Multiple myeloma (MM), a plasma cell malignancy, accounts for approximately 1 percent of all cancers and slightly more than 10 percent of hematologic malignancies in the United States. Approximately 20,000 new cases will be diagnosed this year and over 11,000 people will die from this disease. Current therapies for MM often induce remission, but nearly all patients eventually relapse and die. T-cell mediated anti-tumor therapies using genetically modify T cells with specific chimeric antigen receptors (CARs) have non-overlapping activity, toxicity and tumor resistance profiles compared to conventional chemotherapeutic agents. The main challenge in designing a CAR T cell immunotherapeutic approach is identifying the best antigen for tumor targeting. CS-1 is a cell surface glycoprotein of the signaling lymphocyte activation molecule (SLAM) receptor family that is highly and selectively expressed on normal plasma cells and MM cells, with lower expression on NK cells and little or no expression on normal tissues. This unique expression pattern and proven clinical benefit of CS-1 monoclonal antibody for the treatment of relapsed MM makes CS-1 a good target for CAR T cell therapy. Central memory T cells (TCM) from PBMC were isolated using a two-step process on the AutoMACS device to first deplete CD14+, CD45RA+ and CD25+ cells, then to positively select CD62L+ cells. These TCM undergo anti-CD3/CD28 bead stimulation and transduction with a lentiviral vector encoding CS-1 CAR containing a CD28 co-stimulatory domain and two mutations on IgG4 linker CH-2 portion to ensure enhanced potency and persistence after adoptive transfer. Gene modified CS-1 CAR T cells specifically lysed MM.1S, a MM cell line, in 4-hour 51Cr release assays and all the CAR+ cells expressed 107a upon co-cultured with the MM.1S cells. To investigate the potency of the CS-1CAR T cells, 2x106 MM.1S cells that were engineered to express GFP firefly luciferase were inoculated into NSG mice by tibia injection. 7 days post tumor engraftment, 1x106 CS-1 CAR T cells were intravenously injected into the tumor bearing mice. In contrast to untreated and mock cell treated mice in which tumor progressed rapidly systemically, single intravenous infusion of CS-1 CAR T cells induced dramatic tumor regression and significantly prolonged survival. In addition to CS-1, CD44v6 and BCMA are antigens that have also been shown to be over-expressed on MM tumor cells. We therefore compared the two CARs with CS-1 CAR for their anti-MM activity. Based on our studies, targeting CS-1 resulted in the best efficacy (Figure 1) and would be an attractive strategy for development of a clinical trial. Disclosures No relevant conflicts of interest to declare.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23
Author(s):  
Pinar Ataca Atilla ◽  
Mary K McKenna ◽  
Norihiro Watanabe ◽  
Maksim Mamonkin ◽  
Malcolm K. Brenner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Tumor Control ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Introduction: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia associated antigen with chimeric antigen receptor T (CAR T) cells have had limited success. We determined whether combinatorial expression of chimeric antigen receptors directed to two different AML associated antigens would augment tumor eradication and prevent relapse in targets with heterogeneous expression of myeloid antigens. Methods: We generated CD123 and CD33 targeting CARs; each containing a 4-1BBz or CD28z endodomain. We analyzed the anti-tumor activity of T cells expressing each CAR alone or in co-transduction with a CLL-1 CAR with CD28z endodomain and CD8 hinge previously optimized for use in our open CAR-T cell trial for AML (NCT04219163). We analyzed CAR-T cell phenotype, expansion and transduction efficacy by flow cytometry and assessed function by in vitro and in vivo activity against AML cell lines expressing high, intermediate or low levels of the target antigens (Molm 13= CD123 high, CD33 high, CLL-1 intermediate, KG1a= CD123 low, CD33 low, CLL-1 low and HL60= CD123 low, CD33 intermediate, CLL-1 intermediate/high) For in vivo studies we used NOD.SCID IL-2Rg-/-3/GM/SF (NSGS) mice with established leukemia, determining antitumor activity by bioluminescence imaging. Results: We obtained high levels of gene transfer and expression with both single (CD33.4-1BBʓ, CD123.4-1BBʓ, CD33.CD28ʓ, CD123.CD28ʓ, CLL-1 CAR) and double transduction CD33/CD123.4-1BBʓ or CD33/CD123.CD28ʓ) although single-transductants had marginally higher total CAR expression of 70%-80% versus 60-70% after co-transduction. Constructs containing CD28 co-stimulatory domain exhibited rapid expansion with elevated peak levels compared to 41BB co-stim domain irrespective of the CAR specificity. (p&lt;0.001) (Fig 1a). In 72h co-culture assays, we found consistently improved anti-tumor activity by CAR Ts expressing CLL-1 in combination either with CD33 or with CD123 compared to T cells expressing CLL-1 CAR alone. The benefit of dual expression was most evident when the target cell line expressed low levels of one or both target antigens (e.g. KG1a) (Fig 1b) (P&lt;0.001). No antigen escape was detected in residual tumor. Mechanistically, dual expression was associated with higher pCD3ʓ levels compared to single CAR T cells on exposure to any given tumor (Fig 1c). Increased pCD3ʓ levels were in turn associated with augmented CAR-T degranulation (assessed by CD107a expression) in both CD4 and CD8 T cell populations and with increased TNFα and IFNɣ production (p&lt;0.001 Fig 1d). In vivo, combinatorial targeting with CD123/CD33.CD28ʓ and CLL-1 CAR T cells improved tumor control and animal survival in lines (KG1a, MOLM13 and HL60) expressing diverse levels of the target antigens (Fig 2). Conclusion: Combinatorial targeting of T cells with CD33 or CD123.CD28z CARs and CLL-1-CAR improves CAR T cell activation associated with superior recruitment/phosphorylation of CD3ʓ, producing enhanced effector function and tumor control. The events that lead to increased pCD3ʓ after antigen engagement in the dual transduced cells may in part be due to an overall increase in CAR expression but may also reflect superior CAR recruitment after antigen engagement. We are now comparing the formation, structure, and stability of immune synapses in single and dual targeting CARs for AML. Disclosures Brenner: Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Atilla:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: founder; Marker Therapeuticsa: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Memgen: Membership on an entity's Board of Directors or advisory committees; KUUR: Membership on an entity's Board of Directors or advisory committees.

scholarly journals AAV-Mediated In Vivo CAR Gene Therapy for Targeting Human T Cell Leukemia

2021 ◽  
Author(s):  
Waqas Nawaz ◽  
Bilian Huang ◽  
Shijie Xu ◽  
Yanlei Li ◽  
Linjing Zhu ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Car T Cells ◽  
Car T Cell ◽  
Human T Cell ◽  
Car T

AbstractChimeric antigen receptor (CAR) T cell therapy is the most active field in immuno-oncology and brings substantial benefit to patients with B cell malignancies. However, the complex procedure for CAR T cell generation hampers its widespread applications. Here, we describe a novel approach in which human CAR T cells can be generated within the host upon injecting an Adeno-associated virus (AAV)vector carrying the CAR gene, which we call AAV delivering CAR gene therapy (ACG). Upon single infusion into a humanized NCG tumor mouse model of human T cell leukemia, AAV generates sufficient numbers of potent in vivo CAR cells, resulting in tumor regression; these in vivo generated CAR cells produce antitumor immunological characteristics. This instantaneous generation of in vivo CAR T cells may bypass the need for patient lymphodepletion, as well as the ex vivo processes of traditional CAR T cell production, which may make CAR therapy simpler and less expensive. It may allow the development of intricate, individualized treatments in the form of on-demand and diverse therapies.Significance StatementAAV can generate enough CAR cells within the host. That act as a living drug, distributed throughout the body, and persist for weeks, with the ability to recognize and destroy tumor cells.

scholarly journals 124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A133-A133
Author(s):  
Cheng-Fu Kuo ◽  
Yi-Chiu Kuo ◽  
Miso Park ◽  
Zhen Tong ◽  
Brenda Aguilar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

scholarly journals Next Generation NKG2D-based CAR T-cells (CYAD-02): Co-expression of a Single shRNA Targeting MICA and MICB Improves Cell Persistence and Anti-Tumor Efficacy in vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3931-3931
Author(s):  
Martina Fontaine ◽  
Benjamin Demoulin ◽  
Simon Bornschein ◽  
Susanna Raitano ◽  
Steve Lenger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Activated T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Anti Tumor Activity ◽  
Nkg2d Receptor

Background The Natural Killer Group 2D (NKG2D) receptor is a NK cell activating receptor that binds to eight different ligands (NKG2DL) commonly over-expressed in cancer, including MICA and MICB. The product candidate CYAD-01 are chimeric antigen receptor (CAR) T-cells encoding the full length human NKG2D fused to the intracellular domain of CD3ζ. Data from preclinical models have shown that CYAD-01 cells specifically target solid and hematological tumors. Encouraging preliminary results from the Phase I clinical trial THINK, assessing CYAD-01 safety, showed initial signals of objective clinical responses in patients with r/r AML and MDS. The clinical development of CAR T-cells has been limited by several challenges including achieving sufficient numbers of cells for clinical application. We have previously shown that NKG2D ligands are transiently expressed on activated T cells and that robust cell yields are generated through the addition of a blocking antibody and a PI3K inhibitor during cell manufacture. Here, we investigated the ability of an optimized short hairpin RNA (shRNA) technology to modulate NKG2DL expression on CYAD-01 cells and to determine if there is an increase in the anti-tumor activity of NKG2D-based CAR T-cells (termed CYAD-02). Methods Molecular and cellular analyses identified MICA and MICB as the key NKG2DL expressed on activated T-cells and highly likely to participate in driving fratricide. In silico analysis and in vitro screening allowed the identification of a single shRNA targeting the conserved regions of MICA and MICB, thus downregulating both MICA and MICB expression. The selected shRNA was incorporated in the NKG2D-based CAR vector, creating the next-generation NKG2D-based CAR T-cell candidate, CYAD-02. In addition, truncated versions of the NKG2D receptor were generated to explore the mechanisms of action of NKG2D receptor activity in vivo. The in vivo persistence and anti-tumor activity of CYAD-02 cells was evaluated in an aggressive preclinical model of AML. Results Injection of CAR T-cells bearing truncated forms of the NKG2D-CAR in immunosuppressed mice resulted in similar persistence to the control T-cells. In contrast, CYAD-01 cells had reduced persistence, suggesting that the recognition of the NKG2DL by the NKG2D receptor could contribute to this effect. Analysis of cell phenotype upon CAR T-cell activation showed that MICA and MICB were transiently expressed on T-cells during manufacturing. These results collectively suggested that downregulating MICA and MICB expression in CYAD-01 cells could be a mean to increase CAR T-cell persistence in vivo. Candidate shRNA were screened for efficient targeting of both MICA and MICB at the mRNA and protein level. T-cells transduced with a single vector encoding for the NKG2D-based CAR and the selected shRNA targeting MICA and MICB (CYAD-02) demonstrated 3-fold increased expansion during in vitro culture in the absence of the blocking antibody used to increase cell yield during manufacture. When injected into immunosuppressed mice, CYAD-02 cells generated with the Optimab process showed 10-fold higher engraftment one week after injection and potent anti-tumor activity resulting in 2.6-fold increase of mouse survival in an aggressive AML model. Conclusions By using a single vector encoding the NKG2D-based CAR next to a shRNA targeting MICA and MICB and combined with improved cell culture methods, CYAD-02, the next-generation of NKG2D-based CAR T-cells, demonstrated enhanced in vivo persistence and anti-tumor activity. Following FDA acceptance of the IND application, a Phase 1 dose-escalation trial evaluating the safety and clinical activity of CYAD-02 for the treatment of r/r AML and MDS is scheduled to start in early 2020. Disclosures Fontaine: Celyad: Employment. Demoulin:Celyad: Employment. Bornschein:Celyad: Employment. Raitano:Celyad: Employment. Machado:Horizon Discovery: Employment. Moore:Avvinity Therapeutics: Employment, Other: Relationship at the time the work was performed; Horizon Discovery: Employment, Equity Ownership, Other: Relationship at the time the work was performed; Centauri Therapeutics: Consultancy, Other: Current relationship. Sotiropoulou:Celyad: Employment. Gilham:Celyad: Employment.

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